| anno_start anno_end anno_text entity_type sentence section |
| 12 15 DNA chemical Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE |
| 31 46 Topoisomerase 2 protein_type Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE |
| 47 50 DNA chemical Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE |
| 73 77 Tdp2 protein Reversal of DNA damage induced Topoisomerase 2 DNA–protein crosslinks by Tdp2 TITLE |
| 0 9 Mammalian taxonomy_domain Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 10 41 Tyrosyl-DNA phosphodiesterase 2 protein Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 43 47 Tdp2 protein Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 58 73 Topoisomerase 2 protein_type Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 75 79 Top2 protein_type Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 81 84 DNA chemical Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 117 121 Top2 protein_type Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 136 139 DNA chemical Mammalian Tyrosyl-DNA phosphodiesterase 2 (Tdp2) reverses Topoisomerase 2 (Top2) DNA–protein crosslinks triggered by Top2 engagement of DNA damage or poisoning by anticancer drugs. ABSTRACT |
| 0 4 Tdp2 protein Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. ABSTRACT |
| 81 85 Top2 protein_type Tdp2 deficiencies are linked to neurological disease and cellular sensitivity to Top2 poisons. ABSTRACT |
| 18 42 X-ray crystal structures evidence Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 46 57 ligand-free protein_state Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 58 62 Tdp2 protein Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 67 75 Tdp2-DNA complex_assembly Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 112 115 DNA chemical Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 130 137 dynamic protein_state Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 138 142 Tdp2 protein Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 143 158 active site lid structure_element Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 168 192 substrate binding trench site Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 230 233 DNA chemical Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 262 266 Top2 protein_type Herein, we report X-ray crystal structures of ligand-free Tdp2 and Tdp2-DNA complexes with alkylated and abasic DNA that unveil a dynamic Tdp2 active site lid and deep substrate binding trench well-suited for engaging the diverse DNA damage triggers of abortive Top2 reactions. ABSTRACT |
| 23 27 Tdp2 protein Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT |
| 63 74 mutagenesis experimental_method Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT |
| 79 98 biochemical studies experimental_method Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT |
| 116 120 Mg2+ chemical Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT |
| 149 191 phosphotyrosyl-arginine cation-π interface site Modeling of a proposed Tdp2 reaction coordinate, combined with mutagenesis and biochemical studies support a single Mg2+-ion mechanism assisted by a phosphotyrosyl-arginine cation-π interface. ABSTRACT |
| 22 26 Tdp2 protein We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 27 38 active site site We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 48 55 ablates protein_state We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 56 60 Tdp2 protein We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 61 65 Mg2+ chemical We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 106 110 Tdp2 protein We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 128 136 tyrosine residue_name We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 206 215 etoposide chemical We further identify a Tdp2 active site SNP that ablates Tdp2 Mg2+ binding and catalytic activity, impairs Tdp2 mediated NHEJ of tyrosine blocked termini, and renders cells sensitive to the anticancer agent etoposide. ABSTRACT |
| 61 65 Tdp2 protein Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT |
| 94 97 DNA chemical Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT |
| 117 121 Top2 protein_type Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT |
| 165 169 Tdp2 protein Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT |
| 287 291 Top2 protein_type Collectively, our results provide a structural mechanism for Tdp2 engagement of heterogeneous DNA damage that causes Top2 poisoning, and indicate that evaluation of Tdp2 status may be an important personalized medicine biomarker informing on individual sensitivities to chemotherapeutic Top2 poisons. ABSTRACT |
| 8 11 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO |
| 41 44 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO |
| 49 64 RNA polymerases protein_type Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO |
| 94 97 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO |
| 138 141 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO |
| 205 208 DNA chemical Nuclear DNA compaction and the action of DNA and RNA polymerases create positive and negative DNA supercoiling—over- and under-winding of DNA strands, respectively—and the linking together (catenation) of DNA strands. INTRO |
| 0 14 Topoisomerases protein_type Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO |
| 35 38 DNA chemical Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO |
| 94 97 DNA chemical Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO |
| 121 124 DNA chemical Topoisomerases relieve topological DNA strain and entanglement to facilitate critical nuclear DNA transactions including DNA replication, transcription and cell division. INTRO |
| 4 13 mammalian taxonomy_domain The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO |
| 14 36 type II topoisomerases protein_type The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO |
| 37 42 Top2α protein The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO |
| 47 52 Top2β protein The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO |
| 92 95 DNA chemical The mammalian type II topoisomerases Top2α and Top2β enzymes generate transient, reversible DNA double strand breaks (DSBs) to drive topological transactions. INTRO |
| 17 21 Top2 protein_type Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 22 25 DNA chemical Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 92 115 phosphotyrosyl linkages ptm Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 128 140 5′-phosphate chemical Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 175 186 active site site Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 187 191 Top2 protein_type Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 192 200 tyrosine residue_name Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 215 219 Top2 protein_type Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 240 246 Top2cc complex_assembly Reversibility of Top2 DNA cleavage reactions is facilitated by formation of covalent enzyme phosphotyrosyl linkages between the 5′-phosphate ends of the incised duplex and an active site Top2 tyrosine, resulting in Top2 cleavage complexes (Top2cc). INTRO |
| 4 10 Top2cc complex_assembly The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries. INTRO |
| 19 22 DNA chemical The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries. INTRO |
| 117 123 Top2cc complex_assembly The Top2cc protein–DNA adduct is a unique threat to genomic integrity which must be resolved to prevent catastrophic Top2cc collisions with the cellular replication and transcription machineries. INTRO |
| 30 34 Top2 protein_type To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO |
| 111 120 etoposide chemical To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO |
| 122 132 teniposide chemical To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO |
| 137 148 doxorubicin chemical To promote cancer cell death, Top2 reactions are ‘poisoned’ by keystone pharmacological anticancer agents like etoposide, teniposide and doxorubicin. INTRO |
| 13 17 Top2 protein_type Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A). INTRO |
| 71 74 DNA chemical Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A). INTRO |
| 181 184 DNA chemical Importantly, Top2 is also poisoned when it engages abundant endogenous DNA damage not limited to but including ribonucleotides, abasic sites and alkylation damage such as exocyclic DNA adducts arising from bioactivation of the vinyl chloride carcinogen (Figure 1A). INTRO |
| 15 18 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO |
| 36 42 Top2cc complex_assembly In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO |
| 53 56 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO |
| 117 120 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO |
| 160 164 Top2 protein_type In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO |
| 165 168 DNA chemical In the case of DNA damage-triggered Top2cc, compound DNA lesions arise that consist of the instigating lesion, and a DNA DSB bearing a bulky terminal 5′-linked Top2 DNA–protein crosslink. INTRO |
| 27 30 DNA chemical The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO |
| 46 52 Top2cc complex_assembly The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO |
| 71 74 DNA chemical The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO |
| 145 148 DNA chemical The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO |
| 221 227 Top2cc complex_assembly The chemical complexity of DNA damage-derived Top2cc necessitates that DNA repair machinery dedicated to resolving these lesions recognizes both DNA and protein, whilst accommodating diverse chemical structures that trap Top2cc. INTRO |
| 27 30 DNA chemical Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored. INTRO |
| 106 112 Top2cc complex_assembly Precisely how the cellular DNA repair machinery navigates these complex lesions is an important aspect of Top2cc repair that has not yet been explored. INTRO |
| 0 4 Tdp2 protein Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. FIG |
| 15 38 phosphotyrosyl linkages ptm Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. FIG |
| 50 53 DNA chemical Tdp2 processes phosphotyrosyl linkages in diverse DNA damage contexts. FIG |
| 15 18 DNA chemical (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG |
| 65 68 DNA chemical (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG |
| 121 125 Top2 protein_type (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG |
| 135 139 Top2 protein_type (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG |
| 158 164 Top2cc complex_assembly (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG |
| 196 200 Top2 protein_type (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG |
| 228 243 phosphotyrosine residue_name (A) Unrepaired DNA damage and repair intermediates such as bulky DNA adducts, ribonucleotides or abasic sites can poison Top2 and trap Top2 cleavage complex (Top2cc), resulting in a DSB with a 5′–Top2 protein adduct linked by a phosphotyrosine bond. FIG |
| 0 4 Tdp2 protein Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG |
| 23 38 phosphotyrosine residue_name Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG |
| 68 72 Top2 protein_type Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG |
| 81 84 DNA chemical Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG |
| 97 109 5′-phosphate chemical Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG |
| 129 132 DNA chemical Tdp2 hydrolyzes the 5′–phosphotyrosine adduct derived from poisoned Top2 leaving DNA ends with a 5′-phosphate, which facilitates DNA end joining through the NHEJ pathway. FIG |
| 4 7 DNA chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 85 103 Tdp2 enzyme assays experimental_method (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 112 124 deoxyadenine chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 126 128 dA chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 131 144 Ethenoadenine chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 146 148 ϵA chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 156 167 abasic site site (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 169 172 THF chemical (B) DNA oligonucleotide substrates synthesized by EDC-imidazole coupling and used in Tdp2 enzyme assays contain deoxyadenine (dA), Ethenoadenine (ϵA) or an abasic site (THF) and a 5′–nitrophenol moiety. FIG |
| 0 14 Phosphotyrosyl ptm Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 44 52 mTdp2cat structure_element Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 62 75 p-nitrophenol chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 134 142 mTdp2cat structure_element Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 143 157 reaction rates evidence Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 161 174 p–nitrophenol chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 184 187 DNA chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 252 255 PNP chemical Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 272 280 mTdp2cat structure_element Phosphotyrosyl bond hydrolysis catalyzed by mTdp2cat releases p-nitrophenol, which is detected by measuring absorbance at 415 nm. (C) mTdp2cat reaction rates on p–nitrophenol modified DNA substrates shown in panel B. Rates are reported as molecules of PNP s−1 produced by mTdp2cat. FIG |
| 0 8 P-values evidence P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG |
| 37 43 t-test experimental_method P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG |
| 111 120 Structure evidence P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG |
| 124 132 mTdp2cat structure_element P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG |
| 133 141 bound to protein_state P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG |
| 142 158 5′-phosphate DNA chemical P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG |
| 188 190 ϵA chemical P-values calculated using two-tailed t-test; error bars, s.d. n = 4, n.s. = not statistically significant. (D) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow). FIG |
| 0 22 DNA binding β2Hβ–grasp site DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG |
| 128 131 DNA chemical DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG |
| 0 22 DNA binding β2Hβ–grasp site DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG |
| 128 131 DNA chemical DNA binding β2Hβ–grasp (tan) and cap elements engage the 5′-nucleotide as well as the +2 and +3 nucleotides (blue) of substrate DNA. FIG |
| 51 60 Structure evidence PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG |
| 64 72 mTdp2cat structure_element PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG |
| 73 81 bound to protein_state PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG |
| 82 98 5′-phosphate DNA chemical PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG |
| 128 131 THF chemical PDB entry 5HT2 is displayed, also see Table 1. (E) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow). FIG |
| 51 60 Structure evidence PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 64 72 mTdp2cat structure_element PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 80 90 absence of protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 91 94 DNA chemical PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 107 115 extended protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 116 128 3-helix loop structure_element PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 135 139 open protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 160 177 DNA-binding grasp site PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 189 196 monomer oligomeric_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 197 198 E structure_element PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 206 209 apo protein_state PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 210 219 structure evidence PDB entry 5INK is displayed, also see Table 1. (F) Structure of mTdp2cat in the absence of DNA showing the extended 3-helix loop (tan) open-conformation of the DNA-binding grasp as seen in monomer E of the apo structure. FIG |
| 0 31 Tyrosyl DNA phosphodiesterase 2 protein Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO |
| 33 37 Tdp2 protein Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO |
| 59 76 5′-phosphotyrosyl ptm Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO |
| 78 82 5′-Y ptm Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO |
| 84 92 linkages ptm Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO |
| 160 164 Top2 protein_type Tyrosyl DNA phosphodiesterase 2 (Tdp2) directly hydrolyzes 5′-phosphotyrosyl (5′-Y) linkages, and is a key modulator of cellular resistance to chemotherapeutic Top2 poisons. INTRO |
| 0 4 Tdp2 protein Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO |
| 5 14 knockdown experimental_method Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO |
| 52 61 etoposide chemical Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO |
| 158 162 Tdp2 protein Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO |
| 175 181 Top2cc complex_assembly Tdp2 knockdown sensitizes A549 lung cancer cells to etoposide, and increases formation of nuclear γH2AX foci, a marker of DSBs, underlining the importance of Tdp2 in cellular Top2cc repair. INTRO |
| 0 4 Tdp2 protein Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO |
| 76 82 mutant protein_state Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO |
| 83 86 p53 protein Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO |
| 106 112 mutant protein_state Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO |
| 113 116 p53 protein Tdp2 is overexpressed in lung cancers, is transcriptionally up-regulated in mutant p53 cells and mediates mutant p53 gain of function phenotypes, which can lead to acquisition of therapy resistance during cancer progression. INTRO |
| 18 22 Tdp2 protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO |
| 36 49 topoisomerase protein_type The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO |
| 98 103 human species The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO |
| 104 108 TDP2 protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO |
| 237 244 loss of protein_state The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO |
| 245 249 Tdp2 protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO |
| 259 264 Top2β protein The importance of Tdp2 in mediating topoisomerase biology is further underlined by the facts that human TDP2 inactivating mutations are found in individuals with intellectual disabilities, seizures and ataxia, and at the cellular level, loss of Tdp2 inhibits Top2β-dependent transcription. INTRO |
| 20 24 TDP2 protein It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood. INTRO |
| 93 97 Tdp2 protein It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood. INTRO |
| 149 153 Tdp2 protein It is possible that TDP2 single nucleotide polymorphisms (SNPs) encode mutations that impact Tdp2 function, but the molecular underpinnings for such Tdp2 deficiencies are not understood. INTRO |
| 39 44 X-ray experimental_method Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 45 63 crystal structures evidence Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 71 99 minimal catalytically active protein_state Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 100 136 endonuclease/exonuclease/phosphatase structure_element Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 138 141 EEP structure_element Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 153 158 mouse taxonomy_domain Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 159 163 Tdp2 protein Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 165 173 mTdp2cat structure_element Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 175 183 bound to protein_state Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 186 189 DNA chemical Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 213 230 5′-phosphorylated protein_state Previously we reported high-resolution X-ray crystal structures of the minimal catalytically active endonuclease/exonuclease/phosphatase (EEP) domain of mouse Tdp2 (mTdp2cat) bound to a DNA substrate mimic, and a 5′-phosphorylated reaction product. INTRO |
| 56 60 Tdp2 protein However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain. INTRO |
| 90 93 DNA chemical However, important questions regarding the mechanism of Tdp2 engagement and processing of DNA damage remain. INTRO |
| 24 28 Tdp2 protein First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO |
| 39 62 phosphotyrosyl linkages ptm First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO |
| 81 84 DNA chemical First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO |
| 106 112 Top2cc complex_assembly First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO |
| 169 172 DNA chemical First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO |
| 191 202 active site site First, it is unclear if Tdp2 processes phosphotyrosyl linkages in the context of DNA damage that triggers Top2cc, and if so, how the enzyme can accommodate such complex DNA damage within its active site. INTRO |
| 9 20 metal-bound protein_state Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO |
| 21 25 Tdp2 protein Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO |
| 26 36 structures evidence Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO |
| 64 68 Mg2+ chemical Based on metal-bound Tdp2 structures, we also proposed a single Mg2+ mediated catalytic mechanism, but this mechanism requires further scrutiny and characterization. INTRO |
| 32 56 structure-function study experimental_method Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 64 68 Tdp2 protein Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 120 125 X-ray experimental_method Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 126 136 structures evidence Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 140 151 ligand-free protein_state Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 152 156 Tdp2 protein Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 162 166 Tdp2 protein Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 167 175 bound to protein_state Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 198 217 1-N6-etheno-adenine chemical Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 219 222 DNA chemical Herein, we report an integrated structure-function study of the Tdp2 reaction mechanism, including a description of new X-ray structures of ligand-free Tdp2, and Tdp2 bound to abasic and alkylated (1-N6-etheno-adenine) DNA damage. INTRO |
| 28 47 structural analysis experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 49 60 mutagenesis experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 62 79 functional assays experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 84 121 quanyum mechanics/molecular mechanics experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 123 128 QM/MM experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 130 138 modeling experimental_method Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 146 150 Tdp2 protein Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 194 198 Tdp2 protein Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 227 256 tyrosyl DNA phosphodiesterase protein_type Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 295 298 DNA chemical Our integrated results from structural analysis, mutagenesis, functional assays and quanyum mechanics/molecular mechanics (QM/MM) modeling of the Tdp2 reaction coordinate describe in detail how Tdp2 mediates a single-metal ion tyrosyl DNA phosphodiesterase reaction capable of acting on diverse DNA end damage. INTRO |
| 26 29 DNA chemical We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. INTRO |
| 52 56 Tdp2 protein We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. INTRO |
| 57 68 active site site We further establish that DNA damage binding in the Tdp2 active site is linked to conformational change and binding of metal cofactor. INTRO |
| 27 31 Tdp2 protein Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO |
| 41 48 ablates protein_state Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO |
| 53 57 Tdp2 protein Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO |
| 58 83 single metal binding site site Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO |
| 88 92 Tdp2 protein Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO |
| 147 151 Top2 protein_type Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO |
| 172 181 mammalian taxonomy_domain Finally, we characterize a Tdp2 SNP that ablates the Tdp2 single metal binding site and Tdp2 substrate induced conformational changes, and confers Top2 drug sensitivity in mammalian cells. INTRO |
| 0 4 Tdp2 protein Tdp2 processing of compound DNA damage RESULTS |
| 28 31 DNA chemical Tdp2 processing of compound DNA damage RESULTS |
| 11 15 Top2 protein_type Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS |
| 48 51 DNA chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS |
| 69 72 DNA chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS |
| 92 110 1-N6-ethenoadenine chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS |
| 112 114 ϵA chemical Two potent Top2 poisons include bulky alkylated DNA helix-distorting DNA base adducts (e.g. 1-N6-ethenoadenine, ϵA) and abundant abasic sites (Figure 1A). RESULTS |
| 8 12 Tdp2 protein Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known. RESULTS |
| 23 46 phosphotyrosyl linkages ptm Whether Tdp2 processes phosphotyrosyl linkages within these diverse structural contexts is not known. RESULTS |
| 28 47 EDC coupling method experimental_method To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS |
| 72 85 p-nitrophenol chemical To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS |
| 87 90 PNP chemical To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS |
| 137 140 DNA chemical To test this, we adapted an EDC coupling method to generate 5′-terminal p-nitrophenol (PNP) modified oligonucleotides that also harbored DNA damage at the 5′-nucleotide position (see Materials and Methods). RESULTS |
| 56 61 mouse taxonomy_domain We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 62 66 Tdp2 protein We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 67 83 catalytic domain structure_element We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 85 93 mTdp2cat structure_element We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 106 109 PNP chemical We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 135 148 topoisomerase protein_type We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 149 157 tyrosine residue_name We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 200 203 DNA chemical We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 223 241 colorimetric assay experimental_method We then evaluated the ability of a recombinant purified mouse Tdp2 catalytic domain (mTdp2cat) to release PNP (a structural mimic of a topoisomerase tyrosine) from the 5′-terminus of compound damaged DNA substrates using a colorimetric assay (Figure 1B). RESULTS |
| 18 22 Tdp2 protein We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS |
| 44 47 PNP chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS |
| 100 106 dA-PNP chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS |
| 108 114 ϵA-PNP chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS |
| 141 163 tetrahydrofuran spacer chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS |
| 165 168 THF chemical We observe robust Tdp2-dependent release of PNP from 5′-modified oligonucleotides in the context of dA-PNP, ϵA-PNP or the abasic-site analog tetrahydrofuran spacer (THF) (Figure 1C). RESULTS |
| 6 10 Tdp2 protein Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS |
| 31 54 phosphotyrosyl linkages ptm Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS |
| 123 126 DNA chemical Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS |
| 139 142 DNA chemical Thus, Tdp2 efficiently cleaves phosphotyrosyl linkages in the context of a compound 5′ lesions composed of abasic or bulky DNA base adduct DNA damage. RESULTS |
| 38 42 Tdp2 protein To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 57 63 Top2cc complex_assembly To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 82 85 DNA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 97 124 crystallized and determined experimental_method To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 125 130 X-ray experimental_method To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 131 149 crystal structures evidence To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 153 161 mTdp2cat structure_element To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 162 170 bound to protein_state To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 171 187 5′-phosphate DNA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 213 218 5′-ϵA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 272 275 DNA chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 289 295 5′-THF chemical To understand the molecular basis for Tdp2 processing of Top2cc in the context of DNA damage, we crystallized and determined X-ray crystal structures of mTdp2cat bound to 5′-phosphate DNA (product complex) with a 5′-ϵA at 1.43 Å resolution (PDB entry 5HT2) and the abasic DNA damage mimic 5′-THF at 2.15 Å resolution (PDB entry 5INK; Figure 1D and E, Table 1). RESULTS |
| 9 17 Tdp2-DNA complex_assembly In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS |
| 26 36 structures evidence In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS |
| 38 46 mTdp2cat structure_element In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS |
| 56 70 mixed α-β fold structure_element In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS |
| 93 129 12-stranded anti-parallel β-sandwich structure_element In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS |
| 182 186 Tdp2 protein In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS |
| 187 198 active site site In these Tdp2-DNA complex structures, mTdp2cat adopts a mixed α-β fold typified by a central 12-stranded anti-parallel β-sandwich enveloped by several helical elements that mold the Tdp2 active site. RESULTS |
| 70 87 DNA-binding cleft site One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate. RESULTS |
| 139 142 DNA chemical One half of the molecule contributes to formation of the walls of the DNA-binding cleft that embraces the terminal position of the damaged DNA substrate. RESULTS |
| 7 23 DNA lesion-bound protein_state In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 39 42 DNA chemical In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 65 76 β-2-helix-β structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 78 82 β2Hβ structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 85 90 grasp structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 98 109 helical cap structure_element In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 120 144 substrate binding trench site In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 160 165 ssDNA chemical In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 202 213 active site site In the DNA lesion-bound state, two key DNA binding elements, the β-2-helix-β (β2Hβ) ‘grasp’, and ‘helical cap’ mold the substrate binding trench and direct the ssDNA of a 5′-overhang substrate into the active site. RESULTS |
| 34 43 structure evidence A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS |
| 47 55 DNA-free protein_state A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS |
| 56 60 Tdp2 protein A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS |
| 62 65 apo protein_state A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS |
| 100 104 loop structure_element A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS |
| 108 131 conformationally mobile protein_state A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS |
| 159 162 DNA chemical A comparison to an additional new structure of DNA-free Tdp2 (apo state, Figure 1F) shows that this loop is conformationally mobile and important for engaging DNA substrates. RESULTS |
| 57 59 ϵA chemical The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS |
| 93 97 Tdp2 protein The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS |
| 106 120 5′-tyrosylated protein_state The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS |
| 162 165 DNA chemical The mode of engagement of the 5′-nucleobase of the bulky ϵA adduct describes a mechanism for Tdp2 to bind 5′-tyrosylated substrates that contain diverse forms of DNA damage. RESULTS |
| 4 9 5′-ϵA chemical The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS |
| 50 54 Tdp2 protein The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS |
| 55 88 van Der Waals interaction surface site The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS |
| 115 131 hydrophobic wall site The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS |
| 183 189 Leu315 residue_name_number The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS |
| 194 200 Ile317 residue_name_number The 5′-ϵA nucleobase is recognized by an extended Tdp2 van Der Waals interaction surface, referred to here as the ‘hydrophobic wall’ that is assembled with the sidechains of residues Leu315 and Ile317 (Figure 2A and B). RESULTS |
| 0 10 Structures evidence Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG |
| 14 22 mTdp2cat structure_element Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG |
| 23 31 bound to protein_state Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG |
| 32 35 DNA chemical Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG |
| 57 61 Top2 protein_type Structures of mTdp2cat bound to DNA damage that triggers Top2 poisoning. FIG |
| 4 13 Structure evidence (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 17 25 mTdp2cat structure_element (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 26 34 bound to protein_state (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 35 51 5′-phosphate DNA chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 81 83 ϵA chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 94 98 Mg2+ chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 130 136 waters chemical (A) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing ϵA (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 0 8 mTdp2cat structure_element mTdp2cat is colored by electrostatic surface potential (red = negative, blue = positive, gray = neutral/hydrophobic). FIG |
| 4 44 σ-A weighted 2Fo-Fc electron density map evidence (B) σ-A weighted 2Fo-Fc electron density map (at 1.43 Å resolution, contoured at 2.0 σ) for the ϵA DNA complex. FIG |
| 96 102 ϵA DNA chemical (B) σ-A weighted 2Fo-Fc electron density map (at 1.43 Å resolution, contoured at 2.0 σ) for the ϵA DNA complex. FIG |
| 4 6 ϵA chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 43 56 hydrogen bond bond_interaction The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 66 68 ϵA chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 89 94 water chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 124 133 Structure evidence The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 137 145 mTdp2cat structure_element The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 146 154 bound to protein_state The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 155 171 5′-phosphate DNA chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 201 204 THF chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 215 219 Mg2+ chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 251 257 waters chemical The ϵA nucleotide is shown in yellow and a hydrogen bond from the ϵA O4′ to inner-sphere water is shown as gray dashes. (C) Structure of mTdp2cat bound to 5′-phosphate DNA (product complex) containing THF (yellow), Mg2+ (magenta) and its inner-sphere waters (gray). FIG |
| 0 8 mTdp2cat structure_element mTdp2cat is colored with red (electronegative), blue (electropositive) and gray (hydrophobic) electrostatic surface potential displayed. FIG |
| 33 73 σ-A weighted 2Fo-Fc electron density map evidence PDB entry 5INK is displayed. (D) σ-A weighted 2Fo-Fc electron density map (at 2.15 Å resolution, contoured at 2.0 σ) for THF-DNA complex. FIG |
| 121 128 THF-DNA complex_assembly PDB entry 5INK is displayed. (D) σ-A weighted 2Fo-Fc electron density map (at 2.15 Å resolution, contoured at 2.0 σ) for THF-DNA complex. FIG |
| 4 7 THF chemical The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG |
| 33 46 hydrogen bond bond_interaction The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG |
| 56 59 THF chemical The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG |
| 80 85 water chemical The THF is shown in yellow and a hydrogen bond from the THF O4′ to inner-sphere water is shown as gray dashes. FIG |
| 24 34 determined experimental_method For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS |
| 37 46 structure evidence For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS |
| 63 73 5′-adenine chemical For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS |
| 75 80 5′-dA chemical For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS |
| 82 90 bound to protein_state For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS |
| 91 95 Tdp2 protein For comparison, we also determined a structure of an undamaged 5′-adenine (5′-dA) bound to Tdp2 at 1.55 Å (PDB entry 5INL). RESULTS |
| 2 20 structural overlay experimental_method A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS |
| 127 132 bound protein_state A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS |
| 147 150 DNA chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS |
| 167 169 ϵA chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS |
| 171 173 dA chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS |
| 178 180 dC chemical A structural overlay of damaged and undamaged nucleotides shows no major distortions to nucleotide planarity between different bound sequences and DNA damage (compare ϵA, dA and dC, Supplementary Figure S1A–D). RESULTS |
| 67 69 ϵG chemical Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 71 73 ϵT chemical Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 111 115 Tdp2 protein Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 116 127 active site site Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 132 152 planar base stacking bond_interaction Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 162 173 active site site Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 181 197 hydrophobic wall site Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 205 209 β2Hβ structure_element Therefore, structurally diverse undamaged or alkylated bases (e.g. ϵG, ϵT) could likely be accommodated in the Tdp2 active site via planar base stacking with the active site facing hydrophobic wall of the β2Hβ motif. RESULTS |
| 14 32 abasic deoxyribose chemical Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5′-terminus (Figure 2C). RESULTS |
| 40 43 THF chemical Likewise, the abasic deoxyribose analog THF substrate binds similar to the alkylated and non-alkylated substrates, but with a slight alteration in the approach of the 5′-terminus (Figure 2C). RESULTS |
| 22 32 absence of protein_state Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS |
| 58 61 THF chemical Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS |
| 104 109 water chemical Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS |
| 173 177 Mg2+ chemical Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS |
| 178 200 ion coordination shell bond_interaction Interestingly, in the absence of a nucleobase, O4′ of the THF ring adopts a close approach (2.8 Å) to a water molecule that directly participates in the outer sphere single Mg2+ ion coordination shell (Figure 2D). RESULTS |
| 108 111 THF chemical These collective differences may explain the slight, but statistically significant elevated activity on the THF substrate (Figure 1C). RESULTS |
| 29 33 Tdp2 protein Structural plasticity in the Tdp2 DNA binding trench RESULTS |
| 34 52 DNA binding trench site Structural plasticity in the Tdp2 DNA binding trench RESULTS |
| 29 45 DNA-damage bound protein_state An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS |
| 66 70 Tdp2 protein An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS |
| 71 82 active site site An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS |
| 119 124 water chemical An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS |
| 170 184 catalytic core site An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS |
| 193 215 DNA binding β2Hβ-grasp site An intriguing feature of the DNA-damage bound conformation of the Tdp2 active site is an underlying network of protein–water–protein contacts that span a gap between the catalytic core and the DNA binding β2Hβ-grasp (Supplementary Figure S2). RESULTS |
| 68 78 β2Hβ-grasp site In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS |
| 92 106 hydrogen bonds bond_interaction In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS |
| 138 142 Mg2+ chemical In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS |
| 150 156 Asp358 residue_name_number In this arrangement, six solvent molecules form a channel under the β2Hβ-grasp, ending with hydrogen bonds to the peptide backbone of the Mg2+ ligand Asp358. RESULTS |
| 15 39 hydrophobic interactions bond_interaction The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS |
| 56 60 β2Hβ structure_element The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS |
| 61 70 DNA-bound protein_state The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS |
| 131 135 β2Hβ structure_element The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS |
| 158 161 DNA chemical The paucity of hydrophobic interactions stabilizing the β2Hβ DNA-bound conformation suggests that conformational plasticity in the β2Hβ might be a feature of DNA damage and metal cofactor engagement. RESULTS |
| 28 40 crystallized experimental_method To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS |
| 41 45 Tdp2 protein To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS |
| 53 63 absence of protein_state To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS |
| 64 67 DNA chemical To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS |
| 85 93 DNA free protein_state To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS |
| 94 98 Tdp2 protein To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS |
| 99 108 structure evidence To test this hypothesis, we crystallized Tdp2 in the absence of DNA and determined a DNA free Tdp2 structure to 2.4 Å resolution (PDB entry 5INM; Figures 1F and 3A). RESULTS |
| 33 37 Tdp2 protein Conformational plasticity in the Tdp2 active site. FIG |
| 38 49 active site site Conformational plasticity in the Tdp2 active site. FIG |
| 8 12 open protein_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 14 21 3-helix structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 44 52 flexible protein_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 53 69 active-site loop structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 82 89 monomer oligomeric_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 90 91 E structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 99 107 DNA-free protein_state (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 108 116 mTdp2cat structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 117 126 structure evidence (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 160 164 T309 residue_name_number (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 198 201 EEP structure_element (A) The open, 3-helix conformation (tan) of flexible active-site loop observed in monomer E of the DNA-free mTdp2cat structure (PDB entry 5INM) is supported by T309 (green), which packs against the EEP core. FIG |
| 4 23 β2Hβ docking pocket site The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG |
| 61 65 N312 residue_name_number The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG |
| 67 71 N314 residue_name_number The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG |
| 76 80 L315 residue_name_number The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG |
| 94 109 solvent-exposed protein_state The β2Hβ docking pocket (circled) is unoccupied and residues N312, N314 and L315 (orange) are solvent-exposed. FIG |
| 44 50 closed protein_state Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 51 55 β2Hβ structure_element Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 76 88 mTdp2cat–DNA complex_assembly Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 97 106 structure evidence Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 118 123 5′-ϵA chemical Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 150 154 T309 residue_name_number Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 190 212 β2Hβ DNA-binding grasp site Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 223 237 hydrogen bonds bond_interaction Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 257 261 Y321 residue_name_number Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 269 273 N314 residue_name_number Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 296 315 β2Hβ docking pocket site Wall-eyed stereo view is displayed. (B) The closed β2Hβ conformation in the mTdp2cat–DNA product structure containing 5′-ϵA (yellow, PDB entry 5HT2). T309 (green) is an integral part of the β2Hβ DNA-binding grasp (tan) and hydrogen bonds to the backbone of Y321, while N314 (orange) occupies the β2Hβ docking pocket. FIG |
| 53 69 active site loop structure_element Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 99 108 promoters oligomeric_state Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 116 124 DNA-free protein_state Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 125 133 mTdp2cat structure_element Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 208 226 sequence alignment experimental_method Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 264 280 electron density evidence Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 301 328 Limited trypsin proteolysis experimental_method Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 369 377 flexible protein_state Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 378 394 active-site loop structure_element Wall-eyed stereo view is displayed. (C) Alignment of active site loop conformers observed in the 5 promoters of the DNA-free mTdp2cat (PDB entry 5INM, see Table 1) crystallographic asymmetric unit (left) and sequence alignment showing residues not observed in the electron density as ‘∼’ (right). (D) Limited trypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 0 8 mTdp2cat structure_element mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 9 11 WT protein_state mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 28 36 mTdp2cat structure_element mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 37 42 D358N mutant mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 79 87 presence protein_state mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 91 101 absence of protein_state mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 102 106 Mg2+ chemical mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 156 159 DNA chemical mTdp2cat WT (lanes 1–13) or mTdp2cat D358N (lanes 14–26) were incubated in the presence or absence of Mg2+ and/or a 12 nt self annealing, 5′-phosphorylated DNA (substrate ‘12 nt’ in Supplementary Table S1), then reacted with 0.6, 1.7 or 5 ng μl−1 of trypsin. FIG |
| 28 36 SDS-PAGE experimental_method Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 98 130 Limited chymotrypsin proteolysis experimental_method Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 171 179 flexible protein_state Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 180 196 active-site loop structure_element Reactions were separated by SDS-PAGE and proteins visualized by staining with coomassie blue. (E) Limited chymotrypsin proteolysis probes the solvent accessibility of the flexible active-site loop. FIG |
| 40 48 mTdp2cat structure_element Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG |
| 49 51 WT protein_state Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG |
| 69 77 mTdp2cat structure_element Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG |
| 78 83 D358N mutant Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG |
| 108 120 chymotrypsin experimental_method Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG |
| 132 139 trypsin experimental_method Experiments performed as in panel D for mTdp2cat WT (lanes 27–39) or mTdp2cat D358N (lanes 40–52), but with chymotrypsin instead of trypsin. FIG |
| 29 33 Tdp2 protein This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS |
| 95 106 active site site This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS |
| 107 111 Mg2+ chemical This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS |
| 126 149 substrate binding loops structure_element This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS |
| 178 187 protomers oligomeric_state This crystal form contains 5 Tdp2 protein molecules in the asymmetric unit, with variations in active site Mg2+ occupancy and substrate binding loops observed for the individual protomers. RESULTS |
| 33 48 DNA ligand-free protein_state The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS |
| 67 89 active site β2Hβ-grasp site The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS |
| 150 159 DNA-bound protein_state The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS |
| 161 167 closed protein_state The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS |
| 168 190 β2Hβ DNA binding grasp site The most striking feature of the DNA ligand-free state is that the active site β2Hβ-grasp can adopt alternative structures that are distinct from the DNA-bound, closed β2Hβ DNA binding grasp (Figure 3A and B). RESULTS |
| 7 14 monomer oligomeric_state In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS |
| 16 25 chain ‘E’ structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS |
| 32 37 grasp structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS |
| 49 53 open protein_state In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS |
| 55 67 3-helix loop structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS |
| 109 112 EEP structure_element In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS |
| 113 127 catalytic core site In one monomer (chain ‘E’), the grasp adopts an ‘open’ 3-helix loop conformation that projects away from the EEP catalytic core. RESULTS |
| 4 12 monomers oligomeric_state Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS |
| 27 37 disordered protein_state Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS |
| 67 83 DNA binding loop structure_element Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS |
| 106 122 electron density evidence Two monomers have variable disordered states for which much of the DNA binding loop is not visible in the electron density. RESULTS |
| 35 43 DNA-free protein_state The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS |
| 44 56 crystal form evidence The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS |
| 61 67 closed protein_state The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS |
| 68 72 β2Hβ structure_element The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS |
| 99 108 DNA bound protein_state The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS |
| 109 119 structures evidence The remaining two molecules in the DNA-free crystal form are closed β2Hβ conformers similar to the DNA bound structures (Figure 3C). RESULTS |
| 20 24 Tdp2 protein Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS |
| 25 28 DNA chemical Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS |
| 66 72 closed protein_state Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS |
| 85 95 β2Hβ grasp site Thus, we posit that Tdp2 DNA binding conformationally selects the closed form of the β2Hβ grasp, rather than inducing closure upon binding. RESULTS |
| 27 35 extended protein_state A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. RESULTS |
| 36 43 3-helix structure_element A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. RESULTS |
| 72 94 substrate-binding loop structure_element A detailed analysis of the extended 3-helix conformation shows that the substrate-binding loop is able to undergo metamorphic structural changes. RESULTS |
| 8 12 open protein_state In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 28 41 Asn312-Leu315 residue_range In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 62 73 active site site In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 78 93 solvent-exposed protein_state In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 128 134 Thr309 residue_name_number In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 183 189 pocket site In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 197 200 EEP structure_element In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 220 224 loop structure_element In this open form, residues Asn312-Leu315 are distal from the active site and solvent-exposed (orange sticks, Figure 3A), while Thr309 (green surface, Figure 3A) packs into a shallow pocket of the EEP core to anchor the loop. RESULTS |
| 10 16 Thr309 residue_name_number Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS |
| 53 69 cis–peptide bond bond_interaction Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS |
| 78 84 Asp308 residue_name_number Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS |
| 85 91 Thr309 residue_name_number Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS |
| 115 145 short antiparallel beta-strand structure_element Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS |
| 153 157 β2Hβ structure_element Burial of Thr309 is enabled by an unusual main chain cis–peptide bond between Asp308-Thr309 and disassembly of the short antiparallel beta-strand of the β2Hβ fold. RESULTS |
| 19 25 closed protein_state By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS |
| 26 36 β2Hβ grasp site By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS |
| 64 70 Asn312 residue_name_number By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS |
| 75 81 Asn314 residue_name_number By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS |
| 99 119 β2Hβ docking pockets site By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS |
| 125 131 Leu315 residue_name_number By comparison, the closed β2Hβ grasp conformer is stabilized by Asn312 and Asn314 binding into two β2Hβ docking pockets, and Leu315 engagement of the 5′-terminal nucleobase (Figure 3B). RESULTS |
| 23 29 closed protein_state To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS |
| 30 34 β2Hβ structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS |
| 49 55 Thr309 residue_name_number To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS |
| 76 79 EEP structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS |
| 87 93 pocket site To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS |
| 168 172 β2Hβ structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS |
| 173 193 antiparallel β-sheet structure_element To transition into the closed β2Hβ conformation, Thr309 disengages from the EEP domain pocket, flips peptide backbone conformation cis to trans, and is integral to the β2Hβ antiparallel β-sheet. RESULTS |
| 21 27 closed protein_state Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS |
| 28 38 β2Hβ-grasp site Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS |
| 69 80 active site site Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS |
| 91 104 hydrogen bond bond_interaction Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS |
| 113 119 Trp307 residue_name_number Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS |
| 128 153 Mg2+ coordinating residue site Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS |
| 154 160 Asp358 residue_name_number Stabilization of the closed β2Hβ-grasp conformation is linked to the active site through a hydrogen bond between Trp307 and the Mg2+ coordinating residue Asp358. RESULTS |
| 20 28 DNA free protein_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 29 38 structure evidence Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 71 77 closed protein_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 78 86 monomers oligomeric_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 103 107 Mg2+ chemical Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 121 133 active sites site Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 145 153 monomers oligomeric_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 159 163 open protein_state Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 210 228 metal binding site site Accordingly, in the DNA free structure, we observe a trend where the 2 closed monomers have an ordered Mg2+ ion in their active sites, while the monomers with open conformations have a poorly ordered or vacant metal binding site. RESULTS |
| 71 74 DNA chemical Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS |
| 147 169 β2Hβ DNA binding grasp site Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS |
| 217 221 Mg2+ chemical Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS |
| 237 241 Tdp2 protein Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS |
| 242 253 active site site Overall, these observations suggest that engagement of diverse damaged DNA ends is enabled by an elaborate substrate selected stabilization of the β2Hβ DNA binding grasp, and these rearrangements are coordinated with Mg2+ binding in the Tdp2 active site. RESULTS |
| 12 16 Mg2+ chemical To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS |
| 21 24 DNA chemical To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS |
| 35 39 Tdp2 protein To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS |
| 81 89 mTdp2cat structure_element To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS |
| 110 154 limited trypsin and chymotrypsin proteolysis experimental_method To evaluate Mg2+ and DNA-dependent Tdp2 structural states in solution, we probed mTdp2cat conformations using limited trypsin and chymotrypsin proteolysis (Figure 3C–E). RESULTS |
| 0 17 In the absence of protein_state In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 18 21 DNA chemical In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 25 30 Mg2+, chemical In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 31 39 mTdp2cat structure_element In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 82 99 DNA binding grasp site In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 115 122 trypsin experimental_method In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 124 130 Arg316 residue_name_number In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 156 168 chymotrypsin experimental_method In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 170 176 Trp307 residue_name_number In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 181 187 Leu315 residue_name_number In the absence of DNA or Mg2+, mTdp2cat is efficiently cleaved in the metamorphic DNA binding grasp at one site by trypsin (Arg316), or at two positions by chymotrypsin (Trp307 and Leu315). RESULTS |
| 15 20 Mg2+, chemical By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS |
| 45 49 Mg2+ chemical By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS |
| 50 53 DNA chemical By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS |
| 109 117 mTdp2cat structure_element By comparison, Mg2+, and to a greater extent Mg2+/DNA mixtures (compare Figure 3, lanes 4, 7 and 13) protect mTdp2cat from proteolytic cleavage. RESULTS |
| 27 31 Mg2+ chemical Interestingly, addition of Mg2+ alone protects against proteolysis as well. RESULTS |
| 24 28 Mg2+ chemical This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 45 51 closed protein_state This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 72 82 β2Hβ-grasp site This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 103 127 hydrogen-bonding network bond_interaction This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 133 139 Asp358 residue_name_number This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 167 177 β2Hβ-grasp site This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 186 192 Trp307 residue_name_number This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 219 223 Tdp2 protein This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 224 235 active site site This is consistent with Mg2+ stabilizing the closed conformation of the β2Hβ-grasp through an extended hydrogen-bonding network with Asp358 and the indole ring of the β2Hβ-grasp residue Trp307 (also discussion below on Tdp2 active site SNPs). RESULTS |
| 37 41 Tdp2 protein To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 73 78 human species To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 83 88 mouse taxonomy_domain To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 89 93 Tdp2 protein To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 103 113 determined experimental_method To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 133 142 structure evidence To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 150 155 human species To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 156 163 Tdp2cat structure_element To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 171 179 bound to protein_state To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 182 192 DNA 5′-PO4 chemical To assess structural conservation of Tdp2 conformational changes between human and mouse Tdp2, we also determined a 3.2 Å resolution structure of the human Tdp2cat domain bound to a DNA 5′-PO4 terminus product complex (PDB entry 5INO). RESULTS |
| 19 24 human species Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS |
| 25 37 hTdp2cat-DNA complex_assembly Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS |
| 46 55 structure evidence Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS |
| 63 71 mTdp2cat structure_element Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS |
| 72 81 DNA bound protein_state Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS |
| 129 138 DNA-bound protein_state Comparisons of the human hTdp2cat-DNA complex structure to the mTdp2cat DNA bound state show a high level of conservation of the DNA-bound conformations (Supplementary Figure S3A). RESULTS |
| 21 29 mTdp2cat structure_element Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS |
| 61 69 hTdp2cat structure_element Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS |
| 70 92 substrate binding loop structure_element Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS |
| 117 121 Mg2+ chemical Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS |
| 126 129 DNA chemical Moreover, similar to mTdp2cat, proteolytic protection of the hTdp2cat substrate binding loop occurs with addition of Mg2+ and DNA (Supplementary Figure S3B). RESULTS |
| 6 11 X-ray experimental_method Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS |
| 12 22 structures evidence Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS |
| 27 55 limited proteolysis analysis experimental_method Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS |
| 70 73 DNA chemical Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS |
| 122 131 conserved protein_state Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS |
| 147 157 vertebrate taxonomy_domain Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS |
| 158 162 Tdp2 protein Thus, X-ray structures and limited proteolysis analysis indicate that DNA- and metal-induced conformational changes are a conserved feature of the vertebrate Tdp2-substrate interaction. RESULTS |
| 0 4 Tdp2 protein Tdp2 metal ion dependence RESULTS |
| 32 57 X-ray structural analyses experimental_method Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS |
| 90 94 Mg2+ chemical Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS |
| 101 109 bound in protein_state Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS |
| 114 118 Tdp2 protein Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS |
| 119 130 active site site Consistently in high-resolution X-ray structural analyses we, and others observe a single Mg2+ metal bound in the Tdp2 active site. RESULTS |
| 18 26 DNA-free protein_state This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 40 56 DNA damage bound protein_state This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 73 95 reaction product-bound protein_state This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 96 109 crystal forms evidence This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 113 118 mouse taxonomy_domain This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 138 146 D. rerio species This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 168 178 C. elegans species This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 179 183 Tdp2 protein This includes the DNA-free (Figure 3A), DNA damage bound (Figure 3B) and reaction product-bound crystal forms of mouse, (PDB entry 4GZ1), D. rerio (PDB entry 4FPV) and C. elegans Tdp2 (PDB entry 4FVA). RESULTS |
| 18 38 biochemical analysis experimental_method However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction. RESULTS |
| 100 104 Tdp2 protein However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction. RESULTS |
| 105 137 phosphotyrosyl phosphodiesterase protein_type However, previous biochemical analysis has suggested an alternative two-metal ion mechanism for the Tdp2-phosphotyrosyl phosphodiesterase reaction. RESULTS |
| 34 38 Mg2+ chemical In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity. RESULTS |
| 55 59 Ca2+ chemical In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity. RESULTS |
| 72 76 Tdp2 protein In these experiments, at limiting Mg2+ concentrations, Ca2+ addition to Tdp2 reactions stimulated activity. RESULTS |
| 64 78 phosphotyrosyl ptm While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS |
| 96 100 Tdp2 protein While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS |
| 164 187 metal ion binding sites site While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS |
| 203 214 active site site While this work was suggestive of a two metal ion mechanism for phosphotyrosyl bond cleavage by Tdp2, we note that second metal ion titrations can be influenced by metal ion binding sites outside of the active site. RESULTS |
| 9 24 divalent metals chemical In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS |
| 51 55 Tdp2 protein In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS |
| 64 67 DNA chemical In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS |
| 109 122 active center site In fact, divalent metals have been observed in the Tdp2 protein–DNA complexes (PDB entry 4GZ2) distal to the active center, and we propose this might account for varied results in different studies. RESULTS |
| 49 53 Tdp2 protein To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 54 71 phosphodiesterase protein_type To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 95 119 metal ion binding assays experimental_method To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 132 150 crystal structures evidence To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 158 169 presence of protein_state To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 177 192 divalent metals chemical To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 194 198 Mn2+ chemical To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 203 208 Ca2+) chemical To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 251 255 Tdp2 protein To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 256 288 phosphotyrosyl phosphodiesterase protein_type To further probe the metal ion dependence of the Tdp2 phosphodiesterase reaction, we performed metal ion binding assays, determined crystal structures in the presence of varied divalent metals (Mn2+ and Ca2+), and analyzed metal ion dependence of the Tdp2 phosphotyrosyl phosphodiesterase reaction (Figure 4). RESULTS |
| 33 37 Tdp2 protein Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding. FIG |
| 43 76 Intrinsic tryptophan fluorescence evidence Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding. FIG |
| 80 88 mTdp2cat structure_element Metal cofactor interactions with Tdp2. (A) Intrinsic tryptophan fluorescence of mTdp2cat was used to monitor a conformational response to divalent metal ion binding. FIG |
| 7 11 Mg2+ chemical Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG |
| 15 19 Ca2+ chemical Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG |
| 25 33 titrated experimental_method Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG |
| 41 49 presence protein_state Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG |
| 53 63 absence of protein_state Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG |
| 64 72 5′-P DNA chemical Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG |
| 82 105 tryptophan fluorescence evidence Either Mg2+ or Ca2+ were titrated in the presence or absence of 5′-P DNA, and the tryptophan fluorescence was monitored with an excitation wavelength of 280 nm and emission wavelength of 350 nm using 10 nm band pass filters. FIG |
| 5 9 Mg2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 14 18 Ca2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 79 102 tryptophan fluorescence evidence Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 106 114 mTdp2cat structure_element Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 122 130 presence protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 135 145 absence of protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 146 149 DNA chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 157 162 D358N mutant Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 163 174 active site site Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 175 181 mutant protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 185 193 mTdp2cat structure_element Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 197 209 unresponsive protein_state Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 213 217 Mg2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 223 231 mTdp2cat structure_element Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 254 259 T5PNP chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 287 291 Mg2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 296 300 Ca2+ chemical Both Mg2+ and Ca2+ induce a conformational change which elicits an increase in tryptophan fluorescence of mTdp2cat in the presence and absence of DNA, while D358N active site mutant of mTdp2cat is unresponsive to Mg2+. (B) mTdp2cat activity assayed on a T5PNP substrate as a function of Mg2+ and Ca2+ concentration. FIG |
| 0 3 PNP chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 65 69 Mg2+ chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 95 102 absence protein_state PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 106 117 presence of protein_state PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 129 133 Ca2+ chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 172 212 σ-A weighted 2Fo-Fc electron density map evidence PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 224 265 model-phased anomalous difference Fourier evidence PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 276 280 maps evidence PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 289 306 mTdp2cat–DNA–Mn2+ complex_assembly PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 346 350 Mn2+ chemical PNP release (monitored by absorbance at 415 nm) as a function of Mg2+ concentration and in the absence or presence of 1 or 10 mM Ca2+ is shown; error bars, s.d. n = 4. (C) σ-A weighted 2Fo-Fc electron density map (blue) and model-phased anomalous difference Fourier (magenta) maps for the mTdp2cat–DNA–Mn2+ complex (PDB entry 5INP) show a single Mn2+ (cyan) is bound with expected octahedral coordination geometry. FIG |
| 18 50 anomalous difference Fourier map evidence A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn2+ as the identity of this atom. FIG |
| 93 97 Mn2+ chemical A 53σ peak in the anomalous difference Fourier map (data collected at λ = 1.5418 Å) supports Mn2+ as the identity of this atom. FIG |
| 18 22 Ca2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 30 34 Ca2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 47 50 DNA chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 74 78 Mg2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 88 92 Mg2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 105 108 DNA chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 127 139 mTdp2cat–DNA complex_assembly (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 140 150 structures evidence (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 162 166 Ca2+ chemical (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 180 205 5′-phosphate binding mode site (D) Comparison of Ca2+ (green Ca2+ ion, orange DNA) (PDB entry 5INQ), and Mg2+ (magenta Mg2+ ion, yellow DNA) (PDB entry 4GZ1) mTdp2cat–DNA structures shows that Ca2+ distorts the 5′-phosphate binding mode. FIG |
| 4 15 proteolysis experimental_method Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding. RESULTS |
| 35 39 Mg2+ chemical Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding. RESULTS |
| 50 54 Tdp2 protein Our proteolysis results indicate a Mg2+-dependent Tdp2 conformational response to metal binding. RESULTS |
| 4 8 Tdp2 protein The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS |
| 9 20 active site site The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS |
| 31 41 tryptophan residue_name The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS |
| 70 90 metal binding center site The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS |
| 106 139 intrinsic tryptophan fluorescence evidence The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS |
| 190 198 mTdp2cat structure_element The Tdp2 active site has three tryptophan residues within 10 Å of the metal binding center, so we assayed intrinsic tryptophan fluorescence to detect metal-induced conformational changes in mTdp2cat. RESULTS |
| 76 84 presence protein_state These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A). RESULTS |
| 89 99 absence of protein_state These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A). RESULTS |
| 100 103 DNA chemical These data were an excellent fit to a single-site binding model both in the presence and absence of DNA (Figure 4A). RESULTS |
| 23 27 Mg2+ chemical This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS |
| 28 30 Kd evidence This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS |
| 70 87 Hill coefficients evidence This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS |
| 124 142 metal binding site site This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS |
| 155 163 presence protein_state This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS |
| 168 178 absence of protein_state This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS |
| 179 182 DNA chemical This analysis revealed Mg2+ Kd values in the sub-millimolar range and Hill coefficients which were consistent with a single metal binding site both in the presence and absence of DNA (Supplementary Table S2). RESULTS |
| 56 60 Tdp2 protein We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat. RESULTS |
| 73 109 p-nitrophenyl-thymidine-5′-phosphate chemical We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat. RESULTS |
| 113 121 mTdp2cat structure_element We then measured effects of metal ion concentrations on Tdp2 cleavage of p-nitrophenyl-thymidine-5′-phosphate by mTdp2cat. RESULTS |
| 72 75 DNA chemical This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site. RESULTS |
| 104 115 active site site This small molecule substrate is not expected to be influenced by metal–DNA coordination outside of the active site. RESULTS |
| 23 27 Ca2+ chemical Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B). RESULTS |
| 103 107 Tdp2 protein Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B). RESULTS |
| 149 153 Mg2+ chemical Inclusion of ultrapure Ca2+ (1 mM or 10 mM) results in a dose-dependent inhibition but not stimulation Tdp2 activity, even in conditions of limiting Mg2+ (Figure 4B). RESULTS |
| 22 32 titrations experimental_method We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS |
| 38 43 human species We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS |
| 44 51 hTdp2FL protein We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS |
| 56 64 hTdp2cat structure_element We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS |
| 136 140 Mg2+ chemical We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS |
| 159 163 Ca2+ chemical We performed the same titrations with human hTdp2FL and hTdp2cat (Supplementary Figure S4), and find similar stimulation of activity by Mg2+ and inhibition by Ca2+. RESULTS |
| 15 37 metal binding analyses experimental_method Overall, these metal binding analyses are consistent with a single metal ion mediated reaction. RESULTS |
| 72 76 Tdp2 protein To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 77 88 active site site To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 104 122 crystal structures evidence To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 126 142 soaking crystals experimental_method To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 176 183 support protein_state To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 185 189 Mn2+ chemical To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 194 201 inhibit protein_state To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 203 207 Ca2+ chemical To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 224 228 Tdp2 protein To further evaluate the structural influence of divalent cations on the Tdp2 active site, we determined crystal structures by soaking crystals with metal cofactors that either support (Mn2+) or inhibit (Ca2+, Figure 4B) the Tdp2 reaction (PDB entries 5INP and 5INQ). RESULTS |
| 0 33 Anomalous difference Fourier maps evidence Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 41 54 Tdp2–DNA–Mn2+ complex_assembly Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 77 89 binding site site Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 94 98 Mn2+ chemical Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 107 111 Tdp2 protein Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 112 123 active site site Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 142 165 octahedral coordination bond_interaction Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 195 199 Mn2+ chemical Anomalous difference Fourier maps of the Tdp2–DNA–Mn2+ complex show a single binding site for Mn2+ in each Tdp2 active site (Figure 4C), with octahedral coordination and bond lengths typical for Mn2+ ligands (Supplementary Table S3). RESULTS |
| 4 8 Mn2+ chemical The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS |
| 34 38 Tdp2 protein The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS |
| 39 50 active site site The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS |
| 66 76 Mg2+-bound protein_state The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS |
| 138 142 Mn2+ chemical The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS |
| 161 165 Tdp2 protein The Mn2+ ion is positioned in the Tdp2 active site similar to the Mg2+-bound complex (Figure 2C), which is consistent with the ability of Mn2+ to support robust Tdp2 catalytic activity. RESULTS |
| 19 40 co-complex structures evidence In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS |
| 46 50 Ca2+ chemical In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS |
| 81 85 Ca2+ chemical In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS |
| 148 157 Mg2+ site site In contrast, while co-complex structures with Ca2+ also show a single metal ion, Ca2+ binds in a slightly different position, shifted ∼1 Å from the Mg2+ site. RESULTS |
| 9 13 Ca2+ chemical Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS |
| 22 46 octahedrally coordinated bond_interaction Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS |
| 76 80 Ca2+ chemical Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS |
| 124 128 Ca2+ chemical Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS |
| 149 162 Mg2+ ion site site Although Ca2+ is also octahedrally coordinated, longer bond lengths for the Ca2+ ligands (Supplementary Table S3) shift the Ca2+ ion relative to the Mg2+ ion site. RESULTS |
| 15 53 bi-dentate inner sphere metal contacts bond_interaction Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS |
| 63 67 Ca2+ chemical Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS |
| 75 81 Glu162 residue_name_number Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS |
| 94 128 active site phosphate-binding mode site Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS |
| 147 153 5′-PO4 chemical Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS |
| 165 169 Tdp2 protein Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS |
| 170 181 active site site Interestingly, bi-dentate inner sphere metal contacts from the Ca2+ ion to Glu162 distort the active site phosphate-binding mode, and dislodge the 5′-PO4 out of the Tdp2 active site (Figure 4D). RESULTS |
| 71 75 Ca2+ chemical Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS |
| 112 116 Tdp2 protein Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS |
| 131 151 divalent metal bound protein_state Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS |
| 152 156 Tdp2 protein Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS |
| 157 167 structures evidence Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS |
| 192 196 Ca2+ chemical Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS |
| 224 228 Tdp2 protein Together with results showing that under the conditions examined here, Ca2+ inhibits rather than stimulates the Tdp2 reaction, the divalent metal bound Tdp2 structures provide a mechanism for Ca2+-mediated inhibition of the Tdp2 reaction. RESULTS |
| 13 17 Tdp2 protein Modeling the Tdp2 reaction coordinate RESULTS |
| 56 60 Mg2+ chemical Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 75 84 simulated experimental_method Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 89 93 Tdp2 protein Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 119 140 hybrid QM/MM modeling experimental_method Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 147 151 Tdp2 protein Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 152 169 substrate analog- protein_state Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 174 187 product-bound protein_state Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 188 198 structures evidence Next, to examine the feasibility of our proposed single Mg2+ mechanism, we simulated the Tdp2 reaction coordinate with hybrid QM/MM modeling using Tdp2 substrate analog- and product-bound structures as guides. RESULTS |
| 9 28 structural analyses experimental_method Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS |
| 45 58 superposition experimental_method Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS |
| 64 67 DNA chemical Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS |
| 85 100 5′-aminohexanol chemical Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS |
| 115 121 5′-PO4 chemical Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS |
| 155 159 Tdp2 protein Previous structural analyses showed that the superposition of a DNA substrate mimic (5′-aminohexanol) and product (5′-PO4) complexes delineates a probable Tdp2 reaction trajectory characterized by inversion of stereochemistry about the adducted 5′-phosphorus. RESULTS |
| 53 58 water chemical In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS |
| 76 91 hydrogen bonded bond_interaction In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS |
| 95 101 Asp272 residue_name_number In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS |
| 106 112 Asn274 residue_name_number In this scheme (Figure 5A), a candidate nucleophilic water that is strongly hydrogen bonded to Asp272 and Asn274, is well positioned for the in-line nucleophilic attack ∼180° opposite of the P–O bond of the 5′-Tyr adduct. RESULTS |
| 35 39 Tdp2 protein Structure-function analysis of the Tdp2 reaction mechanism. FIG |
| 41 56 phosphotyrosine residue_name (A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2. FIG |
| 65 69 Tdp2 protein (A) Proposed mechanism for hydrolysis of phosphotyrosine bond by Tdp2. FIG |
| 27 39 binding-site site Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. FIG |
| 48 59 5′-tyrosine residue_name Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. FIG |
| 70 79 phosphate chemical Residues in green form the binding-site for the 5′-tyrosine (red) and phosphate, yellow bind the 5′ nucleotide and blue bind nucleotides 2–3. FIG |
| 34 39 mTdp2 protein Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG |
| 53 64 Free energy evidence Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG |
| 76 92 QM/MM simulation experimental_method Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG |
| 144 149 water chemical Residue numbers shown are for the mTdp2 homolog. (B) Free energy during the QM/MM simulation as a function of distance between the nucleophilic water and 5′-phosphorus atom. FIG |
| 57 69 mTdp2cat-DNA complex_assembly Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states. FIG |
| 89 119 QM/MM reaction path simulation experimental_method Reaction proceeds from right to left. (C) Models for the mTdp2cat-DNA complex during the QM/MM reaction path simulation showing the substrate (left, tan), transition state intermediate (center, cyan) and product (right, pink) states. FIG |
| 34 39 mTdp2 protein Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG |
| 53 84 Electrostatic surface potential evidence Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG |
| 100 118 5′-phosphotyrosine residue_name Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG |
| 157 168 presence of protein_state Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG |
| 171 191 cation–π interaction bond_interaction Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG |
| 222 228 Arg216 residue_name_number Residue numbers shown are for the mTdp2 homolog. (D) Electrostatic surface potential calculated for 5′-phosphotyrosine in isolation (upper panel) and in the presence of a cation–π interaction with the guanidinium group of Arg216 (lower panel) shows electron-withdrawing effect of this interaction. FIG |
| 0 23 Electrostatic potential evidence Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG |
| 161 170 wild-type protein_state Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG |
| 175 181 mutant protein_state Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG |
| 182 187 human species Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG |
| 188 191 MBP experimental_method Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG |
| 192 200 hTdp2cat structure_element Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG |
| 201 216 fusion proteins experimental_method Electrostatic potential color gradient extends from positive (red) through neutral (gray), to negative (blue). (E) Bar graph displaying the relative activity of wild-type and mutant human MBP-hTdp2cat fusion proteins on the three substrates. FIG |
| 11 14 PNP chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG |
| 20 23 PNP chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG |
| 24 33 phosphate chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG |
| 38 43 T5PNP chemical Release of PNP from PNP phosphate and T5PNP was detected as an increase in absorbance at 415 nm. FIG |
| 0 14 Reaction rates evidence Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG |
| 68 76 wildtype protein_state Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG |
| 77 80 MBP experimental_method Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG |
| 81 89 hTdp2cat structure_element Reaction rates are expressed as the percent of activity relative to wildtype MBP-hTdp2cat; error bars, s.d. FIG |
| 11 16 hTdp2 protein Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated. FIG |
| 55 60 mTdp2 protein Mutants of hTdp2 (black) and the equivalent residue in mTdp2 (tan) are indicated. FIG |
| 65 70 water chemical We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C). RESULTS |
| 114 119 water chemical We examined the energy profile of the nucleophilic attack of the water molecule by using the distance between the water oxygen and the P atom on the phosphate moiety as the sole reaction coordinate in the present calculation (Figure 5B and C). RESULTS |
| 62 70 mTdp2cat structure_element A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 71 86 5′–aminohexanol chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 104 113 structure evidence A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 132 140 tyrosine residue_name A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 155 170 5′-aminohexanol chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 187 191 Mg2+ chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 209 215 waters chemical A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 225 234 mTdp2-DNA complex_assembly A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 243 252 structure evidence A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 298 327 molecular dynamics simulation experimental_method A starting model was generated from atomic coordinates of the mTdp2cat 5′–aminohexanol substrate analog structure (PDB 4GZ0) with a tyrosine replacing the 5′-aminohexanol then adding the Mg2+ and inner-sphere waters from the mTdp2-DNA product structure (PDB, 4GZ1), and running an initial round of molecular dynamics simulation (10 ns) to allow the system to reach an equilibrium. RESULTS |
| 6 24 QM/MM optimization experimental_method After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS |
| 160 168 mTdp2cat structure_element After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS |
| 169 184 5′-aminohexanol chemical After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS |
| 202 211 structure evidence After QM/MM optimization of this model (Figure 5C, ‘i-substrate’), the O–P distance is 3.4 Å, which is in agreement with the range of distances observed in the mTdp2cat 5′-aminohexanol substrate analog structure (3.2–3.4 Å). RESULTS |
| 10 15 water chemical Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS |
| 48 54 Asp272 residue_name_number Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS |
| 80 93 hydrogen bond bond_interaction Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS |
| 123 137 phosphotyrosyl ptm Here, the water proton and the neighboring O of Asp272 participates in a strong hydrogen bond (distance of 1.58 Å) and the phosphotyrosyl O–P distance is stretched to 1.77 Å, which is 0.1 Å beyond an equilibrium bond length. RESULTS |
| 35 45 simulation experimental_method In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS |
| 54 59 water chemical In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS |
| 108 121 hydrogen bond bond_interaction In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS |
| 147 152 water chemical In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS |
| 157 163 Asp272 residue_name_number In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS |
| 190 195 water chemical In the subsequent two steps of the simulation, as the water-phosphate O–P distance reduces to 1.98 Å, a key hydrogen bond between the nucleophilic water and Asp272 shortens to 1.38 Å as the water H–O bond approaches the point of dissociation. RESULTS |
| 25 30 water chemical The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS |
| 62 75 hydrogen bond bond_interaction The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS |
| 81 87 Asn274 residue_name_number The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS |
| 155 160 water chemical The second proton on the water nucleophile maintains a strong hydrogen bond with Asn274 throughout the reaction, implicating this residue in orienting the water nucleophile during the reaction. RESULTS |
| 27 41 phosphotyrosyl ptm Concomitant with this, the phosphotyrosyl O–P bond weakens (d = 1.89 Å), and the formation of the penta-covalent transition state (Figure 5C ‘ii-transition state’) is observed. RESULTS |
| 57 66 phosphate chemical The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond. RESULTS |
| 111 125 phosphotyrosyl ptm The final steps show inversion of stereochemistry at the phosphate, along with lengthening and breaking of the phosphotyrosyl O–P bond. RESULTS |
| 78 83 water chemical Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base. RESULTS |
| 87 93 Asp272 residue_name_number Product formation is coupled to a transfer of a proton from the nucleophillic water to Asp272, consistent with the proposed function for this residue as the catalytic base. RESULTS |
| 55 62 His 359 residue_name_number Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation. RESULTS |
| 104 114 simulation experimental_method Of note, both nitrogens of the imidazole side chain of His 359 require protonation for stability of the simulation. RESULTS |
| 0 7 Asp 326 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 16 29 hydrogen bond bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 40 46 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 69 80 salt bridge bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 101 111 protonated protein_state Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 120 126 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 170 173 Asp residue_name Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 174 177 His residue_name Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 190 193 EEP structure_element Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 204 208 APE1 protein Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 229 232 pKa evidence Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 241 244 His residue_name Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 302 315 hydrogen bond bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 328 345 doubly protonated protein_state Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 346 352 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 361 370 phosphate chemical Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 426 430 Mg2+ chemical Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 449 455 His359 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 485 491 H-bond bond_interaction Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 501 507 Asp326 residue_name_number Asp 326 makes a hydrogen bond to N∂1 of His359, suggesting that this salt bridge could stabilize the protonated form of His359 as has been demonstrated for the analogous Asp-His pair in the EEP domain of APE1, which elevates the pKa of this His above 8.0. In our model, the transition state contains a hydrogen bond between the doubly protonated His359 and the phosphate oxygen that also coordinates with the single catalytic Mg2+, while the second His359 imidazole proton maintains a H-bond with the Asp326 residue throughout the reaction. RESULTS |
| 23 32 structure evidence In the final optimized structure, the observed product state (Figure 5C, ‘iii-product’) is found in a conformation that is 7.4 kcal mol−1 more stable than the initial reactive state (Figure 5B). RESULTS |
| 4 12 tyrosine residue_name The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 34 48 coordinated to bond_interaction The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 53 57 Mg2+ chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 118 122 Mg2+ chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 148 153 water chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 196 205 phosphate chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 220 226 Glu162 residue_name_number The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 259 263 Mg2+ chemical The tyrosine oxy-anion product is coordinated to the Mg2+ ion with a 2.0 Å distance, which is the shortest of the six Mg2+ ligands (including three water molecules, one of the free oxygens on the phosphate group and the Glu162 residue), indicating the single Mg2+ greatly stabilizes the product oxy-anion. RESULTS |
| 48 62 QM/MM modeling experimental_method An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. RESULTS |
| 99 103 Top2 protein_type An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. RESULTS |
| 104 112 tyrosine residue_name An additional striking feature gleaned from the QM/MM modeling is the putative binding mode of the Top2 tyrosine-leaving group. RESULTS |
| 10 19 conserved protein_state A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS |
| 30 37 Tyr 188 residue_name_number A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS |
| 39 46 Arg 216 residue_name_number A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS |
| 51 58 Ser 239 residue_name_number A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS |
| 81 90 conserved protein_state A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS |
| 91 95 Top2 protein_type A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS |
| 96 119 tyrosine binding pocket site A trio of conserved residues (Tyr 188, Arg 216 and Ser 239) forms the walls of a conserved Top2 tyrosine binding pocket. RESULTS |
| 16 36 cation–π interaction bond_interaction We propose this cation–π interaction further contributes to tuned stabilization of the negatively charged phenolate reaction product. RESULTS |
| 34 57 electrostatic potential evidence Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS |
| 65 79 phosphotyrosyl ptm Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS |
| 116 124 presence protein_state Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS |
| 129 139 absence of protein_state Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS |
| 144 150 Arg216 residue_name_number Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS |
| 171 177 Arg216 residue_name_number Consistent with this, analysis of electrostatic potential of the phosphotyrosyl moiety using Gaussian 09.D01 in the presence and absence of the Arg216 guanidinium reveals Arg216 is strongly electron withdrawing (Figure 5D). RESULTS |
| 45 65 cation–π interaction bond_interaction We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS |
| 127 133 Arg216 residue_name_number We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS |
| 143 145 QM experimental_method We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS |
| 160 162 MM experimental_method We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS |
| 223 239 energy penalties evidence We further examined the contribution of this cation–π interaction to the reaction chemistry by moving the guanidinium group of Arg216 from the QM system to the MM system as either a +1 or ∼0 charge species, and re-computed energy penalties for each step in the reaction coordinate (Supplementary Figure S5A). RESULTS |
| 9 15 Arg216 residue_name_number Removing Arg216 from the quantum subsystem incurs an ∼2 kcal mol−1 penalty in the transition state and product complex. RESULTS |
| 30 36 Arg216 residue_name_number Removing the +1 charge on the Arg216 has a minimal impact on the transition state, but incurs an additional ∼2 kcal mol−1 penalty in the product complex. RESULTS |
| 12 26 QM/MM modeling experimental_method Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS |
| 62 66 Tdp2 protein Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS |
| 114 118 Mg2+ chemical Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS |
| 170 174 Tdp2 protein Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS |
| 185 203 5′-phosphotyrosine residue_name Altogether, QM/MM modeling identifies new determinants of the Tdp2 reaction, and demonstrates our proposed single Mg2+ catalyzed reaction model is a viable mechanism for Tdp2-catalyzed 5′-phosphotyrosine bond hydrolysis. RESULTS |
| 0 4 Tdp2 protein Tdp2 mutational analysis RESULTS |
| 5 24 mutational analysis experimental_method Tdp2 mutational analysis RESULTS |
| 27 31 Tdp2 protein To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 95 100 mouse taxonomy_domain To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 101 105 Tdp2 protein To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 106 124 crystal structures evidence To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 186 191 mouse taxonomy_domain To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 205 228 engineered and purified experimental_method To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 238 243 human species To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 244 247 MBP experimental_method To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 248 256 hTdp2cat structure_element To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 257 263 mutant protein_state To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 325 330 human species To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 367 376 mutations experimental_method To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 380 384 Tdp2 protein To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 457 468 tyrosylated protein_state To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 469 472 DNA chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 484 488 5′-Y ptm To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 491 514 p-nitrophenyl phosphate chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 516 520 PNPP chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 526 572 thymidine 5′-monophosphate p-nitrophenyl ester chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 574 579 T5PNP chemical To test the aspects of the Tdp2 reaction mechanism described here derived from high-resolution mouse Tdp2 crystal structures (denoted with superscript numbering ‘m’ for numbering of the mouse protein), we engineered and purified thirteen human MBP-hTdp2cat mutant proteins (denoted with superscript numbering and ‘h’ for the human protein) and assayed the impacts of mutations on Tdp2 catalytic activity using three in vitro reporter substrates including a tyrosylated DNA substrate (5′-Y), p-nitrophenyl phosphate (PNPP) and thymidine 5′-monophosphate p-nitrophenyl ester (T5PNP) (Figure 5E, Supplementary Figures S5B and S5C). RESULTS |
| 132 136 Tdp2 protein By analyzing activities on this nested set of chemically related substrates we aimed to dissect structure-activity relationships of Tdp2 catalysis. RESULTS |
| 13 22 mutations experimental_method For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 33 37 Tdp2 protein For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 38 49 active site site For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 64 78 phosphotyrosyl ptm For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 174 177 DNA chemical For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 224 228 5′-Y ptm For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 233 238 T5PNP chemical For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 247 251 PNPP chemical For example, mutations impacting Tdp2 active site chemistry and phosphotyrosyl bond cleavage should similarly affect catalysis on all three substrates, but mutants impacting DNA damage binding might only impair catalysis on 5′-Y and T5PNP but not PNPP that lacks a nucleobase. RESULTS |
| 0 18 Structural results evidence Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS |
| 23 37 QM/MM modeling experimental_method Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS |
| 47 54 mAsp272 residue_name_number Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS |
| 67 72 water chemical Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS |
| 130 152 phosphotyrosyl linkage ptm Structural results and QM/MM modeling indicate mAsp272 activates a water molecule for in-line nucleophilic attack of the scissile phosphotyrosyl linkage. RESULTS |
| 74 81 mutated experimental_method To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS |
| 85 87 to experimental_method To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS |
| 90 93 His residue_name To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS |
| 183 186 Leu residue_name To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS |
| 191 194 Met residue_name To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS |
| 228 246 water-binding site site To test if this proposed Lewis base is critical for reaction chemistry we mutated it to a His, which could alternatively support metal binding, as well as bulky hydrophobic residues (Leu and Met) that we predict would block the water-binding site. RESULTS |
| 38 44 hD262N mutant Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS |
| 65 78 substitutions experimental_method Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS |
| 127 134 hAsp262 residue_name_number Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS |
| 136 143 mAsp272 residue_name_number Similar to a previously characterized hD262N mutation, all three substitutions ablate activity, supporting essential roles for hAsp262 (mAsp272) in catalysis. RESULTS |
| 9 16 mutated experimental_method Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C). RESULTS |
| 50 71 β2Hβ hydrophobic wall site Next, we mutated key elements of the mobile loop (β2Hβ hydrophobic wall, Figure 2A and C). RESULTS |
| 0 9 Mutations experimental_method Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS |
| 10 16 hI307A mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS |
| 18 24 hL305A mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS |
| 26 32 hL305F mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS |
| 37 43 hL305W mutant Mutations hI307A, hL305A, hL305F and hL305W all impaired catalysis on both nucleotide-containing substrates (<50% activity). RESULTS |
| 4 10 hL305W mutant The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS |
| 97 118 β2Hβ hydrophobic wall site The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS |
| 167 170 DNA chemical The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS |
| 181 185 5′-Y ptm The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS |
| 234 238 β2Hβ structure_element The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS |
| 318 331 substitutions experimental_method The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS |
| 354 358 PNPP chemical The hL305W substitution that we expect to have the most distorting impact on conformation of the β2Hβ hydrophobic wall also has the largest impact on catalysis of the DNA substrate 5′-Y. By comparison, as predicted by our model where β2Hβ dictates key interactions with undamaged and damaged nucleobases, all of these substitutions have little impact on PNPP (>90% activity). RESULTS |
| 45 80 enzyme substrate cation–π interface site Third, we altered properties of the proposed enzyme substrate cation–π interface. RESULTS |
| 31 37 mutant protein_state No activity was detected for a mutant that removes the positive charge at this position (hR206A). RESULTS |
| 89 95 hR206A mutant No activity was detected for a mutant that removes the positive charge at this position (hR206A). RESULTS |
| 29 35 pocket site The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 70 81 replacement experimental_method The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 85 92 hArg206 residue_name_number The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 94 101 mArg216 residue_name_number The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 110 116 lysine residue_name The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 183 187 5′-Y ptm The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 215 220 T5PNP chemical The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 224 228 PNPP chemical The precise geometry of this pocket is also critical for catalysis as replacement of hArg206 (mArg216) with a lysine also results in a profound decrease in catalysis (<5% activity on 5′-Y, no detectable activity on T5PNP or PNPP). RESULTS |
| 11 19 mutation experimental_method Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS |
| 23 30 hTyr178 residue_name_number Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS |
| 63 70 hArg206 residue_name_number Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS |
| 72 79 mArg216 residue_name_number Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS |
| 135 140 Y178F mutant Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS |
| 145 150 Y178W mutant Similarly, mutation of hTyr178 that structurally scaffolds the hArg206 (mArg216) guanidinium also significantly impacts activity, with Y178F and Y178W having <25% activity on all substrates. RESULTS |
| 35 42 hHis351 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS |
| 43 50 hAsp316 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS |
| 52 59 mAsp326 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS |
| 60 67 mHis359 residue_name_number Fourth, we evaluated roles for the hHis351–hAsp316 (mAsp326–mHis359) transition state stabilization charge pair. RESULTS |
| 14 23 mutations experimental_method We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS |
| 29 36 removed experimental_method We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS |
| 76 89 hydrogen bond bond_interaction We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS |
| 91 97 hH351Q mutant We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS |
| 131 134 pKa evidence We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS |
| 142 151 Histidine residue_name We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS |
| 153 159 hD316N mutant We found that mutations that removed the charge yet retained the ability to hydrogen bond (hH351Q) or should abrogate the elevated pKa of the Histidine (hD316N) had severe impacts on catalysis. RESULTS |
| 63 74 active site site Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 86 95 aspartate residue_name Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 97 103 mobile protein_state Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 104 130 substrate engagement loops structure_element Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 149 170 cation–π interactions bond_interaction Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 176 187 active site site Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 217 235 charge interaction bond_interaction Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 250 254 Tdp2 protein Thus altogether, our mutational data support key roles for the active site Lewis base aspartate, mobile substrate engagement loops, enzyme–substrate cation–π interactions, and active site transition state stabilizing charge interaction in supporting Tdp2 catalysis. RESULTS |
| 2 6 Tdp2 protein A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function RESULTS |
| 7 18 active site site A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function RESULTS |
| 58 62 Tdp2 protein A Tdp2 active site single nucleotide polymorphism impairs Tdp2 function RESULTS |
| 44 48 TDP2 protein Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons. RESULTS |
| 147 151 Top2 protein_type Recently, it was found that inactivation of TDP2 by a splice-site mutation is associated with neurological disease and confers hypersensitivity to Top2 poisons. RESULTS |
| 22 27 human species We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS |
| 78 82 Tdp2 protein We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS |
| 83 86 DNA chemical We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS |
| 150 154 Tdp2 protein We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS |
| 180 183 DNA chemical We considered whether human SNPs causing missense mutations might also impact Tdp2 DNA–protein crosslink repair functions established here as well as Tdp2-mediated NHEJ of blocked DNA termini. RESULTS |
| 26 31 human species We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 32 36 TDP2 protein We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 115 141 DNA processing active site site We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 143 154 rs199602263 gene We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 206 213 hAsp350 residue_name_number We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 218 221 Asn residue_name We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 227 237 rs77273535 gene We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 287 294 hIle307 residue_name_number We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 299 302 Val residue_name We identified two SNPs in human TDP2 curated in the NCBI SNP database that result in missense mutations within the DNA processing active site: rs199602263 (minor allele frequency 0.0002), which substitutes hAsp350 for Asn, and rs77273535 (minor allele frequency 0.004, which substitutes hIle307 for Val) (Figure 6A). RESULTS |
| 12 18 hD350N mutant We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D). RESULTS |
| 19 31 substitution experimental_method We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D). RESULTS |
| 101 107 hI307V mutant We show the hD350N substitution severely impairs activity on all substrates tested in vitro, whereas hI307V only has a mild impact on catalysis (Figure 6B–D). RESULTS |
| 39 44 D350N mutant To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A). RESULTS |
| 152 162 structures evidence To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A). RESULTS |
| 166 174 mTdp2cat structure_element To better understand the basis for the D350N catalytic defect, we analyzed the structural environment of this substitution based on the high-resolution structures of mTdp2cat (Figure 6A). RESULTS |
| 19 23 Tdp2 protein Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 31 35 Mg2+ chemical Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 40 69 octahedral coordination shell bond_interaction Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 96 120 hydrogen-bonding network bond_interaction Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 133 140 hAsp350 residue_name_number Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 142 149 mAsp358 residue_name_number Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 171 180 DNA-bound protein_state Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 201 228 β2Hβ substrate-binding loop structure_element Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 237 253 hydrogen bonding bond_interaction Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 257 264 mTrp307 residue_name_number Interestingly, the Tdp2 single Mg2+ ion octahedral coordination shell also involves an extended hydrogen-bonding network mediated by hAsp350 (mAsp358) that stabilizes the DNA-bound conformation of the β2Hβ substrate-binding loop through hydrogen bonding to mTrp307. RESULTS |
| 6 13 hAsp350 residue_name_number Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS |
| 15 22 mAsp358 residue_name_number Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS |
| 61 72 active site site Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS |
| 90 112 substrate binding loop structure_element Here, hAsp350 (mAsp358) serves as a structural nexus linking active site metal binding to substrate binding loop conformations. RESULTS |
| 0 4 Tdp2 protein Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs. FIG |
| 31 42 Active site site Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs. FIG |
| 63 67 TDP2 protein Tdp2 SNPs impair function. (A) Active site residues mutated by TDP2 SNPs. FIG |
| 0 5 D350N mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 7 12 mTdp2 protein D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 13 18 D358N mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 24 29 I307V mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 31 36 mTdp2 protein D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 37 42 I317V mutant D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 44 57 substitutions experimental_method D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 78 82 Tdp2 protein D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 83 94 active site site D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 118 126 mTdp2cat structure_element D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 127 136 structure evidence D350N (mTdp2 D358N) and I307V (mTdp2 I317V) substitutions are mapped onto the Tdp2 active site of the high-resolution mTdp2cat structure (4GZ1). FIG |
| 27 35 SDS-PAGE experimental_method (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 52 54 WT protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 59 65 mutant protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 66 69 MBP experimental_method (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 70 78 hTdp2cat structure_element (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 139 141 WT protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 146 152 mutant protein_state (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 153 156 MBP experimental_method (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 157 165 hTdp2cat structure_element (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 180 218 5′–phosphotyrosyl–DNA oligonucleotides chemical (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 227 238 fluorescein chemical (B) Coomassie blue stained SDS-PAGE gel of purified WT and mutant MBP-hTdp2cat proteins used for assays in panels C and D. (C) Activity of WT and mutant MBP-hTdp2cat proteins on a 5′–phosphotyrosyl–DNA oligonucleotides with 3′-fluorescein label. FIG |
| 135 148 TBE-urea PAGE experimental_method Samples were withdrawn from reactions, neutralized with TBE-urea loading dye at the indicated timepoints, and electrophoresed on a 20% TBE-urea PAGE. FIG |
| 25 27 WT protein_state (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG |
| 42 48 mutant protein_state (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG |
| 49 54 human species (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG |
| 55 58 MBP experimental_method (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG |
| 59 67 hTdp2cat structure_element (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG |
| 99 103 Tdp2 protein (D) Relative activity of WT and indicated mutant human MBP-hTdp2cat fusion proteins on three model Tdp2 substrates. FIG |
| 26 29 MBP experimental_method Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG |
| 30 38 hTdp2cat structure_element Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG |
| 60 62 WT protein_state Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG |
| 79 103 5′-Y DNA oligonucleotide chemical Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG |
| 127 132 T5PNP chemical Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG |
| 148 152 PNPP chemical Quantification of percent MBP-hTdp2cat activity relative to WT protein for the 5′-Y DNA oligonucleotide substrate (blue bars), T5PNP (red bars) and PNPP (green bars) is displayed. FIG |
| 11 14 PNP chemical Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG |
| 20 33 PNP phosphate chemical Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG |
| 35 39 PNPP chemical Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG |
| 110 114 5′-Y ptm Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG |
| 160 175 gel based assay experimental_method Release of PNP from PNP phosphate (PNPP) and was detected as an increase in absorbance at 415 nm, whereas the 5′-Y substrate is quantification of activity in a gel based assay shown in Figure 6C. FIG |
| 38 44 hD350N mutant To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS |
| 46 52 mD358N mutant To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS |
| 65 92 crystallized and determined experimental_method To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS |
| 97 106 structure evidence To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS |
| 114 122 DNA-free protein_state To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS |
| 135 141 mD358N mutant To define the molecular basis for the hD350N (mD358N) defect, we crystallized and determined the structure of the DNA-free form of the mD358N protein to 2.8Å resolution (PDB entry 5INN). RESULTS |
| 5 14 structure evidence This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 25 30 D358N mutant This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 31 39 mutation experimental_method This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 53 66 hydrogen bond bond_interaction This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 75 81 Asp358 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 86 92 Trp307 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 117 123 Asn358 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 141 147 Trp307 residue_name_number This structure shows the D358N mutation disrupts the hydrogen bond between Asp358 and Trp307, shifts the position of Asn358 and destabilizes Trp307. RESULTS |
| 19 35 electron density evidence Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6). RESULTS |
| 55 64 β2Hβ loop structure_element Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6). RESULTS |
| 81 91 disordered protein_state Consequently, poor electron density is visible for the β2Hβ loop which is mostly disordered (Supplementary Figure S6). RESULTS |
| 9 13 Mg2+ chemical Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 58 60 WT protein_state Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 61 68 mTdpcat protein Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 69 77 crystals evidence Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 99 109 metal site site Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 113 123 unoccupied protein_state Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 131 137 mD358N mutant Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 138 146 crystals evidence Although Mg2+ is present at the same concentration as the WT-mTdpcat crystals (10 mM), we find the metal site is unoccupied in the mD358N crystals. RESULTS |
| 50 61 active site site Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. RESULTS |
| 75 79 Tdp2 protein Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. RESULTS |
| 94 99 D350N mutant Therefore, metal-regulated opening/closure of the active site may modulate Tdp2 activity, and D350N is sufficient to block both metal binding and conformational change. RESULTS |
| 38 44 hD350N mutant In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 46 52 mD358N mutant In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 62 66 Mg2+ chemical In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 90 123 intrinsic tryptophan fluorescence evidence In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 151 155 Mg2+ chemical In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 167 178 active site site In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 258 262 Tdp2 protein In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 275 279 loop structure_element In support of this, we also find that hD350N (mD358N) impairs Mg2+ binding as measured by intrinsic tryptophan fluorescence (Figure 4A), and abrogates Mg2+-stimulated active site conformational changes detected by trypsin and chymotrypsin sensitivity of the Tdp2 metamorphic loop (Figure 3D). RESULTS |
| 0 4 Tdp2 protein Tdp2 facilitates NHEJ repair of 5′-phosphotyrosine adducted DSBs RESULTS |
| 32 50 5′-phosphotyrosine residue_name Tdp2 facilitates NHEJ repair of 5′-phosphotyrosine adducted DSBs RESULTS |
| 13 17 Tdp2 protein Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS |
| 18 44 structure/activity studies experimental_method Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS |
| 61 78 5′-detyrosylation ptm Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS |
| 79 82 DNA chemical Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS |
| 141 145 Tdp2 protein Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS |
| 169 173 Top2 protein_type Overall, our Tdp2 structure/activity studies reveal a tuned, 5′-detyrosylation DNA end processing activity and it has been demonstrated that Tdp2 could enable repair of Top2 damage by the non-homologous end-joining (NHEJ) pathway. RESULTS |
| 38 52 5′-tyrosylated protein_state Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8). RESULTS |
| 118 127 mammalian taxonomy_domain Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8). RESULTS |
| 219 226 hTdp2FL protein Accordingly, we demonstrate here that 5′-tyrosylated ends are sufficient to severely impair an in vitro reconstituted mammalian NHEJ reaction (Figure 7A, lanes 3 and 6), unless supplemented with catalytic quantities of hTdp2FL (Figure 7A, lane 8). RESULTS |
| 15 23 hTdp2cat structure_element Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 56 63 hTdp2FL protein Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 108 131 catalytically deficient protein_state Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 132 137 E152Q mutant Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 138 144 mutant protein_state Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 149 157 inactive protein_state Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 200 204 Tdp2 protein Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 255 269 phosphotyrosyl ptm Interestingly, hTdp2cat is slightly more effective than hTdp2FL in promoting NHEJ of adducted ends, while a catalytically deficient E152Q mutant was inactive in this assay, supporting the notion that Tdp2 catalytic activity is required to support NHEJ of phosphotyrosyl blocked DSBs (Supplementary Figure S7A). RESULTS |
| 48 56 tyrosine residue_name We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). RESULTS |
| 215 219 Tdp2 protein We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). RESULTS |
| 239 243 Tdp2 protein We confirmed that efficient joining of the same tyrosine-adducted substrate in cells (Figure 7B) was dependent on both NHEJ (reduced over 10-fold in ligase IV deficient HCT 116 cells; Supplementary Figure S7B), and Tdp2 (reduced 5-fold in Tdp2 deficient MEFs; Figure 7C). RESULTS |
| 144 148 Tdp2 protein Moreover, products with error (i.e. junctions have missing sequence flanking the adducted terminus) are twice as frequent in cells deficient in Tdp2 (Figure 7D). RESULTS |
| 52 60 tyrosine residue_name Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS |
| 81 85 Tdp2 protein Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS |
| 95 109 detyrosylation ptm Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS |
| 213 220 Artemis protein Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS |
| 228 244 Mre11/Rad50/Nbs1 complex_assembly Therefore, in accord with previous work, joining of tyrosine adducted ends after Tdp2-mediated detyrosylation is both more efficient and more accurate than joining after endonucleolytic excision (e.g. mediated by Artemis or the Mre11/Rad50/Nbs1 complex). RESULTS |
| 11 15 Tdp2 protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 16 27 active site site Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 60 64 Tdp2 protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 80 83 Cy5 chemical Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 108 120 5′-phosphate chemical Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 144 158 5′-tyrosylated protein_state Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 199 201 Ku protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 207 218 NHEJ ligase protein_type Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 220 225 XRCC4 protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 227 236 ligase IV protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 241 244 XLF protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 262 269 hTdp2FL protein Effects of Tdp2 active site SNP-encoded mutants on cellular Tdp2 functions. (A) Cy5 labeled substrates with 5′-phosphate termini (Lanes 1–4) or 5′-tyrosylated termini (Lanes 5–9) were incubated with Ku, the NHEJ ligase (XRCC4, ligase IV and XLF; X-L-X) and 1 nM hTdp2FL as indicated (+) for 5 min at 37°C. FIG |
| 49 60 native PAGE experimental_method Concatemer ligation products were detected by 5% native PAGE. FIG |
| 24 51 cellular end joining assays experimental_method (B) Workflow diagram of cellular end joining assays. FIG |
| 0 3 DNA chemical DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. FIG |
| 20 38 5′-phosphotyrosine residue_name DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. FIG |
| 111 120 mammalian taxonomy_domain DNA substrates with 5′-phosphotyrosine adducts and 4 nucleotide 5′ overhangs were electroporated into cultured mammalian cells. FIG |
| 11 14 DNA chemical After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 65 69 qPCR experimental_method After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 73 83 sequencing experimental_method After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 102 106 qPCR experimental_method After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 160 171 tyrosylated protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 205 213 wildtype protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 227 231 Tdp2 protein After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 245 249 Tdp2 protein After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 277 285 wildtype protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 299 306 hTDP2FL protein After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 390 392 WT protein_state After 1 h, DNA was recovered from cells and repair efficiency by qPCR or sequencing as indicated. (C) qPCR assessment of cellular end joining efficiency of the tyrosylated substrate comparing results from wildtype MEF cells to Tdp2−/− cells and Tdp2−/− cells complemented with wildtype or the noted hTDP2FL variants; Joining efficiency shown is the ratio of junctions recovered relative to WT cells. FIG |
| 53 80 cellular end-joining assays experimental_method Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. FIG |
| 127 137 sequencing experimental_method Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. FIG |
| 152 174 end-joining error rate evidence Error bars, s.d, n = 3. (D) Junctions recovered from cellular end-joining assays in the noted cell types were characterized by sequencing to assess the end-joining error rate. FIG |
| 28 53 Clonogenic survival assay experimental_method Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG |
| 57 59 WT protein_state Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG |
| 61 65 Tdp2 protein Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG |
| 151 160 etoposide chemical Error bars, s.d, n = 3. (E) Clonogenic survival assay of WT, Tdp2 knockout and complemented MEF cells after treatment with indicated concentrations of etoposide for 3 h; error bars, s.d, n = 3. FIG |
| 32 41 wild-type protein_state We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS |
| 46 52 mutant protein_state We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS |
| 53 60 hTdp2FL protein We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS |
| 84 88 Tdp2 protein We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS |
| 99 104 mouse taxonomy_domain We next compared the ability of wild-type and mutant hTdp2FL variants to complement Tdp2 deficient mouse embryonic fibroblasts (Supplementary Figure S7C). RESULTS |
| 28 31 DNA chemical Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 37 52 phosphotyrosine residue_name Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 177 186 wild-type protein_state Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 187 192 mouse taxonomy_domain Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 206 210 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 214 219 mouse taxonomy_domain Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 235 244 wild-type protein_state Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 245 250 human species Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 251 255 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 261 265 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 294 299 I307V mutant Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 300 307 variant protein_state Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 308 313 human species Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 314 318 Tdp2 protein Joining of extrachromosomal DNA with phosphotyrosine blocked ends, both in terms of efficiency (Figure 7C) and fidelity (Figure 7D), was indistinguishable comparing MEFs from a wild-type mouse, MEFs from a Tdp2-/- mouse overexpressing wild-type human Tdp2, and Tdp2 -/- MEFs overexpressing the I307V variant human Tdp2. RESULTS |
| 78 82 Tdp2 protein In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS |
| 133 137 Tdp2 protein In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS |
| 161 165 Tdp2 protein In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS |
| 166 171 D350N mutant In contrast, joining of 5′ phosphotyrosine-blocked ends was reduced 5-fold in Tdp2-/- MEFs, and an equivalent defect was observed in Tdp2-/- MEFs overexpressing Tdp2 D350N. RESULTS |
| 71 75 Tdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS |
| 96 100 Tdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS |
| 132 137 D350N mutant Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS |
| 168 177 wild type protein_state Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS |
| 178 182 Tdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS |
| 186 191 hTdp2 protein Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS |
| 192 197 I307V mutant Moreover, the frequency of inaccurate repair was 2-fold higher in both Tdp2 deficient cells and Tdp2 deficient cells overexpressing D350N, relative to cells expressing wild type Tdp2 or hTdp2 I307V (Figure 7D). RESULTS |
| 14 23 wild type protein_state Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 27 32 I307V mutant Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 33 38 human species Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 39 43 Tdp2 protein Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 47 51 Tdp2 protein Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 114 123 etoposide chemical Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 150 159 wild-type protein_state Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 176 190 overexpression experimental_method Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 194 199 human species Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 200 205 D350N mutant Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 206 210 Tdp2 protein Expression of wild type or I307V human Tdp2 in Tdp2-/- MEFs was also sufficient to confer levels of resistance to etoposide comparable to the matched wild-type MEF line, while overexpression of human D350N Tdp2 had no apparent complementation activity (Figure 7E). RESULTS |
| 9 14 D350N mutant The rare D350N variant is thus inactive by all metrics analyzed. RESULTS |
| 15 22 variant protein_state The rare D350N variant is thus inactive by all metrics analyzed. RESULTS |
| 31 39 inactive protein_state The rare D350N variant is thus inactive by all metrics analyzed. RESULTS |
| 32 37 I307V mutant By comparison the more frequent I307V has only mild effects on in vitro activity, and no detectable impact on cellular assays. RESULTS |
| 0 4 Top2 protein_type Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS |
| 99 102 DNA chemical Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS |
| 115 119 Top2 protein_type Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS |
| 120 123 DNA chemical Top2 chemotherapeutic agents remain frontline treatments, and exposure to the chemical and damaged DNA triggers of Top2-DNA protein crosslink formation are unavoidable. DISCUSS |
| 42 45 DNA chemical Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. DISCUSS |
| 75 78 DNA chemical Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. DISCUSS |
| 203 207 Top2 protein_type Understanding how cells cope with complex DNA breaks bearing topoisomerase–DNA protein crosslinks is key to deciphering individual responses to chemotherapeutic outcomes and genotoxic agents that poison Top2. DISCUSS |
| 14 25 mutagenesis experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 30 47 functional assays experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 57 61 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 62 72 structures evidence Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 73 90 in the absence of protein_state Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 91 98 ligands chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 103 118 in complex with protein_state Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 119 122 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 158 162 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 163 166 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 205 209 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 210 221 active site site Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 268 271 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 320 323 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 345 349 Top2 protein_type Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 382 401 structural analysis experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 415 433 mutational studies experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 438 462 QM/MM molecular modeling experimental_method Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 470 474 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 557 560 EEP structure_element Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 578 598 phosphoryl hydrolase protein_type Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 620 624 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 625 636 active site site Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 640 664 conformationally plastic protein_state Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 710 713 DNA chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 718 722 Mg2+ chemical Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 769 773 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 793 797 Tdp2 protein Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 798 809 active site site Together with mutagenesis and functional assays, our new Tdp2 structures in the absence of ligands and in complex with DNA damage reveal four novel facets of Tdp2 DNA-protein conjugate processing: (i) The Tdp2 active site is well-suited for accommodating a variety of DNA structures including abasic and bulky alkylated DNA lesions that trigger Top2 poisoning, (ii) High-resolution structural analysis coupled with mutational studies and QM/MM molecular modeling of the Tdp2 reaction coordinate support a single metal-ion mechanism for the diverse clade of EEP domain catalyzed phosphoryl hydrolase reactions, (iii) The Tdp2 active site is conformationally plastic, and undergoes intricate rearrangements upon DNA and Mg2+ cofactor binding and (iv) Naturally occurring Tdp2 variants undermine Tdp2 active site chemistry, cellular and biochemical activities. DISCUSS |
| 31 35 Tdp2 protein This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS |
| 62 65 DNA chemical This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS |
| 149 153 Tdp2 protein This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS |
| 154 157 DNA chemical This mechanistic dissection of Tdp2 interactions with damaged DNA and metal cofactor provides a detailed molecular understanding of the mechanism of Tdp2 DNA protein crosslink processing. DISCUSS |
| 0 4 Tdp2 protein Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 74 78 Top1 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 83 87 Top2 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 188 192 Tdp2 protein Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 215 218 DNA chemical Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 238 242 Top2 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 257 261 Top2 protein_type Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 262 265 DNA chemical Tdp2 was originally identified as a protein conferring resistance to both Top1 and Top2 anti-cancer drugs, however it is hypothesized that the predominant natural source of substrates for Tdp2 are likely the potent DNA damage triggers of Top2 poisoning and Top2 DNA protein crosslinks encountered during transcription. DISCUSS |
| 26 29 DNA chemical The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS |
| 52 56 Top2 protein_type The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS |
| 57 60 DNA chemical The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS |
| 97 101 Tdp2 protein The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS |
| 156 169 topoisomerase protein_type The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS |
| 185 196 active site site The properties of complex DNA strand breaks bearing Top2-DNA protein crosslinks necessitate that Tdp2 accommodates both damaged nucleic acid as well as the topoisomerase protein in its active site for catalysis. DISCUSS |
| 4 8 Tdp2 protein The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. DISCUSS |
| 9 37 substrate interaction groove site The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. DISCUSS |
| 50 53 DNA chemical The Tdp2 substrate interaction groove facilitates DNA-protein conjugate recognition in two important ways. DISCUSS |
| 11 38 nucleic acid binding trench site First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 57 64 dynamic protein_state First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 65 93 β2Hβ DNA damage-binding loop structure_element First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 148 171 phosphotyrosyl linkages ptm First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 217 219 ϵA chemical First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 292 305 base-stacking bond_interaction First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 306 322 hydrophobic wall site First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 330 339 β2Hβ-loop structure_element First, the nucleic acid binding trench is assembled by a dynamic β2Hβ DNA damage-binding loop that is capable of recognizing and processing diverse phosphotyrosyl linkages even in the context of bulky adducts such as ϵA. This is achieved by binding of nucleic acid ‘bases out’ by an extended base-stacking hydrophobic wall of the β2Hβ-loop. DISCUSS |
| 14 19 QM/MM experimental_method Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS |
| 68 88 cation–π interaction bond_interaction Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS |
| 125 129 Tdp2 protein Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS |
| 138 141 DNA chemical Secondly, our QM/MM analysis further highlights an enzyme–substrate cation–π interaction as an additional key feature of the Tdp2 protein–DNA crosslink binding and reversal. DISCUSS |
| 4 22 strictly conserved protein_state The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS |
| 23 34 active site site The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS |
| 35 41 Arg216 residue_name_number The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS |
| 137 151 phosphotyrosyl ptm The strictly conserved active site Arg216 appears optimally positioned to stabilize a delocalized charge on the phenolate product of the phosphotyrosyl cleavage reaction through molecular orbital overlap and polarization of the leaving group. DISCUSS |
| 58 86 substrate cation–π interface site To our knowledge, this is the first proposed example of a substrate cation–π interface exploited to promote a phosphoryl-transfer reaction. DISCUSS |
| 85 89 Tdp2 protein This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS |
| 166 172 Top2cc complex_assembly This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS |
| 174 186 picornaviral taxonomy_domain This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS |
| 195 198 RNA chemical This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS |
| 203 220 Hepatitis B Virus taxonomy_domain This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS |
| 222 225 HBV taxonomy_domain This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS |
| 235 238 DNA chemical This unique feature likely provides an additional level of substrate-specificity for Tdp2 by restricting activity to hydrolysis of aromatic adducts characteristic of Top2cc, picornaviral protein–RNA and Hepatitis B Virus (HBV) protein–DNA processing intermediates. DISCUSS |
| 21 24 EEP structure_element By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS |
| 25 34 nucleases protein_type By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS |
| 43 47 Ape1 protein By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS |
| 52 56 Ape2 protein By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS |
| 77 80 DNA chemical By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS |
| 159 163 Tdp2 protein By comparison, other EEP nucleases such as Ape1 and Ape2 have evolved robust DNA damage specific endonucleolytic and exonucleolytic activities not shared with Tdp2. DISCUSS |
| 26 30 Tdp2 protein The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation. DISCUSS |
| 31 42 active site site The dynamic nature of the Tdp2 active site presents opportunities for enzyme regulation. DISCUSS |
| 56 60 Tdp2 protein However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown. DISCUSS |
| 102 106 Tdp2 protein However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown. DISCUSS |
| 107 116 β2Hβ-loop structure_element However, whether additional protein factors can bind to Tdp2 and modulate assembly/disassembly of the Tdp2 β2Hβ-loop is unknown. DISCUSS |
| 35 39 Top2 protein_type We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS |
| 63 66 DNA chemical We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS |
| 132 136 Tdp2 protein We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS |
| 137 148 active site site We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS |
| 178 182 Tdp2 protein We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS |
| 222 226 Top2 protein_type We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS |
| 306 310 Top2 protein_type We hypothesize that binding of the Top2 protein component of a DNA–protein crosslink and/or other protein-regulated assembly of the Tdp2 active site might also serve to regulate Tdp2 activity to restrict it from misplaced Top2 processing events, such that it cleaves only topologically trapped or poisoned Top2 molecules when needed. DISCUSS |
| 29 39 structures evidence Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 43 48 mouse taxonomy_domain Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 71 81 C. elegans species Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 82 86 Tdp2 protein Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 129 133 Tdp2 protein Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 134 145 active site site Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 151 156 worms taxonomy_domain Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 160 163 man taxonomy_domain Furthermore, high-resolution structures of mouse (Figures 3 and 4) and C. elegans Tdp2 show that a single metal ion typifies the Tdp2 active site from worms to man. DISCUSS |
| 83 116 intrinsic tryptophan fluorescence experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS |
| 118 143 crystallographic analysis experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS |
| 180 191 mutagenesis experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS |
| 193 216 Ca2+ inhibition studies experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS |
| 221 235 QM/MM analysis experimental_method Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS |
| 271 275 Mg2+ chemical Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS |
| 285 289 Tdp2 protein Herein, we report five additional lines of evidence from metal binding detected by intrinsic tryptophan fluorescence, crystallographic analysis of varied metal cofactor complexes, mutagenesis, Ca2+ inhibition studies and QM/MM analysis that all support a feasible single Mg2+ mediated Tdp2 catalytic mechanism. DISCUSS |
| 0 9 Etoposide chemical Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS |
| 20 24 Top2 protein_type Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS |
| 74 78 Tdp2 protein Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS |
| 107 112 human species Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS |
| 151 155 Top2 protein_type Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS |
| 174 183 etoposide chemical Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS |
| 188 199 doxyrubicin chemical Etoposide and other Top2 poisons remain front line anti-cancer drugs, and Tdp2 frameshift mutations in the human population confer hypersensitivity to Top2 poisons including etoposide and doxyrubicin. DISCUSS |
| 6 10 Tdp2 protein Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS |
| 28 33 human species Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS |
| 117 121 TDP2 protein Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS |
| 259 263 Top2 protein_type Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS |
| 277 282 viral taxonomy_domain Given Tdp2 variation in the human population, links to neurological disease and viral pathogenesis, our finding that TDP2 SNPs ablate catalytic activity has probable implications for modulation of cancer chemotherapy, susceptibility to environmentally linked Top2 poisons, and viral infection. DISCUSS |
| 8 12 Tdp2 protein Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2. DISCUSS |
| 115 119 Tdp2 protein Lastly, Tdp2 inhibitors may synergize or potentiate cytotoxic effects of current anticancer treatments that target Tdp2. DISCUSS |
| 98 102 Tdp2 protein Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions. DISCUSS |
| 150 153 DNA chemical Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions. DISCUSS |
| 248 252 Tdp2 protein Thus, we anticipate this atomic-level and mechanistic definition of the molecular determinants of Tdp2 catalysis and conformational changes driven by DNA–protein and protein–protein interactions will foster unique strategies for the development of Tdp2 targeted small molecule interventions. DISCUSS |
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