Q1.
As seen in the figure below, the difference between the attenuated radiation, i.e., the radiation lost from the beam, and the absorbed dose is much larger for the energies where the Compton process dominates. Can you explain this?

A graph of mass absorption in centimeters squared per gram versus energy in mega-electron volts The values are plotted for photon, pair, Compton, absorption, and attenuation. The lines for attenuation and photon have the highest values.

Absorption and attenuation in water for photons with different energies [Figure from Kiefer, J. (1990). Biological radiation effects. Germany: Springer.]

 Q2.
Can you tell why people living at high altitudes are more exposed to cosmic radiation? Can you tell which is the treatment at hospitals which is of most concern for radiation exposure?
 Q3.
Which of the following is the most harmful to cells?(a)
H2O2
 (b)
H⋅
 (c)
OḤ
 (d)
e−aq

 Q4.
Name the four stages of indirect effects of ionizing radiation.
 Q5.
Low LET radiation mostly induces direct effects: true or false?
 Q6.
Fill in the missing items in the table (modes of radioactive decay).
Mode of radioactive decay

Released particles

General reaction

Example

α-Decay

92238U → 90234Th + 24He

Two fragment nuclei

100256Fm → 54140Xe + 46112Pd

ZAP → ZA − 1D + n0***

413Be → 412Be + n0***

 Q7.
Describe the difference between the well-known periodic table and a chart of nuclides (chart of nuclides).
 Q8.
The unit of effective dose is:(a)
Gy
 (b)
Sv
 (c)
Bq
 (d)
J

 Q9.
The dose that takes into account both the quality of the radiation and the radiosensitivity of the tissue, and is thus a direct measure of the likelihood of developing cancer, is called:(a)
Absorbed dose
 (b)
Equivalent dose
 (c)
Effective dose
 (d)
Dose rate

 Q10.
X-rays and beta particles have been given a radiation weighting factor of 1 because they produce:(a)
Virtually the same biological effect in tissue for equal absorbed doses
 (b)
No biological effect in tissues for equal absorbed doses
 (c)
Varying degrees of biological effect in body tissue for equal absorbed doses
 (d)
None of the answers above

 Q11.
During flash radiotherapy, an ultralow dose rate is used. True or false?
 Q12.
Arrange the following radiations in order of increasing LET in water:(a)
5 MeV alpha particle
 (b)
100 MeV carbon ion
 (c)
10 MeV proton
 (d)
Cobalt-60 γ-rays
 (e)
200 MeV iron ion

 Q13.
Explain why high LET irradiation exerts a relatively larger RBE in the low-dose range.
 Q14.
With decreasing dose rate, a discriminative biological effect can be obtained between late-responding normal tissues and tumors. Please explain.
 Q15.
The consequences for human exposure to ionizing radiation can be classified into two categories—stochastic or deterministic effects/tissue reactions. Explain the reasoning behind this classification and describe the main features of these effects, giving examples.


As seen in the figure below, the difference between the attenuated radiation, i.e., the radiation lost from the beam, and the absorbed dose is much larger for the energies where the Compton process dominates. Can you explain this?

A graph of mass absorption in centimeters squared per gram versus energy in mega-electron volts The values are plotted for photon, pair, Compton, absorption, and attenuation. The lines for attenuation and photon have the highest values.

Absorption and attenuation in water for photons with different energies [Figure from Kiefer, J. (1990). Biological radiation effects. Germany: Springer.]


A graph of mass absorption in centimeters squared per gram versus energy in mega-electron volts The values are plotted for photon, pair, Compton, absorption, and attenuation. The lines for attenuation and photon have the highest values.


Can you tell why people living at high altitudes are more exposed to cosmic radiation? Can you tell which is the treatment at hospitals which is of most concern for radiation exposure?


Which of the following is the most harmful to cells?(a)
H2O2
 (b)
H⋅
 (c)
OḤ
 (d)
e−aq


Fill in the missing items in the table (modes of radioactive decay).
Mode of radioactive decay

Released particles

General reaction

Example

α-Decay

92238U → 90234Th + 24He

Two fragment nuclei

100256Fm → 54140Xe + 46112Pd

ZAP → ZA − 1D + n0***

413Be → 412Be + n0***


Describe the difference between the well-known periodic table and a chart of nuclides (chart of nuclides).


The unit of effective dose is:(a)
Gy
 (b)
Sv
 (c)
Bq
 (d)
J


The dose that takes into account both the quality of the radiation and the radiosensitivity of the tissue, and is thus a direct measure of the likelihood of developing cancer, is called:(a)
Absorbed dose
 (b)
Equivalent dose
 (c)
Effective dose
 (d)
Dose rate


X-rays and beta particles have been given a radiation weighting factor of 1 because they produce:(a)
Virtually the same biological effect in tissue for equal absorbed doses
 (b)
No biological effect in tissues for equal absorbed doses
 (c)
Varying degrees of biological effect in body tissue for equal absorbed doses
 (d)
None of the answers above


Virtually the same biological effect in tissue for equal absorbed doses


Varying degrees of biological effect in body tissue for equal absorbed doses


During flash radiotherapy, an ultralow dose rate is used. True or false?


Arrange the following radiations in order of increasing LET in water:(a)
5 MeV alpha particle
 (b)
100 MeV carbon ion
 (c)
10 MeV proton
 (d)
Cobalt-60 γ-rays
 (e)
200 MeV iron ion


Explain why high LET irradiation exerts a relatively larger RBE in the low-dose range.


With decreasing dose rate, a discriminative biological effect can be obtained between late-responding normal tissues and tumors. Please explain.


The consequences for human exposure to ionizing radiation can be classified into two categories—stochastic or deterministic effects/tissue reactions. Explain the reasoning behind this classification and describe the main features of these effects, giving examples.


SQ1.
The Compton process results in a secondary photon, which has its own track, and an electron, which may also have enough energy to move away from where the primary ionization took place. In both cases, some of the dose is deposited in a different position than where the energy was lost from the beam.
 SQ2.
When going higher in altitude, the amount of atmosphere shielding us from incoming radiation is smaller than at the Earth’s surface. Thus, at higher altitudes, the “shielding” provided by the atmosphere against the incoming radiation from space is less efficient. Radionuclide-based treatments are the main concern in terms of radiation exposure at hospitals. There is the need to protect healthcare staff and to keep dose to caregivers and the public within the acceptable levels.
 SQ3.
OḤ.
 SQ4.
Physical, physicochemical, chemical, biological.
 SQ5.
False.
Mode of radioactive decay

Released particles

General reaction

Example

α-Decay

Helium nucleus

ZAP → Z − 2A − 4P + 24He

92238U → 90234Th + 24He

Spontaneous fission (SF)

Two fragment nuclei

ZAP → Z1A1D1 + Z2A2D2

100256Fm → 54140Xe + 46112Pd

Neutron emission (NE)

Neutron

ZAP → ZA − 1D + n0***

413Be → 412Be + n0***

 SQ6.
The periodic table organizes chemical elements by their respective atomic number, while a chart of nuclides organizes nuclides according to the number of protons (Y-axis) and neutrons (X-axis) present in the nucleus.
 SQ7.
B.
 SQ8.
C.
 SQ9.
A.
 SQ10.
False.
 SQ11.
Cobalt-60 γ-rays (0.2 keV/μm) < 10 MeV proton (~5 keV/μm) < 5 MeV alpha particle (~100 keV/μm) < 100 MeV carbon ion (~200 keV/μm) < 200 MeV iron ion (>300 keV/μm).
 SQ12.
The RBE is defined as the ratio of the high LET dose and the low LET reference dose (generally 250 kV X-rays) at isoeffect. The high LET dose-effect cell survival relation is a straight line over the full dose range. The low LET cell survival curve is however characterized by a broad shoulder in the low-dose range, followed by a straight, parallel steep downward curve in the higher dose range. Hence, the RBE in the low-dose range is higher than in the high-dose range.
 SQ13.
Late-responding normal tissues (low alpha/beta ratio) are better spared than tumors and early-responding normal tissues (high alpha/beta ratio) by decreasing the dose rate. Lowering the dose rate can be considered as decreasing the “fraction size,” with larger sparing of late-responding normal tissues than of tumors, hence a therapeutic beneficial effect.
 SQ14.
Deterministic effects or tissue reactions are those for which there is a threshold (varying between different effects), below which the effect is not seen. Above the threshold, the severity of the effect increases with dose. The syndromes of ARS are examples of deterministic effects.
Stochastic effects are the probabilistic ones, for which there is no threshold—any increase in dose slightly increases the risk of the effect, and severity does not increase with increasing dose. Radiation cancers and genetic/hereditary effects are classified as stochastic effects.


The Compton process results in a secondary photon, which has its own track, and an electron, which may also have enough energy to move away from where the primary ionization took place. In both cases, some of the dose is deposited in a different position than where the energy was lost from the beam.


When going higher in altitude, the amount of atmosphere shielding us from incoming radiation is smaller than at the Earth’s surface. Thus, at higher altitudes, the “shielding” provided by the atmosphere against the incoming radiation from space is less efficient. Radionuclide-based treatments are the main concern in terms of radiation exposure at hospitals. There is the need to protect healthcare staff and to keep dose to caregivers and the public within the acceptable levels.


False.
Mode of radioactive decay

Released particles

General reaction

Example

α-Decay

Helium nucleus

ZAP → Z − 2A − 4P + 24He

92238U → 90234Th + 24He

Spontaneous fission (SF)

Two fragment nuclei

ZAP → Z1A1D1 + Z2A2D2

100256Fm → 54140Xe + 46112Pd

Neutron emission (NE)

Neutron

ZAP → ZA − 1D + n0***

413Be → 412Be + n0***


The periodic table organizes chemical elements by their respective atomic number, while a chart of nuclides organizes nuclides according to the number of protons (Y-axis) and neutrons (X-axis) present in the nucleus.


Cobalt-60 γ-rays (0.2 keV/μm) < 10 MeV proton (~5 keV/μm) < 5 MeV alpha particle (~100 keV/μm) < 100 MeV carbon ion (~200 keV/μm) < 200 MeV iron ion (>300 keV/μm).


The RBE is defined as the ratio of the high LET dose and the low LET reference dose (generally 250 kV X-rays) at isoeffect. The high LET dose-effect cell survival relation is a straight line over the full dose range. The low LET cell survival curve is however characterized by a broad shoulder in the low-dose range, followed by a straight, parallel steep downward curve in the higher dose range. Hence, the RBE in the low-dose range is higher than in the high-dose range.


Late-responding normal tissues (low alpha/beta ratio) are better spared than tumors and early-responding normal tissues (high alpha/beta ratio) by decreasing the dose rate. Lowering the dose rate can be considered as decreasing the “fraction size,” with larger sparing of late-responding normal tissues than of tumors, hence a therapeutic beneficial effect.


Deterministic effects or tissue reactions are those for which there is a threshold (varying between different effects), below which the effect is not seen. Above the threshold, the severity of the effect increases with dose. The syndromes of ARS are examples of deterministic effects.


Stochastic effects are the probabilistic ones, for which there is no threshold—any increase in dose slightly increases the risk of the effect, and severity does not increase with increasing dose. Radiation cancers and genetic/hereditary effects are classified as stochastic effects.


As described in Chap. 2, ionizing radiation (IR) can interact with matter directly, via molecule ionization, or indirectly, via the radiolysis of water. The result of this interaction is highly reactive ionized molecules that undergo a rapid cascade of chemical reactions, which leads to the breaking of chemical bonds. The radiolytic damage of biomolecules, such as carbohydrates, lipids, and proteins, is described as an indirect effect following water radiolysis and depends on biomolecule concentration in the irradiated medium. The products of water radiolysis—radicals—are often found in clusters and react with the biomolecules present within cells before they have a chance to diffuse and form a homogeneous distribution of products. To date, the studies on radiation-induced damage of these biomolecules are mainly based on the radical analysis of model molecules or on the molecular analysis of cellular mixtures after irradiation. Figure 3.1 shows an overview of the radiolysis products described in this chapter. The description of radiolysis products of the different biomolecules clearly demonstrates possible interactions and reactions between radicals and subcellular targets [1].

A diagram of the membrane bilayer has the carbohydrate, protein, lipid, gated channel, and glycoprotein processes. Radioactive material causes lipid peroxidation, protein radical formation, and carbohydrate rearrangement.

Fig. 3.1
Summary of the radicals produced with proteins, lipids, and carbohydrates following external IR exposure. Cellular exposure to IR leads to dissociation of biological macromolecules. Radiolysis of carbohydrates, proteins, and lipids is explained in their respective blue boxes. PO protein radicals, CO carbohydrate radicals, LO lipid radicals, OOH hydroxyl radicals, POOO protein peroxyl radicals, Trp tryptophan, Tyr tyrosine, His histidine, Met methionine, Cys cysteine, Gly glycine, ROH alcohol—an analog of water where R is alkyl group, O is oxygen atom, and H is hydrogen atom


A diagram of the membrane bilayer has the carbohydrate, protein, lipid, gated channel, and glycoprotein processes. Radioactive material causes lipid peroxidation, protein radical formation, and carbohydrate rearrangement.


Carbohydrates are hydrated organic molecules consisting of carbon (C), hydrogen (H), and oxygen (O), characterized by the formula Cx(H2O)y, where x and y denote the numbers of carbon or water in the molecule. Chemically, most carbohydrates are polyhydroxy aldehydes, ketones, alcohols, and acids, which can polymerize, form connected chains of molecules, and, therefore, become more complex [2]. In biological media, such as cells, some carbohydrates are a major energy source for all non-photosynthetic organisms (e.g., glycogen), and others have vital structural functions (e.g., chitin, cellulose) or are essential components of RNA, DNA, and biochemical cofactor synthesis (e.g., adenosine mono/di/triphosphate).


Investigations of ionization damage to carbohydrates were done mainly in the fields of food and DNA [3]. Food irradiation can be used to extend shelf life (0.5–3.0 kGy), to inhibit sprouting (0.03–0.12 kGy), for insect disinfestation (0.2–0.8 kGy) and parasite disinfestation (0.1–3.0 kGy), and to eliminate pathogenic bacteria that do not form spores (1.5–7.0 kGy). In this context, it is important to know the chemical transformations occurring at a molecular level, including carbohydrates, that might have an adverse impact on the nutritional, sensory, or functional state of food [4]. In DNA, the sugar moiety plays an important role in the radiation-induced strand breaking process, even if not all the carbohydrate alterations are implied [3].


Model molecules of carbohydrates, such as ethylene glycol, glycerol, and glucose, were used to understand radiation products yielded from carbohydrates. Furthermore, they were used to study the formation of radicals via electron spin resonance (ESR) and electron paramagnetic resonance (EPR) or molecular products via high-performance liquid chromatography-mass spectrometry (HPLC-MS2) [4].


The radiolysis of carbohydrates in aqueous system is pH dependent and occurs mainly by an indirect interaction of hydroxyl radical (°OH) with C–H bonds producing carbohydrate radicals. In contrast, carbohydrates react slowly with superoxide radicals (coming from solvated electrons) and scarcely with °H radicals [3, 4]. The carbohydrate radicals readily react with molecular oxygen or experience dismutation, dimerization, and elimination of alcohol or water (the most ubiquitous). Thus, radiolysis of carbohydrate inside the DNA molecule can lead to a degradation of the sugar structure and a loss of the base.


Lipids are small organic molecules, representing 21% of the eukaryotic cell content. Biochemically, they originate entirely or in part from carbanion-based condensations of thioesters, forming fatty acids, which are components of triacylglycerols (TAGs), phospholipids, and sphingolipids, or by carbocation-based condensation of isoprene units, forming isoprenol derivatives including sterols [2]. Lipids perform many essential functions in the cell including signaling and energy storage (due to their highly reduced state) and are the hydrophobic units of bilayers that form cellular and organellar membranes, which contribute to their function and topology.


In aqueous biological media, during IR, lipids (mostly polyunsaturated acids) are likely to undergo lipid peroxidation. This is initiated by some water radiolysis species and presence of endogenous transition metals [5] and propagates the chain reaction and produces several other organic reactive radicals. These primary and secondary radicals, being able to penetrate the membrane interior, may react either with the lipid matrix or with integral membrane proteins.


This radio-induced lipid peroxidation can thus contribute to the loss of cellular function through the inactivation of membrane enzymes and even of cytoplasmic (i.e., water soluble) proteins. Moreover, consequences include also perturbation of membrane function itself (thinning, change of structure or charge distribution, polarity) and consequently some carrier ion complexes and ion channels: efficiency can increase due to accumulation of polar oxidation products, but also be inhibited due to depolarization following conductance leakage [6].


Proteins are biomolecules made of many linear chains of amino acid residues arranged in a three-dimensional structure, with various binding types (covalent or weak electrostatic bonds). Proteins constitute about 74% of the eukaryotic cell organic content. Amino acids, peptides, and proteins undergo a variety of reactions with radio-induced radicals which in most cases are pH dependent. These reactions involve mostly hydrogen abstraction at the α position of the amino acid, electron transfer, addition, fragmentation and rearrangement, dimerization, disproportionation, and substitution [7]. Many studies showed that the most reactive amino acids are the aromatic (Trp, Tyr, His) and sulfur-containing (Met, Cys) amino acids, whereas the least reactive is glycine (Gly) [7, 8]. Once generated, the formed protein radicals can interact with oxygen, yielding a peroxyl radical, and with other biological components for instance yielding other reactive radicals or initiating lipid peroxidation.


Some of the most commonly measured oxidative protein modifications are protein carbonyl groups originating from the oxidation of the amino acid residues or their side chains [9]. This leads to the formation of carbonyl derivatives, protein backbone cleavage, or beta scission of side-chain alkoxyl radicals of aliphatic residues (e.g., Ala, Val). In addition, oxidation of the sulfur of cysteine residues can lead to disulfur bond rearrangement.


Studies performed in biological media, e.g., cells, tend to show that in case of hydroxyl radicals coming from external irradiation, damage to DNA and lipids is a secondary process and proteins are more likely the initial targets, due to their relative amount and reactivity [7, 8] (Box 3.1).


Radiolysis of carbohydrates and proteins occurs mostly via OH, begins with an abstraction of one hydrogen atom, and is pH dependent.

Radiolysis of the carbohydrates within DNA may result in the loss of the base and thus DNA damage.

Lipids are likely to undergo peroxidation following IR processes, initiating a chain reaction leading to the production of organic reactive radicals.

Lipid peroxidation may lead to the loss of cellular functions including those associated with membranes.

In proteins, the most reactive amino acids are the aromatic (Trp, Tyr, His) and sulfur-containing (Met, Cys) ones, whereas the least reactive is glycine (Gly).

Protein radicals may react with oxygen-yielding peroxyl radicals or with other biological compounds such as lipids, leading to lipid peroxidation or formation of other reactive radicals.

Some of the most measured oxidative protein modifications are protein carbonyl groups.

In cells, proteins are the initial targets, due to their relative amount and reactivity.


Radiolysis of carbohydrates and proteins occurs mostly via OH, begins with an abstraction of one hydrogen atom, and is pH dependent.


Radiolysis of the carbohydrates within DNA may result in the loss of the base and thus DNA damage.


Lipids are likely to undergo peroxidation following IR processes, initiating a chain reaction leading to the production of organic reactive radicals.


Lipid peroxidation may lead to the loss of cellular functions including those associated with membranes.


In proteins, the most reactive amino acids are the aromatic (Trp, Tyr, His) and sulfur-containing (Met, Cys) ones, whereas the least reactive is glycine (Gly).


Protein radicals may react with oxygen-yielding peroxyl radicals or with other biological compounds such as lipids, leading to lipid peroxidation or formation of other reactive radicals.


Some of the most measured oxidative protein modifications are protein carbonyl groups.


In cells, proteins are the initial targets, due to their relative amount and reactivity.


In contrast to the above-described effects of IR in carbohydrates, lipids, and proteins, DNA radiolytic lesions occur both directly and indirectly, with the proportion being dependent on radiation type (α, β, γ, heavier ions). Deoxyribonucleic acid (DNA) molecules are, unlike other biomolecules within a cell, unique, and if they get damaged and stay unrepaired, this may lead to serious and often lethal consequences.


Due to the importance of DNA, cells have a complex DNA damage response system, consisting of several interrelated signaling pathways, which can recognize the damage and initiate its repair. DNA can be damaged by different mutagens, such as oxidizing agents and alkylating agents, as well as by IR or UV light. However, the type of DNA damage depends on the type of mutagen, as well as the type, dose, and energy of radiation.


DNA is a large molecule composed of two polynucleotide chains that coil around each other to constitute a double-stranded helix structure. DNA molecules carry the genetic information for most biological processes. The two antiparallel DNA strands are connected by hydrogen bonds, and the backbone of each strand is composed of nucleotides. Each nucleotide consists of an alternating sugar (2-deoxyribose), a phosphate group, and one of the four nitrogen-containing nucleobases [adenine (A), cytosine (C), guanine (G), or thymine (T)]. The structure of the bases is shown in Fig. 3.2. Two of the bases, thymine and cytosine, are single-ring groups (pyrimidines), whereas two other bases, adenine and guanine, are double-ring groups (purines).

Chemical structures of guanine-cytosine and adenine-thymine base pairs. The G-C base pair has three hydrogen bonds and the A-T base pair has two hydrogen bonds.

Fig. 3.2
The four DNA bases with respective hydrogen bonds (dashed lines). G guanine, C cytosine, A adenine, T thymine


Chemical structures of guanine-cytosine and adenine-thymine base pairs. The G-C base pair has three hydrogen bonds and the A-T base pair has two hydrogen bonds.


On one strand, nucleotides are joined to another by covalent bonds between the sugar of one nucleotide and the phosphate group of the next one (phosphodiester bond). The bases on the opposite strands are complementary, adenine pairs with thymine and guanine pairs with cytosine through hydrogen bonds [10].


A base lesion is defined as a modification (oxidation, alkylation, and deamination) of the chemical structure of one of the four DNA bases. Modification can occur through the loss of an electron, called oxidation, the transfer of an alkyl group, called alkylation, or the removal of an amino group, called deamination. After the break of the N-glycosidic bond between the DNA base and the 2-deoxyribose, a base can get lost and an abasic site can be created [11]. A representation of base lesion and abasic site is shown in Fig. 3.3. Sugar and base damages are quite easy for the cell to repair, as will be shown in Sect. 3.4.

Two chemical structures of double-stranded D N A depict a base lesion indicated by an alteration of the base and abasic site indicated by the gap between the bond of the D N A base and the 2-deoxyribose.

Fig. 3.3
Examples of DNA base damages. In base lesions, the chemical structure of any DNA base is modified (highlighted with yellow and red), whereas in abasic sites, the N-glycosidic bond between the DNA base and the 2-deoxyribose is broken (as shown with red arrow). G guanine, C cytosine, A adenine, T thymine, H-bond hydrogen bond, P phosphate


Two chemical structures of double-stranded D N A depict a base lesion indicated by an alteration of the base and abasic site indicated by the gap between the bond of the D N A base and the 2-deoxyribose.


Most of the sugar and base modifications are due to the hydroxyl radical (OH°). This radical reacts with the bases by addition to double bonds and by abstraction of hydrogen from the methyl group of thymine or from any C–H bond, but more likely from the C4 and C5 positions of the deoxyribose [12]. Pyrimidine base modifications are more readily formed after radiation compared with purines. The main radiation-induced base degradation products can be found in the work of Cadet and Wagner [13].


A DNA–DNA intrastrand cross-link (intra CL) is formed when chemical bonds are created between two DNA bases of the same DNA strand, while a DNA–DNA interstrand cross-link (inter CL) is created when the chemical bonds are between bases of opposing strands. A chemical cross-link can also be generated with another endo- or exogenous molecule such as surrounding proteins to produce a DNA-protein cross-link (DPC). A DPC is formed as a covalent linkage between the protein and DNA after radiation-induced generation of DNA base radicals and amino acid radicals, mostly via hydroxyl radicals, which interact with each other [12]. A representation of the cross-links is given in Fig. 3.4.

Three chemical structures of double-stranded D N A depict intra crosslink within a strand, inter crosslink between two strands, and D N A protein crosslink in a strand.

Fig. 3.4
Examples of DNA cross-links. Chemical bonds (yellow) are created between two DNA bases within the same DNA strand (intra cross-link) or opposite strands of double-stranded DNA (inter cross-link). Proteins (blue) can become cross-linked to DNA to form DNA-protein cross-link (DPC). G guanine, C cytosine, A adenine, T thymine, H-bond hydrogen bond, P phosphate


Three chemical structures of double-stranded D N A depict intra crosslink within a strand, inter crosslink between two strands, and D N A protein crosslink in a strand.


They are problematic since replication and transcription mechanisms require a separation of the DNA strands. The most frequent cross-links observed are between tyrosine and thymine, tyrosine and cytosine, or lysine and thymine.


Single-strand breaks (SSBs) result from endogenous processes and exposure to exogenous agents such as radiation and chemicals. A representation of this process is given in Fig. 3.5. More frequently, IR creates free highly reactive radicals, especially hydroxyl radicals (OH°), which may react with nearby DNA and produce an SSB. The repair of SSB is rather simple, as it will be discussed in Sect. 3.4, and thus most of the time, an SSB does not cause any serious problems to the cell. The quantity of SSBs increases linearly with the IR dose applied, and their formation decreases when the linear energy transfer (LET) increases [14].

A chemical structure of double-stranded D N A depicts a single-strand break or S S B, which is indicated by the absence of a phosphate group in a strand. G and C are linked by the H bond.

Fig. 3.5
Single-strand breaks (SSB): an illustration of a single-strand break in DNA. G guanine, C cytosine, A adenine, T thymine, H-bond hydrogen bond, P phosphate


A chemical structure of double-stranded D N A depicts a single-strand break or S S B, which is indicated by the absence of a phosphate group in a strand. G and C are linked by the H bond.


Double-strand breaks (DSBs) are produced when two SSBs on the two opposite DNA strands appear in close vicinity (one or two helix turns, thus about 15–20 DNA base pairs apart) [11]. Since DSBs are considered as the most important cause of cell death after IR, understanding their mechanisms of formation is essential. Radiation-induced DSBs increase linearly with radiation doses up to several hundred Gray (Gy) and have been detected at as low as 1 mGy [15]. As explained in Chap. 2, low linear energy transfer (LET) IR consists of electrons and photons that liberate secondary electrons and produce reactive oxygen species (ROS). However, even if they can create closely spaced lesions, the collision between particles and atoms in tissues is infrequent, thus leading to less, randomly distributed DSBs. On the contrary, the damages induced by high-LET particles are distributed along the particle tracks, which exhibit higher rates of collision and lead to nonrandom DSB distributions. Furthermore, there is a complexity of the nature of the DSBs formed according to the dose and the type of radiations, which influence the DNA damage response (DDR) and its efficacy. One can talk about “clean DSBs,” produced by hydrolysis of the phosphodiester bonds, which are easier to repair compared to “dirty DSBs,” which contain residual modified sugar residues produced by reaction of the 2-deoxyribose with hydroxyl radicals [11] (see Fig. 3.6). “Dirty” DSBs are more frequently created by high-LET heavy ions or α particles.

Two chemical structures of double-stranded D N A depict clean and dirty double-strand breaks or D S B, indicated by the absence of the phosphate group in both strands and the sugar-phosphate bond in both strands.

Fig. 3.6
Double-strand breaks (DSB): an illustration depicting different types of double-strand breaks in DNA. G guanine, C cytosine, A adenine, T thymine, H-bond hydrogen bond, P phosphate


Two chemical structures of double-stranded D N A depict clean and dirty double-strand breaks or D S B, indicated by the absence of the phosphate group in both strands and the sugar-phosphate bond in both strands.


Induction of DSB lesions by radiation is reviewed by Sage and Shikazono [16]. The ROS produced by the water radiolysis mediated by irradiation induces oxidized bases and loss of bases. Both lesions are repaired by base excision repair (BER, see Sect. 3.4), which can lead to DSB formation. Usually, DNA gaps of 1 or 2 nucleotides are filled by DNA polymerase and sealed by DNA ligase IIIα. During this process, SSBs can be generated in both DNA strands, and when they are close enough lead to a DSB. Moreover, the repair of a cluster lesion, e.g., an SSB opposite to an oxidative DNA lesion, could also result in the formation of a DSB as a result of irradiation. Additionally, through replication, if a damage is complex, e.g., effect on DNA secondary structures, formation of abasic sites, cross-links, and effect on DNA-binding proteins, the replication fork can stall and a DSB might occur. Moreover, conformational variables of the chromatin, which is a dynamic entity, and nuclear factors might affect DSB formation caused by radiation-induced radicals across the genome and according to the different points of the cell cycle.


Complex DNA damages, described as clustered DNA damages, are also named “locally multiple damaged sites” (LMDSs). LMDSs consist of closely spaced DNA lesions within a short DNA segment and are responsible for an increased cellular lethality since they are more difficult to repair. Two or more DNA lesions of the same or different type may be induced by IR within one or two helical turns of the DNA molecule, on the opposite strand. This clustered bistranded damage can be SSBs, DSBs, oxidized bases, and abasic sites. For example, at a dose of 1 Gy of IR, all this damage can be generated isolated or up to 10 bp apart [17]. Furthermore, the number of lesions per cluster depends on the radiation type and dose [18]. Experimental and theoretical studies have evidenced an increased complexity of the DNA damage induced by high-LET IR due to clustered ionizations, making complex DNA damage the signature of high-LET IR. Indeed, such lesions are considered the most important ones in terms of biological effects since they are the most challenging for the DNA repair machinery.


Not all cellular DNA damage is caused by exogenous factors; it can also be the result of cell metabolism as well as other normal cell processes. An overview of the average yield of DNA damage by endogenous factors per day and by low- and high-LET IR by 1 Gy is given in Table 3.1. One can see that even though the number of particles in the nucleus for high-LET radiation is much lower compared to low-LET radiation, the number of ionizations is the same. The dose deposition profile of high-LET IR induces more localized, complex, and clustered damages, which are more difficult to repair.Table 3.1
Comparison of DNA damage for endogenous factors and low- or high-LET radiations

Endogenous/cell/day

Low-LET IR/Gy

High-LET IR/Gy

Tracks in nucleus

–

1000

A few <1

Ionizations in nucleus

–

100,000

100,000

Ionizations in DNA

–

1500

1500

Base damage

16,000

10,000

10,000

DNA single-strand breaks

10,000–55,000

700–1000

300–600

DNA double-strand breaks

8

40

>40

Cross-link DNA/DNA

8

30

–

Cross-link DNA/protein

A few

150

–

Locally multiple damaged sites

A few

Increased with LET

The number of tracks in the cell nucleus as well as the number of induced damages for high-LET IR depends on the particle type and energy; therefore, the given values represent only an estimate


Ultraviolet (UV) light (100–400 nm) is a natural genotoxic agent able to induce deleterious effects affecting biological processes and structures, but also DNA structure, leading to a genomic instability [19]. DNA damage induced by UV is mainly pyrimidine dimers, oxidized bases, as well as SSBs and DSBs. Nucleotides absorb UV radiations, which raise the DNA base to a highly reactive singlet or triplet state, leading therefore to photochemical reactions. The chemical nature and the amount of DNA damage strongly depend on the wavelength of the incident photons. Three main types of DNA lesions are formed involving two successive pyrimidine bases (CC, TT, TC, and CT) and leading to a DNA double-helix distortion: cyclobutane pyrimidine dimers (CPDs), pyrimidine 6-4 pyrimidone photoproducts (6-4PPs), and their Dewar isomers. The most energetic part of the solar spectrum corresponding to UVB (290–320 nm) leads to the formation of CPDs and 6-4PPs, whereas less energetic but 20 times more intense UVA (320–400 nm) also induces the formation of CPDs associated with a wide variety of lesions such as single-strand breaks and oxidized bases. Furthermore, in addition of the direct photolesions induced, some indirect DNA damage can occur through the production of ROS, especially hydroxyl radicals (OH°) and RNS. ROS can induce the oxidation of pyrimidine and purine bases, and also the deoxyribose backbone of DNA, such as the induction of the most frequent, i.e., the 8-hydroxyguanine (8-oxo-G) and in a smaller extent SSBs and DSBs. Moreover, the ROS induced by UV can lead to the alkylation of bases and to cross-linking of DNA–DNA or DNA-protein. CPDs and 6-4PPs are mostly formed between TT and TC, and in less proportion for CT and CC sequences. Additionally, the chromatin structure, as well as the composition of the neighboring nucleotide sequence of pyrimidine dimers, also influences the formation of UV-induced DNA damage. More recently, some studies discussed the influence of the epigenetic markers (DNA methylation, histone posttranslational modifications) in the induction of UV-induced lesions at a particular locus. Indeed, the methylation of DNA at C5 of cytosine (5-mC) was associated with an increase by 80% of the CPD yield and a decrease by 3 of the 6-4PP [20] (Box 3.2).


Deoxyribonucleic acid (DNA) is a large molecule composed of two polynucleotide chains that coil around each other to constitute a double-stranded helix structure.

IR can cause DNA base or sugar damage, single- or double-strand breaks, DNA interstrand, intrastrand, or protein cross-links.

DSBs are considered to be one of the most serious DNA lesions.

High-LET IR induces more localized, complex, as well as clustered damage, which has the most serious potential biological consequences.


Deoxyribonucleic acid (DNA) is a large molecule composed of two polynucleotide chains that coil around each other to constitute a double-stranded helix structure.


IR can cause DNA base or sugar damage, single- or double-strand breaks, DNA interstrand, intrastrand, or protein cross-links.


DSBs are considered to be one of the most serious DNA lesions.


High-LET IR induces more localized, complex, as well as clustered damage, which has the most serious potential biological consequences.


As described above, various types of DNA lesions occur through endogenous and exogeneous factors frequently in a human cell. Depending on the complexity, these lesions challenge cellular genomic integrity. At the time of cell division, many cellular processes are coordinated to ensure the maintenance of the stable genome and ascertain the preservation of the nuclear material [21]. These processes are known as the DNA damage response (DDR). The types of DNA damage and their primary repair pathway are listed in Table 3.2. The DDR signaling capacity can, if not sufficient, cause problems for the cell to maintain genome stable, which may result in a mutation. This may, as a last consequence, trigger transformation into a tumor or cancer cell. As DNA damage occurs physically, it can be repaired; however, when the mutation is established, the alterations that took place in the base sequence cannot be repaired. Accordingly, it is essential for normal cells to maintain DDR function to avoid such process.Table 3.2
DNA damage repair mechanisms

DNA repair mechanism

DNA damaging/genotoxic agents

DNA lesion feature

DNA damage example

DNA repair features

Base excision repair (BER)

Reactive oxygen species, X-rays, alkylating agents

Oxidative lesion

Oxidation (8-oxo-G) uracil, single-strand break

Removal of base by N-glycosylase abasic sugar removal, replacement

Nucleotide excision repair (NER)

UV lights and polycyclic aromatic hydrocarbons

Helix-distortion lesion

Bulky adducts, intrastrand cross-link

Removal of DNA fragment and replacement

Mismatch repair (MMR)

Replication

Replication error

A–G mismatch, T–C mismatch, insertion, deletion

Removal of strand by exonuclease, digestion, and replacement

Double-strand break repair (DSBR)

X-rays, ionizing radiations, reactive oxygen species, anti-tumor agents

Double-strand DNA breaks

Double-strand break, interstrand cross-link

Unwinding, alignment, ligation


Base excision repair (BER) is the most common and important DNA repair process involved in removing minor DNA base defects. Many BER genes are extremely maintained from bacteria to humans demonstrating that BER is a fundamental repair process [22]. BER is a well-studied pathway for damage repair caused by respiration, spontaneous hydrolysis, and alkylation events, such as single-nucleotide bases (small, non-helix-distorting base lesions), that occur hundreds of times every day in each cell [23]. Thus, the BER system is critical to eliminate damaged bases that could otherwise produce mispair mutations or DNA replication breakdowns. In BER, SSBs are formed and repaired in an organized chain of events involving multiple proteins. Within BER, two pathways are simultaneously active: short patch repair (SP-BER), which is used to eliminate a broken base which has a non-bulky character, and long patch repair (LP-BER), which can replace the area in which the damaged DNA base is found. A schematical view of SP- and LP-BER can be found in Fig. 3.7.

A flow diagram depicts short and long patch repair carried out by the activities of glycosylase, A P lyase, polymerase, and standard displacement. The hemieacetal and aldehyde formations lead to short and long patch repairs, respectively.

Fig. 3.7
Short and long patch base excision repair: recognition of the DNA lesion occurs by a specific DNA glycosylase which removes the damaged base by hydrolyzing the N-glycosidic bond. The remaining AP site is processed by APE. Depending on the cleavability of the resulting 5′dRP by Polβ, repair is performed via the short or long patch BER pathway. Reproduced with permission from [24]. AP-endonuclease apurinic/apyrimidinic endonuclease, AP-lyase apurinic/apyrimidinic lyase, OH hydroxide, P phosphate, 5’dRP 5′ deoxyribose phosphate, Lig III ligase III, XRCC1 X-ray repair cross-complementing 1, RF-C replication factor C, Fen1 flap structure-specific endonuclease 1, PCNA proliferating cell nuclear antigen, Lig I ligase I


A flow diagram depicts short and long patch repair carried out by the activities of glycosylase, A P lyase, polymerase, and standard displacement. The hemieacetal and aldehyde formations lead to short and long patch repairs, respectively.


In BER, specialized proteins called glycosylases recognize and remove the majority of the damaged DNA bases. There are multiple glycosylases, each of which is unique to a certain form of base damage. All these enzymes have, as their primary function, to cut out the base which got damaged yet without impacting the DNA backbone, causing further damage in an abasic place in the DNA (either apurinic or apyrimidinic site) [25]. Although each DNA glycosylase is specialized to a certain substrate and works in a distinct manner, they all have a single principal way of action: first, taking the damaged base outside the DNA helix, thus assisting the detection of bases with minute alterations, and, second, triggering the cutting of an N-glycosidic bond, which in turn enables the formation of an abasic site [22]. Humans have 11 DNA glycosylases, which are classified as monofunctional (removing a base which results in formation of an AP site), bifunctional (removing a base and cutting the DNA backbone close to the damaged base), or Nei-like (which removes the base but also cuts each side of it).


Once the monofunctional DNA glycosylase has created the AP site, another repair enzyme, AP endonuclease 1 (APE1), incises and hydrolyzes the AP site, removing the base followed by the sugar residue, cutting the DNA backbone, and as a result an SSB is formed. APE1 also operates on bifunctional glycosylase products, creating a one-nucleotide gap product after hydrolysis. Polynucleotide kinase phosphatase (PNKP), whose product is suitable for DNA polymerase action, is required for the repair of oxidized DNA bases. When there is a gap or SSB is formed, poly(ADP-ribose) polymerase 1 is activated (PARP1) [23]. In this way, the integrity of the break can be maintained. PARP1 also orchestrates, via its poly(ADP-ribosyl)ation activity, a cascade of proteins binding to the SSBs with the main aim to detect and promote its further repair.


The most common polymerase used in BER is DNA polymerase (Pol), which fills the gap with the proper nucleotide and catalyzes a lyase reaction. SP-BER is linked by the DNA ligase III-XRCC1-mediated mechanism to complete the process [25]. In contrast to SP-BER, LP-BER occurs when a lesion is resistant to Pol cleavage, and polymerases such as PCNA, flap endonuclease 1 (FEN1), and PARP are recruited. While displacing the broken strand, the polymerase synthesizes DNA and inserts a repair patch consisting of 2–12 of the correct nucleotides into the gap. The repair synthesis is carried out by the T complex of the replication factor C (RFC)/proliferating cell nuclear antigen (PCNA)/DNA polymerase δ/ε. Here, the lap endonuclease 1 (FEN1) acts by taking out the flap structure that is overhanging the damaged base site, and the nick that is formed is ligated by DNA ligase I [14]. SP-BER and LP-BER primarily differ in how many of the DNA bases are cut out during the repair (see Fig. 3.8). SP-BER only replaces the bases which are damaged, whereas LP-BER cuts out and replaces up to ten nucleotides.

Two flow diagrams compare G G R or global genomic repair and T R or transcription-coupled repair. The common steps are incision, excision, polymerization, and ligation.

Fig. 3.8
Nucleotide excision repair (NER) pathway: during global genomic repair (GGR), recognition of the DNA lesion occurs by XPC–HR23B, RPA–XPA, or DDB1–DDB2. DNA unwinding is performed by the transcription factor TFIIH and excision of the lesion by XPG and XPF–ERCC1. Finally, resynthesis occurs by Polδ or Polε and ligation by DNA ligase I. During transcription-coupled repair (TCR), the induction of the lesion results in blockage of RNAPII. This leads to assembly of CSA, CSB, and/or TFIIS at the site of the lesion, by which RNAPII is removed from the DNA or displaced from the lesion, making it accessible to the exonucleases XPF–Ercc1 and XPG cleaving the lesion-containing DNA strand. Resynthesis again occurs by Polδ or Polε and ligation by DNA ligase I. 23B: Reproduced with permission from Christmann et al. [24]. DDB1 DNA damage-binding protein 1, DDB2 DNA damage-binding protein 2, RPA replication protein A, TFIIH transcription factor IIH, ERCC1 excision repair cross-complementing group 1 protein, Polyδ/ε DNA polymerase delta/epsilon, PCNA proliferating cell nuclear antigen, Lig1 DNA ligase 1, RNAPII RNA polymerase II, CSA and CSB Cockayne syndrome factors A and B, TFIIS transcription initiation factor IIS, HR23B homologous recombinational repair group 23B


Two flow diagrams compare G G R or global genomic repair and T R or transcription-coupled repair. The common steps are incision, excision, polymerization, and ligation.


IR-induced base damage is effectively repaired by BER. BER deficiencies can result in a higher mutation rate but seldom cause cellular radiosensitivity [26]. The X-ray cross-complementing factor 1 (XRCC1) gene mutation, which causes a 1.7-fold increase in radiation sensitivity, is an exception. The radiation sensitivity of XRCC1-deficient cells, on the other hand, could be due to XRCC1’s involvement in other repair processes, such as SSB repair. Reduced repair and radiosensitization can be caused by mutations, deletions, or inhibition of either of these genes.


In both BER and SSB repair, DNA polymerase beta (pol) is a key enzyme. Under some situations, cells lacking pol or expressing a dominant negative construct to pol, which inhibits its function, have been demonstrated to be more vulnerable to ionizing radiation in vitro [27]. Small-molecule medicines that block PARP1 have also been produced. The PARP inhibitors are a medication that targets BER and SSB repair and are now being tested in clinical trials for cancer treatment, as described in Chap. 6 (Box 3.3).


BER is a specific repair mechanism that is used to handle DNA base damage.

BER removes single-nucleotide base lesions (small, non-helix-distorting base lesions) from the genome.

SP-BER and LP-BER are two complementary BER systems essential for removing base damage and fixing SSB in DNA, minimizing mutagenesis but differing in what base damages they can handle.

BER inhibitors have showed potential as radio/chemosensitizers in a variety of malignancies, or they can create synthetic deadly alliances with common cancer mutations.


BER is a specific repair mechanism that is used to handle DNA base damage.


BER removes single-nucleotide base lesions (small, non-helix-distorting base lesions) from the genome.


SP-BER and LP-BER are two complementary BER systems essential for removing base damage and fixing SSB in DNA, minimizing mutagenesis but differing in what base damages they can handle.


BER inhibitors have showed potential as radio/chemosensitizers in a variety of malignancies, or they can create synthetic deadly alliances with common cancer mutations.


From unicellular bacteria to complex humans and plants, nucleotide excision repair (NER) works in a similar way. In humans, NER is known for its one-of-a-kind repair process to remove photolesions caused by UV radiation. However, there is one circumstance in which NER genes can influence the IR response. More DNA cross-links are formed when cells are irradiated under hypoxia than when irradiated under normoxic circumstances. Excision activity of two NER genes, DNA excision repair protein (ERCC1) and DNA repair endonuclease (XPF), is required for such cross-links, among other things. Defects in either of these genes may cause hypoxic cells to become more radiosensitive. As a result, the status of the NER pathway is relevant to radiotherapy in combination with specific chemotherapeutic drugs, as well as hypoxic tumors treated only with radiotherapy [28].


The principle of NER is shown in Fig. 3.8. The lesion-recognizing NER factors look for unpaired single-stranded DNA on the other side of the damaged strand [22]. The oligonucleotide that contains the lesion is eliminated, and to restore the DNA to its original form, a repair patch is created using the opposite undamaged complementary strand as a template. With varied degrees of success, NER eliminates lesions from the entire genome and can be separated into two paths [24]:1.
Global Genome Repair (GGR or GG-NER): GG-NER is a genome-wide process, i.e., lesions can be eliminated from DNA that encodes, or not, for genes.
 2.
Transcription-Coupled Repair (TCR or TC-NER): TC-NER exclusively eliminates lesions in the DNA strands of genes that are actively transcribed. If a DNA strand that is actively transcribed is broken, the RNA polymerase could inhibit DNA repair by blocking access to damage sites. TC-NER has evolved to overcome RNA polymerase’s barrier by essentially eliminating it from the damage site, allowing repair proteins access.


Global Genome Repair (GGR or GG-NER): GG-NER is a genome-wide process, i.e., lesions can be eliminated from DNA that encodes, or not, for genes.


Transcription-Coupled Repair (TCR or TC-NER): TC-NER exclusively eliminates lesions in the DNA strands of genes that are actively transcribed. If a DNA strand that is actively transcribed is broken, the RNA polymerase could inhibit DNA repair by blocking access to damage sites. TC-NER has evolved to overcome RNA polymerase’s barrier by essentially eliminating it from the damage site, allowing repair proteins access.


In the early damage recognition phase, the two NER subpathways vary. In GGR, the NER proteins are recruited by the stalled RNA polymerase in collaboration with Cockayne syndrome protein B and A (CSB and CSA). In TCR, the NER proteins are engaged by the stalled RNA polymerase in collaboration with CSB and CSA [14].


Mutations in the NER genes do not cause IR sensitivity. However, defective NER increases sensitivity to UV-induced DNA damage and anticancer drugs that create bulky adducts, such as alkylating agents. Human DNA repair deficiency such as xeroderma pigmentosum, in which individuals are hypersensitive to UV radiation, is caused by germline mutations in the NER genes [14] (Box 3.4).


Nucleotide excision repair (NER) is a technique for removing bulky adducts from DNA, chiefly those caused by UV.

Defects in certain NER proteins may result in enhanced radiosensitivity of hypoxic cells.

Large DNA lesions like thymine dimers and cisplatin adducts are repaired using a DNA repair pathway.

The two types of NER pathways are global genome repair (GGR or GG-NER) and transcription-coupled repair (TCR) (TCR or TC-NER).


Nucleotide excision repair (NER) is a technique for removing bulky adducts from DNA, chiefly those caused by UV.


Defects in certain NER proteins may result in enhanced radiosensitivity of hypoxic cells.


Large DNA lesions like thymine dimers and cisplatin adducts are repaired using a DNA repair pathway.


The two types of NER pathways are global genome repair (GGR or GG-NER) and transcription-coupled repair (TCR) (TCR or TC-NER).


The mismatch repair (MMR) system has a role after the cellul replication process, where sometimes incorrect bases pair with each other (which is called a mismatch). Therefore, MMR aids in keeping DNA homeostasis and plays a major role in evolutionary genomic stability [29]. Its basic purpose is to rectify the small insertion-deletion loops (indels) and the base-base mispairs that are spontaneously generated at the time of DNA replication. These mis-incorporated bases have escaped the proofreading action of replication polymerase. Usually, the polymerase that carries out the DNA synthesis process is not completely error-free. The DNA polymerase on average makes one mistake for every 105 nucleotides [29], which implies that ~100,000 errors arise through each S phase of the cell. Even though the DNA polymerase is there to ascertain that such mistakes do not occur, a few mutations can go unnoticed by it and hence the MMR-associated genes act as the second line of defense. However, if the cell is deficient in the MMR process, these errors remain uncorrected. Therefore, the mutational rate and sequence length modification in the microsatellites, which is a known trait of tumor cells, increase. The relevance of MMR in radiation-induced damage and cellular radiosensitivity is a matter of controversy. The mismatch repair (MMR) pathway was first discovered in E. coli cells [30]. Researchers have explored and understood that the MMR pathways and its associated proteins are evolutionarily conserved in almost all organisms including humans [31]. MMR works by inserting or deleting the mispaired bases by recognizing the mispaired lesion; excision, i.e., removal of the erroneous strand; and DNA resynthesis and gap repair by filling it with the correct resynthesized DNA.


The parent strand, which includes a palindrome DNA sequence “GATC” and adenine, is methylated by the enzyme deoxy-adenine-methylase. However, after replication when there are two new incorrect strands, methylation in the newly formed daughter strand is not seen [32] (Fig. 3.9). Such alterations are recognized and repaired by the methyl mismatch repair. The specific region of mispairing is recognized by the Mut S protein, which is coupled by the MutL. The activity of MutS is stimulated by the heterodimer MSH2–MSH6, along with MutSα. The MutSα recognizes small IDLS comprising 1–2 nucleotides, whereas the MSH2–MSH6 identifies longer insertion-deletion loop-type mismatches. After the binding of MutS to the DNA, it is followed by the ATP-dependent prerequisite of MutL homolog (MSH) complex. The parent strand is recognized by the MutL, which brings the misrepaired region nearer and leads to a loop formation around the area. Another protein, MutH, an endonuclease enzyme, performs the activity of cleaving. Next, UVr-D, a helicase, releases the cut strand leading to the formation of a gap where the new error-free or accurate nucleotide sequence is included by the polymerase 1 and joined by ligase. Cells that are deficient in the MMR proteins exhibit a high frequency of mutations and also irreversible microsatellite instability. Accordingly, individuals with germline mutations in MMR genes are more susceptible to various types of cancers [33] (Box 3.5).

A flow diagram depicts the steps of mismatch detection in double-stranded D N A, nicking A T P to A D P plus P i. excision, re-synthesis, and ligation.

Fig. 3.9
Overview of eukaryotic mismatch repair system. In the human cell, the predominantly found MutSα (MSH2–MSH6) or the MutSβ recognizes the DNA mismatch repair and initiates its repair. Some of the crucial molecules which participate in the repair are the MutLα (MLH1-PMS2), the proliferating cell nuclear antigen (PCNA), and the replication factor (RCF). EXO1 catalyzes the repair, and ligase finally ligates the repaired DNA


A flow diagram depicts the steps of mismatch detection in double-stranded D N A, nicking A T P to A D P plus P i. excision, re-synthesis, and ligation.


MMR targets DNA mismatches that arise mainly during replication, as well as repairing mismatches that occur in DNA following treatment with alkylating agents.

The MMR pathway detects and repairs erroneous insertions, deletions, and base substitutions that have not been detected by the proofreading function of DNA polymerase during DNA replication, thus maintaining the genome stability.

It works by recognition of mispair, excision of the affected strand, and filling of the gap.


MMR targets DNA mismatches that arise mainly during replication, as well as repairing mismatches that occur in DNA following treatment with alkylating agents.


The MMR pathway detects and repairs erroneous insertions, deletions, and base substitutions that have not been detected by the proofreading function of DNA polymerase during DNA replication, thus maintaining the genome stability.


It works by recognition of mispair, excision of the affected strand, and filling of the gap.


Double-strand breaks (DSBs) are the most lethal kind of DNA damage because even one uncorrected DSB can result in loss of genetic information and finally lead to cell death. Moreover, such unrepaired or misrepaired DSBs can lead to augmented genomic instability and eventually tumorigenesis [21]. Accordingly, for a cell to pursue its genetic information, a functional DSB repair system is of major importance. As a result, cells have evolved a dedicated response to identify and mend DSBs. For repair of DNA DSBs, two principal pathways are used, namely homologous recombination (HR) and Non-homologous end joining (NHEJ).


These pathways differ with respect to the use of homologous template DNA as well as in DNA repair fidelity. HR utilizes undamaged sister chromatid as its template to repair the damage, and therefore it is error-free. However, NHEJ works by eliminating the damaged DNA followed by direct ligation and hence is error-prone. As HR needs an undamaged template, it only operates in late S and G2, in contrast to NHEJ, which has the capacity for DSB repair regardless of the cell’s position in the cell cycle phase [33].


The homologous recombination (HR) molecular pathway is associated with a large number of cellular processes, from imparting genetic diversity to DNA repair or replication. HR is evolutionarily conserved from bacteria to mammalian cells. This pathway is essential for fixing DNA damages with high accuracy by using the genomic code of the chromosomal copy which was not damaged [34]. HR works by precisely repairing the DSB, shielding cells from any chromosomal abnormalities such as those observed in many cancers. Throughout the process of DNA replication, HR-associated proteins endorse the faithfulness and restoring of distressed DNA replication forks. This adds sturdiness, serving the replication machinery to circumvent under replication and succeeding segregation tribulations of the chromosome. Inherent HR insufficiency in cells can persuade instability in the genome and further lead to cancer. Conversely, discrepancy in the HR pathway also sensitizes tumors not only to DNA damage treatment but also to other potential DNA repair inhibitors for remedial repair pathways.


For the commencement of the HR pathway, the break site 5′–3′ end resection is a requirement, which not only exposes the single-stranded DNA (ssDNA) overhangs but also averts the NHEJ pathway to repair the DNA breaks (Fig. 3.10) [36]. The repair proteins MRE11 (meiotic recombination 11), RAD50 (RAD50 double-strand break repair protein), and NBS1 (nibrin) form the MRN complex, and together with the ataxia-telangiectasia mutated (ATM) kinase, they are the first to recognize the DNA damage. By attaching to the DNA ends, the MRN complex instigates the process of DNA end resection. Next C-terminal binding protein 1 interacting protein (CtIP) is employed so as to produce the overhangs at the 3′ end of the single-stranded DNA [36]. The preference of the choice of repair pathway is governed by the p53-binding protein 1 (53BP1) and breast cancer-associated protein 1 (BRCA1) contrasting activity in addition to the MRE11 resection activity. Whenever a DNA break is identified, both BRCA1 and 53BP1 compete to govern the commitment of the cell to undergo NHEJ or HR, respectively. By hindering the DNA end resection and concurrently securing two double-stranded DNA (dsDNA) ends, facilitating their successive ligation, 53BP1 supports the NHEJ pathway [37]. The mechanism by which BRCA1 suppresses 53BP1 still remains uncertain. Ubiquitination of CtIP occurs when BRCA1 interacts with BRCA1-associated RING domain protein 1 (BARD1). This subsequently enhances the affinity of CtIP for DNA and as a consequence promotes resection [37]. At this time, the DNA ends are protected and prevented from resection by replication timing regulatory factor 1 (RIF1), which is a 53BP1-interacting partner and a Shieldin complex. The increased HR activity can be attributed to either the loss of 53BP1 or the Shieldin complex that weakens the NHEJ pathway. Blocking wide-ranging end resection is central, meant for preventing the hyper-recombination by HR and stopping the loss of genetic material. Some other lethal repairing pathways like break-induced replication (BIR) or single-strand annealing (SSA) can lead to wide-ranging resection whose outcome is loss of heterozygosity [35].

A flow diagram depicts the following steps. Pathway selection, resection, formation of RAD 51 filament, homology search and strand invasion, synthesis, S D S A, heteroduplex extension, resolution, and dissolution.

Fig. 3.10
Overview of homologous recombination (HR) pathways in double-strand break repair. When cells suffer a DSB (purple lines), they can repair them either by HR, with the help of a template that is homologous (turquoise lines), or by the NHEJ pathway. (a) BRCA1 promotes the HR pathways, whereas the Shieldin complex, RIF1, and 53BP1 promote the NHEJ pathway. (b) The resection process is performed by the MRN complex along with CtIP, EXO1, BLM, and DNA2 that form the 3′ ssDNA overhangs. These overhangs are then coated with the RPA (green boxes), which is later shifted by the RAD51 (brown circles). On the other hand, single-strand annealing occurs in case of the RAD-independent repair process, where annealing of the complementary DNA sequences takes place followed by overhangs cleaved by the flap endonuclease and finally the ends of the DNA are ligated. (c) Positive regulators of RAD51 such as RAD51 paralogs, BRCA2, and PALB2 aid in the formation of the RAD51 filament, whereas RECQL5 and FBH2 negatively regulate RAD51. (d) The RAD51 paralogs and RAD54A-B support the RAD51-mediated homology searching and strand invasion. At the same time, FANCM and RTEL negatively govern the RAD51-mediated D loops. (e) The homologous template in the form of sister chromatid or a homologous chromosome is used by the DNA polymerases to copy the missing sequence. (f) The DNA is resolved into a noncrossover product when SDSA dislodges the D loop. (g) In case there is an extension of the heteroduplex and development of Holliday junction created by the second-end capture, the intermediate states can be resolved by either resolution or dissolution. (h) The outcome of resolution is both the crossover and noncrossover products. (i) The outcome of dissolution is a noncrossover product. Adapted with permission (CCBY) from Sullivan and Bernstein [35]. Abbreviations: DSB double-strand DNA break, HR homologous recombination, NHEJ Non-homologous end joining, BRCA1 breast cancer gene 1, RIF1 Rap1-interacting factor 1, 53BP1 p53-binding protein 1, MRN MRE11–RAD51–NBS1 complex, CtIP CtBP-interacting protein, EXO1 exonuclease 1, BLM Bloom’s syndrome helicase, RecQ helicase-like gene, DNA2 DNA replication helicase/nuclease 2, ssDNA single-stranded DNA, RPA replication protein A, RAD51 RAD51 recombinase, PALB2 partner and localizer of BRCA2, RECQL5 RecQ-like helicase 5, FBH2 also GNA11, G protein subunit alpha 11, FANCM FA complementation group M, RTEL regulator of telomere elongation helicase 1, SDSA synthesis-dependent strand annealing


A flow diagram depicts the following steps. Pathway selection, resection, formation of RAD 51 filament, homology search and strand invasion, synthesis, S D S A, heteroduplex extension, resolution, and dissolution.


A full functional HR pathway can be utilized after the DNA end resection. A detailed review of this process can be found in the work of Ranjha et al. [38]. The canonical HR pathway not only restores a direct DSB, but also repairs damage created by stalled or collapsed replication forks [21]. As soon as an extensive resection is executed by the action of several nucleases, cells are obligated to follow a homology-governed mode of repair. The DSB goes through a nuclease-driven progression known as DNA end resection in order to produce 3′-end ssDNA segments all through HR. This is crucial for the searching and strand invasion that occurs later during the recombination process. Along with the CtIP nuclease, DNA end resection is instigated by the MRE11 nuclease within the MRN complex. MRN/CtIP in combination with Bloom syndrome protein (BLM) or exonuclease 1 (EXO1) and DNA replication helicase/nuclease (DNA2) arbitrates the short- as well as long-term resections. During this resection, the 3′ ends of ssDNA get exposed that are rapidly covered by replication protein A (RPA) complex. The ssDNA region covered by RPA further recruits and stimulates the ataxia-telangiectasia and Rad3-related (ATR) kinase. This in turn triggers the checkpoint kinase 1 (Chk1) kinase. The RPA coating not only ascertains the nondegradation of ssDNA overhangs but also avoids the formation of secondary structures. To form the presynaptic filament, RAD51 dislocates RPA, which is then involved in the action of several RAD51 mediator proteins. To construct a displacement loop (D-loop), the RAD51 nucleoprotein filament explores a homologous sequence to occupy and dislocate one strand of the homologous template. This structure aids in the formation of a heteroduplex by pairing the broken strand with the displaced strand, and DNA synthesis at the break site repairs for any missing nucleotides. The outcome of the second end capture leads to the configuration of a double-Holliday junction (dHJ). The resolution of such an intermediate occurs either by a resolution mechanism or by a dissolution, which makes it susceptible to crossover (CO) or noncrossover (NCO). On the other hand, at the time of synthesis-dependent strand annealing (SDSA), no more than one-end invasion takes place, therefore leading to the formation of a single-Holliday junction. This transitional structure is suspended into an NCO. The HR repair pathway is known to also involve chromatin modifiers, remodelers, and even integration of histone variant so as to deal with the obstructions that the nucleosomes produce to the resection machinery. HR is active during the late S phase and the G2 phase and therefore is able to utilize the sister chromatid as a guiding template to repair the DSBs. Hence, this pathway is error-free [38].


The Nonhomologous end joining pathway (NHEJ) pathway (Fig. 3.11) has long been demonstrated to be central in repairing DNA DSBs, and cells deficient in some of these signaling components are known to be very IR sensitive [39]. Moreover, NHEJ has a critical role in V(D)J-recombination when B and T lymphocytes are developed in the immune system. This is also illustrated by severe combined immunodeficiency (SCID) patients who, due to lack or alteration in some of the NHEJ components including the catalytical subunit of DNA-PK (DNA-PKcs) as well as others, have T and B lymphocytes that do not have proper function [39]. Importantly, cells from such patients also display high IR sensitivity.

A 7-step flow diagram depicts that the repair of I R induced D-N-A D-S-B starts with I R indued D N A D S B and ends with the association of the ligase four complex to seal the break.

Fig. 3.11
Schematic of the principal steps of NHEJ. (I) IR triggers the formation of DNA DSB in the cell nucleus. (II) To act on these, the NHEJ pathway commences with the movement of Ku (Ku70/Ku80) proteins towards the loose ends in the DNA DSB. (III) Ku70/Ku80 forms a complex embracing the ends protecting DNA integrity. DNA DSBs with noncomplex termini can be ligated directly after this step as end processing is not required. (IV) When the ends in the DSB require end trimming, the DNA-PKcs is recruited onto DNA via association to the Ku70/Ku80 complex forming a platform for subsequent steps. (V) Once associated to Ku proteins and DNA, DNA-PKcs undergoes autophosphorylation which changes its conformation. (VI) In this way, DNA-PKcs is active as a kinase and regulates the association of multiple DNA end-trimming proteins (e.g., Artemis, WRN, Polμ/λ, PNK), which restores the nucleotides at the termini allowing ligation to take place. (VII) The ligation step is controlled by the DNA ligase IV complexes, which apart from ligase IV also include XRCC4, XLF, and PAXX. At the end of the trimming and ligation step, some bases may be lost causing loss of genomic information which may cause mutations. Abbreviations: DNA DSB DNA double-strand break, NHEJ Non-homologous end joining, Ku dimeric Ku70/Ku80 protein complex, DNA-PKcs DNA-dependent protein kinase catalytic subunit, WRN protein deleted in Werner syndrome, Polμ/λ DNA polymerase μ/λ, PNK polynucleotide kinase, XRCC4 X-ray repair cross-complementing protein 4, XLF XRCC4-like factor, PAXX paralog of XRCC4 and XLF


A 7-step flow diagram depicts that the repair of I R induced D-N-A D-S-B starts with I R indued D N A D S B and ends with the association of the ligase four complex to seal the break.


The NHEJ process starts at the DNA end termini, also known as the break synapsis, where a heteromeric complex of the Ku proteins, Ku70/Ku80, forms a ringlike structure around the DNA. The Ku70/Ku80 complex then moves towards the break to bring the free DNA ends together and protect them from nuclease digestion (Fig. 3.11). This is critical for NHEJ function and for IR sensitivity as cells deficient in either Ku subunits have impaired NHEJ and also are IR sensitive [41].

A flow diagram depicts D N A double helix, nucleosomes, 30-nanometer fiber, and higher-order chromatin fiber, divided into chromosome territory in the interphase and condensed chromosome in the metaphase.

Fig. 3.12
Structure of DNA organization. The DNA forms a double-helix structure, which is wrapped around histones forming so-called nucleosomes. The nucleosomes form complex fibers of 30 nm size, which themselves form the higher order chromatin fibers, which are in the range of 300 nm. In the interphase, these fibers build the chromatin territories, where territories from different chromosomes can overlap, forming so-called networks. In the metaphase, the higher order chromatin fibers are condensed to form chromosomes. (Adapted with permission (CCBY) from Liu et al. [40])


A flow diagram depicts D N A double helix, nucleosomes, 30-nanometer fiber, and higher-order chromatin fiber, divided into chromosome territory in the interphase and condensed chromosome in the metaphase.


The end structures within the DNA DSB which are sensed and protected by the Ku protein complexes are 3′ or 5′ overhangs, blunt ends, closed hairpin, and complex structures including those found in IR-induced DSBs [41]. The current understanding is that the Ku complex heterodimer slides along the DNA strand and multiple subunits align onto DNA to form a protein scaffold. The end structure in the DSB, i.e., the blunt ends, 3′ or 5′ overhangs, thereafter dictates what route the NHEJ takes as some proteins are required for certain end termini to be processed prior to ligation while others are not [41, 42]. For example, when the end termini have some regions with certain nucleotides that overlap, the ends are ligated by the DNA ligase IV and X-ray repair cross-complementing 4 (XRCC4) complex alone. However, in the majority of the cases, the DNA protein kinase catalytic subunit (DNA-PKcs) orchestrates the reactions forming a holocomplex with the Ku proteins on the DNA [42] (Fig. 3.11).


DNA-PKcs is a kinase with the capacity to phosphorylate proteins on serine or threonine resides. It belongs to a protein family also named the PIK kinases to which also ATM and ATR belong. DNA-PKcs requires DNA binding for its kinase activity to control the end-processing activity within NHEJ as well as inactivation of its own function [42]. Thus, when the Ku complex binds DNA-PKcs, it causes autophosphorylation of multiple residues in the kinase domain and thereafter DNA-PKcs can phosphorylate its downstream substrates.


Multiple studies in rodent and human cells using various genetic approaches have shown that a defective DNA-PKcs activity impairs the repair of some but not all IR-induced DNA DSBs, but nevertheless causes increased radiation sensitivity [39]. To further study the function of DNA-PKcs for repair of IR or chemotherapy-induced DNA damage, inhibitors towards the kinase pocket have been developed, some of which have also been demonstrated to function as IR sensitizers of tumor cells and in tumor-bearing mice (reviewed in the work of Myers et al. [43]). All in all, it is clear that DNA-PKcs orchestrates the NHEJ pathway, but despite decades of research, the understanding of the entire molecular mechanisms is still not complete.


The end processing of the nucleotides is required as a DNA DSB seldom has the 3′OH and 5′P termini that are required for ligation. Therefore, the ends in the DNA DSB need to be processed by exonucleases such as Artemis, which has intrinsic 5′ exonuclease function and 5′ exonuclease acquired once in complex with DNA-PKcs [44]. The critical role for Artemis in the NHEJ processing has been shown as cells deficient in Artemis are sensitive to IR. However, Artemis is only required for repair of a subset of ~10–20% of the DNA DSBs, while the others are rejoined efficiently in the absence of Artemis. Therefore, it has been suggested that Artemis is responsible for repair of DNA DSBs that display slow repair kinetics. Apart from Artemis, there are also other proteins involved in the end-processing activity including Werner syndrome ATP-dependent helicase (WRN). It exhibits helicase and exonuclease function and suppresses 5′ end resection as well as HR by blocking MRE11 and CtlP association. Other examples are the polynucleotide phosphatase/kinase (PNKP) and tyrosyl-DNA phosphodiesterase 1 (TDP1) that modify the phosphorylation of the nucleotides and trim the ends to a state allowing ligation to take place. As some nucleotides may be lost in the end termini, the DNA polymerase μ and DNA polymerase λ are also part of the end-trimming activity in NHEJ.


Ligation of broken ends by NHEJ is carried out in a protein complex, which bridges around the DNA end in the DSB. The complex contains, among other proteins, XRCC4, DNA ligase IV, and XRCC4-like factor (XLF). Out of all the proteins involved in NHEJ, DNA ligase IV stands out when it comes to repair of DNA DSBs because mice, in which this gene is disrupted, experience lethality as embryos and dissection of such embryos have revealed extensive apoptosis, in particular in the nervous system [45]. Both ligase IV and XLF mutations, that impair their function, are reported in humans in different tumor types, e.g., leukemias and lymphomas, with the patients showing various degrees of deficiency in B and T lymphocyte function [46] (Box 3.6).


The NHEJ pathway plays a crucial role in the repair of DNA DSBs generated endogenously and by IR.

NHEJ has less fidelity in repair than HR and may therefore in certain circumstances cause mutations.

NHEJ deficiency results in increased radiation sensitivity.

Some of the NHEJ pathway components, e.g., DNA ligase IV, are essential for NHEJ repair, while others are required for efficient repair of certain subsets of DNA DSBs.

NHEJ components, e.g., DNA-PKcs, offer a target that can be used for radiation sensitization purposes in various tumor types.


The NHEJ pathway plays a crucial role in the repair of DNA DSBs generated endogenously and by IR.


NHEJ has less fidelity in repair than HR and may therefore in certain circumstances cause mutations.


Some of the NHEJ pathway components, e.g., DNA ligase IV, are essential for NHEJ repair, while others are required for efficient repair of certain subsets of DNA DSBs.


NHEJ components, e.g., DNA-PKcs, offer a target that can be used for radiation sensitization purposes in various tumor types.


Cells fundamentally utilize two conventional mechanisms to repair their DSBs, i.e., the HR and the NHEJ pathways. However, in recent times, a third pathway is discovered which is known as the alternative NHEJ (alt-NHEJ or aNHEJ), microhomology-mediated end joining (MMEJ), and B (backup)-NHEJ. This is an extremely error-prone pathway that operates in NHEJ-proficient as well as -deficient cells. Unlike HR, this pathway does not require any long homologous DNA templates and is therefore called as “alternative end-joining” pathways. This mechanism typically but not always depends on the microhomologies that exist at or near the DNA DSB ends, which implicates that it might not be completely divergent from the mechanism of HR. The junctions of this repair pathway demonstrated overlapping microhomologies of 3–16 nucleotides as well as nucleotide deletions. Earlier, it was known that the NHEJ pathway could recover short microhomologous region of up to five nucleotides in mammalian cells. However, the alt- NHEJ can operate even in the NHEJ-deficient cells [47]. It is a unique pathway that is seen to be ongoing throughout the cell cycle but found to be augmented in the G2 phase when compared to the G1 phase. Although it is arguable if there are other alt-NHEJ overlapping pathways, there is evidence of a microhomology-mediated end joining (MMEJ) that involves the arrangement of microhomologous series on the inner side of the broken ends prior to fusion and is linked with deletion adjoining the original DSB. This is also an error-prone pathway leading to chromosomal translocations.


One of the characteristics of alt-NHEJ is the excessive deletions and frequent microhomologies at the junction, while such microhomologies are not always present. The exclusivity of alt-NHEJ products implicates the usage of end resection-promoting enzymes, their association of proteins that get benefitted from the microhomologies that can support the intermediates to stabilize, nucleases competent of eliminating the noncompatible 5′ and 3′ overhangs, and finally ligation. The MRE11 complex and CtIP in end resection are known to facilitate the alt-NHEJ, and DNA ligase III emerges to uphold the ligation step.


It is observed that the microhomology-mediated DNA repair proceedings take place via RAD52-dependent single-strand annealing (SSA)-type machinery where the minimum SSA-dependent DSB repair lies between 5 and 29 base pairs of homology. In this mechanism, it is mandatory to have direct repeats on both the sides of the DNA break. Since SSA does not involve any strand invasion events, it is independent of RAD51. As MMEJ depends on the already existing microhomologies around the break, its probable mode of action is associated with SSA. Finally, for the sealing event, MMEJ depends on ligase III [47].


Although repair processes have been intensively investigated for decades, many principal questions concerning the mechanisms of radiation DNA damage induction and repair remain open [reviewed in the work of Falk and Hausmann [48]]. Chromatin in the cell nucleus is arranged into numerous hierarchical levels (Fig. 3.12) from micrometer to nanometer, which leads to the formation of a three-dimensional (3D) architectural chromatin network.


This network is dynamic and influenced by the cellular status and ongoing processes in the cell nucleus. Chromatin architecture is precisely regulated by physical and biochemical regulation systems and, in turn, regulates global and local genome functions. Local chromatin arrangement thus both reflects and determines the functions of the particular genetic locus, such as its transcriptional activity. Importantly in the context of radiobiology, nonrandom chromatin architecture seems to co-determine the response of cells to irradiation in numerous ways: First, in a tight interplay with physical characteristics of the radiation, functional chromatin structure states increase or decrease DNA susceptibility to DNA damage induction. Second, the chromatin architecture acts as an additional level of DSB repair regulation, cooperating with “standard” biochemical genetic and epigenetic regulation systems. Chromatin architecture may regulate DSB repair at individual DSB sites and also globally, via tuning the transcription intensity of genes involved in DNA repair and other processes related to the complex response of cells to radiation DNA damage (e.g., cell cycle progression or apoptosis). Theoretically, chromatin architecture might collect and unify signals of other different signaling networks (biochemical, epigenetic) and transfer these heterogeneous signals into single integrated output signal represented by a specific architectural status of the chromatin network that can be easily interpreted by the cell. Chromatin architecture might thus impersonate a “roofing” regulatory system based on simple physical laws, which allows for a sufficiently fast decision-making process for the optimal repair mechanism at each individual DNA damage site.


Different types (low LET vs. high LET) of IR interact with chromatin in specific ways. Therefore, the relationship between the radiation quality, architecture of structurally and functionally distinct chromatin domains, and DSB induction, repair, and misrepair play a role in the cellular radiation response. Genetically active, decondensed euchromatin and mostly inactive, condensed heterochromatin are the two traditionally recognized structurally and functionally distinct chromatin domains, which affect radiation response. However, it should be noted that radiation response differences may be even more prominent for other chromatin architectural and functional counterparts [49], such as RIDGE (regions of increased gene expression) and anti-RIDGE domains [50], which have even more precisely defined function and more homogenous architecture as compared to euchromatin and heterochromatin (Box 3.7).


DNA is organized in structural units ranging from micrometers to nanometers, forming 3D chromatin architecture.

Chromatin architecture is a key factor determining local damage induction by radiation.

Chromatin architecture operates with genetic and epigenetic regulatory factors orchestrating DNA damage response.


DNA is organized in structural units ranging from micrometers to nanometers, forming 3D chromatin architecture.


Chromatin architecture is a key factor determining local damage induction by radiation.


Chromatin architecture operates with genetic and epigenetic regulatory factors orchestrating DNA damage response.


DNA damage and repair processes can be related to specific cell states and chromatin architectures. The spatiotemporal sequence of repair protein binding to DSB and surrounding phosphorylated and thus activated H2AX histone (called γH2AX) sites can be analyzed using microscopy (Fig. 3.13). The analysis of the formation and subsequent dissociation of repair complexes, and the structure of these complexes, brought deep insights into the mechanisms of the two main DSB repair pathways in human cells, nonhomologous end-joining (NHEJ) and homologous recombination (HR)—as discussed above.

Three micrographs with two fluorescent dyes depict the presence of gamma H 2 A X, chromatin, and gamma H 2 A X with chromatin, respectively, at 5-micrometer scales.

Fig. 3.13
Localization of DNA damage on chromatin: radiation damage induced by high-LET alpha particle radiation microscopically visualized by γH2AX as a biomarker for double-strand breaks (left, magenta), chromatin labeling (middle, green), and merge of the two (right)


Three micrographs with two fluorescent dyes depict the presence of gamma H 2 A X, chromatin, and gamma H 2 A X with chromatin, respectively, at 5-micrometer scales.


The most obvious architectonical chromatin types are condensed (hetero)chromatin with only a low number of active genes and decondensed (eu)chromatin, which is generally considered as genetically (transcriptionally) active. It has been shown that condensed chromatin protects DNA from free radicals generated by ionizing radiation [51], but, at the same time, it is this condensed architecture and a high content of repetitive sequences that complicate and slow down the repair of DSBs located in heterochromatic domains. The protective function against free radicals of the heterochromatic status does not seem to simply result from high condensation of heterochromatin domains but rather from a high amount of proteins that specifically bind to heterochromatin and interact with radiation-induced free radicals before they can damage DNA [51]. However, if a DSB occurs in heterochromatin, its condensed architecture must decondense first in order to allow the formation of huge repair complexes and continuation of repair processes [52]. Moreover, numerous studies indicate that the slower repair of heterochromatic DSBs not only reflects this necessity for the decondensation of a damaged chromatin domain but also points to a slower repair mechanism, specifically homologous recombination (HR) [48]. HR in heterochromatin could be superior over NHEJ for numerous structural reasons and therefore preferred by the architecture of heterochromatin domain; however, at the same time, repetitive sequences present in heterochromatin are a clear contraindication for this repair mechanism. This paradox can be again explained and overcome by the already described heterochromatin decondensation at the beginning of repair. The RAD51 recombinase, which is responsible for complementary DNA strand search and exchange, can bind to heterochromatic DSB sites only upon heterochromatin decondensation and protrusion of a DSB to the domain surface, which ensures spatial separation of the damaged DNA ends from repeats remaining embedded within the heterochromatin domain. HR is thus evidently regulated by chromatin architecture changes, which also ensure the fidelity of this repair mechanism [48]. It remains unknown whether NHEJ or other repair pathways are also associated with some specific chromatin architecture requirements and rearrangements, similar to HR. However, some recent studies suggest that epigenetic and structural regulations are involved in repair pathway selection at individual DSB sites, as it is discussed later. The key properties of hetero- and euchromatin as mentioned here are summarized in Table 3.3.Table 3.3
Properties of hetero- and euchromatin

Heterochromatin

Euchromatin

Condensed DNA
Low amount of active genes
Protection of DNA from radicals through condensed structure and high amount of radical catching proteins clustering around DNA
Slow repair due to necessary decondensation
Homologous recombination superior to nonhomologous end joining

Decondensed DNA
Transcriptionally active
No radical protection
No decondensation necessary and therefore fast repair
No preference of repair mechanisms defined by chromatin architecture


A serious consequence of irradiation is the formation of chromosomal aberrations, and the chromatin architecture significantly participates in this process. The severity and complexity of the genetic damage are related to the complexity of the underlying DNA damage. The connection between damage complexity and radiation type was discussed in Sect. 3.2. An additional factor defining the complexity is the chromatin state, and radiation interacts with this. These interactions can be illustrated on the example of chromosomal translocation formation upon irradiation of euchromatin and heterochromatin with low-LET and high-LET radiation, respectively. The type of radiation, chromatin architecture, and consequently initiated DSB repair processes participate in a specific way in free DNA-end misrejoining (review [53, 54]).


The probability of a chromosomal translocation formation between two specific genetic loci, i.e., the linking of the ends of different chromosomes after induction of DSB in both chromosomes at the same time, depends on spatial (3D) separation of these loci in the cell nucleus. Chromatin is nonrandomly organized in the cell nucleus, though on the probabilistic basis, this means that chromosomal translocations between some genetic locus pairs appear more frequently than translocations between other pairs. This expectation was confirmed by experiments with interphase cells exposed to neutrons or high-LET particles where translocations appeared most frequently between the neighboring chromosomal territories or even genetic loci statistically located in close proximity [55]. Overall, there are two hypotheses used to explain the processes related to repair of DSB in the context of chromatin organization:1.
Position-first hypothesis: It considers DSBs as immobile structures and emphasizes the role of (preset) chromatin architecture in determining the probability of a chromatin exchange between two specific genetic loci.
 2.
Breakage-first hypothesis: It considers DSBs as mobile and gives the chromatin architecture a subsidiary role.


Position-first hypothesis: It considers DSBs as immobile structures and emphasizes the role of (preset) chromatin architecture in determining the probability of a chromatin exchange between two specific genetic loci.


Breakage-first hypothesis: It considers DSBs as mobile and gives the chromatin architecture a subsidiary role.


Both hypotheses explain different phenomena occurring. While the position-first hypothesis works well in explaining the enhanced probability of translocations to be formed by neighboring chromosomes, it does not allow chromatin exchanges between spatially more distant genetic loci, though such translocations were experimentally observed. Furthermore, although complex chromosomal translocations are only occasional events upon cell exposure to photonic (low-LET) radiation, they do occur. As DSBs are dispersed through the cell nucleus and thus spatially separated in cells irradiated with low-LET radiation, formation of complex translocation between three or more DSBs can hardly be explained without involving DSB movement. Both observations can be explained by the breakage-first hypothesis. However, the idea of highly mobile chromatin at DSB sites in cells exposed to low-LET radiation, where chromatin is not locally fragmented as in cells exposed to high-LET particle radiation, has not been generally confirmed. The explanation of this paradox came with the spatiotemporal tracking of individual radiation-induced protein accumulations (foci) [52], showing the majority of “immobile” DSBs accompanied with a small proportion of highly mobile DSB lesions or by subdiffusive nature of DSB loci [56]. The increased mobility correlated with DSB localization in heterochromatin and can thus be attributed to chromatin decondensation at the beginning of heterochromatin repair process, leading to the protrusion of DSBs onto the surface of heterochromatin domains. Numerous DSBs thus accumulate in nuclear subcompartments of a limited volume, which increases the probability of their mutual interactions and consequently chromatin exchanges even among multiple DSBs.


After irradiation with high-LET particles, on the other hand, locally concentrated energy deposition causes serious chromatin fragmentation and mobilization within cell nucleus micro-volumes along the particle tracks. This situation allows mutual contacts of many short chromatin fragments from one or several neighboring chromosomes and thus easy formation of complex chromatin translocations, irrespectively of the original chromatin architecture and chromatin architecture changes during repair. Chromosomal translocations in cells exposed to high-LET radiation thus occur due to physical rather than biological (repair) processes. We have already mentioned that heterochromatin architecture protects DNA from low-LET radiation as heterochromatin-binding proteins prevent DNA interaction with free radicals, mostly mediating harmful effects of low-LET radiation. With high-LET radiation, however, most damage to DNA is caused by the direct effect of radiation particles or emitted secondary electrons. In this case, heterochromatin represents a more dangerous chromatin architecture, as particles cannot be stopped by any chromatin architecture and heterochromatin provides more DNA targets per a volume unit compared to euchromatin. Hence, in cells exposed to high-LET radiation, translocations in heterochromatin tend to be more complex than in euchromatin (Box 3.8).


Hetero- and euchromatin form different chromatin architectural regions within a cell nucleus resulting in different consequences of radiation damage induction.

Chromosomal aberrations after low-LET radiation can be explained through the “position-first hypothesis” in combination with chromatin decondensation in heterochromatic regions.

Chromosomal aberrations after high-LET radiation occur due to physical fragmentation of DNA rather due to biological processes.

Heterochromatin protects DNA from indirect damage (mainly induced by low-LET radiation) but is more sensitive to direct damage (mainly induced by high-LET radiation).


Hetero- and euchromatin form different chromatin architectural regions within a cell nucleus resulting in different consequences of radiation damage induction.


Chromosomal aberrations after low-LET radiation can be explained through the “position-first hypothesis” in combination with chromatin decondensation in heterochromatic regions.


Chromosomal aberrations after high-LET radiation occur due to physical fragmentation of DNA rather due to biological processes.


Heterochromatin protects DNA from indirect damage (mainly induced by low-LET radiation) but is more sensitive to direct damage (mainly induced by high-LET radiation).


Using a variety of tools of super-resolution microscopy and image data computing has revealed that γH2AX foci in cell nuclei exposed to low-LET X-rays are subdivided into several equally sized, functionally relevant clusters. The number of clusters increased with the radiation dose according to the well-known linear-quadratic dependence and decreased at later time periods postirradiation. Calculations of the persistence of homology revealed a highly similar topology of γH2AX and other repair protein clusters, especially when these clusters were closely associated with heterochromatin regions. During the repair period, size and topology of these clusters seem to be maintained as long as they are attached to chromatin at actively repairing DSB sites. These findings suggest a functional relevance of the focus/cluster topology [57].


For instance, while the γH2AX clusters had a typical diameter of about 400 nm–600 m, the MRE11 clusters were smaller (about 200 nm) and usually completely embedded within γH2AX clusters [58]. The sizes of clusters were independent of repair time and cell type. On the other hand, the topological similarity of clusters followed the dynamics of the repair protein interaction with chromatin; that is, binding to damage sites was accompanied by ordering while detachments caused the relaxation of topological arrangements. In contrast, γH2AX and MRE11 clusters spontaneously occurring in the nonirradiated cells (e.g., due to replication defects) did not show this topological similarity.


Recent studies discovered spatial distribution changes of tri-methylated H3K9 histone (H3K9me3), ALU repeat sequences (ALU), or long interspersed nuclear element (LINE)-like L1 sequences, indicating chromatin reorganization or movement and DNA strand relaxation after radiation exposure, followed by recovery during repair [59]. Altogether, described results suggest a functional relevance of chromatin and repair focus nano-architecture in DSB repair process and their regulation (Box 3.9).


DNA repair locations marked by γH2AX and 53BP1 are subdivided into functional clusters at the nanoscale, in a manner which is cell type and radiation type specific.

Other repair protein clusters are smaller and are embedded in the γH2AX and 53BP1 clusters.

After damage induction, chromatin is reorganized accompanied by DNA movement.

Chromatin reorganization is recovered during DNA repair.


DNA repair locations marked by γH2AX and 53BP1 are subdivided into functional clusters at the nanoscale, in a manner which is cell type and radiation type specific.


Other repair protein clusters are smaller and are embedded in the γH2AX and 53BP1 clusters.


Lack of repair (unrepair) and misrepair of DNA damage can lead to increased chromosome breaks or rearrangements and mutations usually referred to as a status of genomic or genetic instability (GI). GI is usually associated with loss of cell cycle control, senescence, and cell death and in humans with pathological disorders including premature aging and predisposition to various types of cancer and inherited diseases [60]. On the other hand, GI is also fundamental for evolution and induction of genetic diversity. It is known that genomic integrity is carefully supervised by specific surveillance mechanisms like DNA damage checkpoint, DNA repair, or mitotic checkpoint. A deficiency in the regulation of any of these mechanisms often leads to GI, which can predispose a cell to malignant transformation [61].