24.2: DNA Mutations, Damage, and Repair

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Learning Goals 

(Learning goals written by Claude, Sonnet 4.6, Anthropic)

Types of Mutations and DNA Damage

• Distinguish between the major categories of mutations by scale and mechanism: explain how chromosomal alterations (amplifications, deletions, translocations, inversions, insertions) differ from small-scale mutations including point mutations (silent—same amino acid; missense—different amino acid; nonsense—premature stop codon) and insertions/deletions, and explain why frameshift mutations caused by insertions or deletions not in multiples of three alter every downstream codon and are almost invariably catastrophic for protein function.
• Describe the six major classes of DNA damage—oxidative damage (8-oxo-dG formation from ROS acting on guanine), alkylation (N7-methylguanine, O6-methylguanine, N3-methyladenine), AP site formation (spontaneous depurination/depyrimidination generating abasic sites at ~10,000 apurinic and 500 apyrimidinic sites per cell per day), bulky adduct formation (benzo[a]pyrene epoxide-guanine adducts causing G→T transversions), DNA crosslinking (UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts between adjacent pyrimidines), and strand breaks (single and double-stranded breaks from ionizing radiation or abortive topoisomerase catalysis)—and for each, identify the chemical basis of the lesion and its mutagenic consequence during replication.

DNA Damage Response and Cell Cycle Checkpoints

• Explain the DNA damage checkpoint signaling cascade: describe how ATM (activated by DSBs) and ATR (activated by ssDNA at stalled forks) phosphorylate and activate CHK2 and CHK1 respectively; how CHK2 activates p53, which induces p21^CIP1 expression to inhibit cyclin E/CDK2 and arrest the cell in G1; how CHK1 inactivates CDC25A (causing S-phase arrest) and CDC25C (causing G2 arrest) while activating WEE1 to inhibit CDK1 and CDK2 through Tyr15 phosphorylation; and how, if damage is irreparable, p53-dependent apoptosis is activated rather than cell cycle re-entry.

DNA Repair Pathways

• Explain base excision repair (BER): describe how damage-specific DNA glycosylases (e.g., OGG1 for 8-oxo-dG) cleave the N-glycosidic bond to create an AP site, how AP endonuclease 1 (APE1) incises the backbone at the AP site to create a single-strand break, and contrast short-patch BER (Pol β replaces one nucleotide, ligated by Lig III/XRCC1) with long-patch BER (Pol δ/ε displaces a flap of several nucleotides, removed by FEN1, ligated by Lig I).
• Explain nucleotide excision repair (NER): distinguish global genome NER (GG-NER, initiated by XPC-RAD23B recognizing helix-distorting bulky lesions throughout the genome) from transcription-coupled NER (TC-NER, initiated when RNA Pol II stalls at a damaged template strand); describe how both pathways converge on TFIIH-mediated local unwinding, dual incision by XPF-ERCC1 (5′) and XPG (3′) removing a 24–32 nt oligonucleotide, gap-filling synthesis by Pol δ/ε, and ligation—and explain why mutations in XP genes cause xeroderma pigmentosum with extreme UV photosensitivity and >1000-fold elevated skin cancer risk.
• Explain mismatch repair (MMR): describe how MutS (bacteria) or MutSα/MutSβ (eukaryotes) recognizes mismatches and small insertions/deletions by inserting Phe36 into the minor groove adjacent to the mismatch and kinked DNA; how strand discrimination is achieved (GATC methylation/MutH nicking in E. coli; strand discontinuities in eukaryotes); how MutL coordinates MutH endonuclease activity, UvrD helicase unwinding, and exonuclease excision of the error-containing strand; and how MMR deficiency is implicated in up to 90% of hereditary nonpolyposis colon cancer cases.
• Compare the two mechanisms of double-strand break (DSB) repair: explain how NHEJ—active throughout the cell cycle but predominantly in G1—uses Ku70/Ku80 to protect and align DNA ends, DNA-PKcs to bridge the break, and Lig IV/XRCC4 to religate, noting that NHEJ is error-prone and may introduce small insertions or deletions; and how HR—restricted to S/G2/M when sister chromatids are available—uses the MRN complex for 5′→3′ resection, RPA and RAD51 (loaded with BRCA1/BRCA2 assistance) for strand invasion, and BLM helicase for Holliday junction resolution, producing high-fidelity repair at the cost of greater mechanistic complexity.
• Explain translesion synthesis (TLS): describe how PCNA monoubiquitination at stalled replication forks recruits specialized Y-family translesion polymerases with spacious active sites that accommodate distorted DNA, enabling bypass of lesions that block replicative polymerases, and explain why TLS is inherently error-prone (inserting the nucleotide causing least steric repulsion, often adenine by the "A rule") and may produce transition mutations, transversion mutations, or frameshift mutations that contribute to somatic mutation accumulation and cancer development over a lifetime.

The integrity of the DNA structure is crucial for cell viability, as underscored by the extensive cellular machinery dedicated to its accurate replication, repair, and storage. Even so, mutations in DNA are fairly common.

DNA Mutations

Mutations are random changes that occur within the sequence of bases in DNA. They can be large-scale, altering chromosome structure, or small-scale, altering only a few bases or even a single base. Mutations can occur for many reasons. For example, DNA mutations can be caused by mistakes made by the DNA polymerase during replication. DNA polymerases are highly processive enzymes that contain proofreading and editing functions. With these safeguards, their error rates are typically very low, ranging from one in a million bases to one in a billion bases. Even with such high fidelity, this error rate will lead to between 3 and 3,000 errors in the human genome per cell undergoing DNA replication. DNA mutations can also result from the replication of DNA that has been damaged by endogenous or exogenous agents. The next section will highlight common types of DNA damage and their effects. If a DNA polymerase encounters a damaged DNA base in the template DNA during replication, it may place a random nucleotide base across from the lesion. For example, an adenine-containing nucleotide will often be added across a lesion, regardless of what the correct match should be. This can lead to the formation of transition or transversion mutations.

A transition mutation is a point mutation that changes a purine nucleotide to another purine (A ↔ G) or a pyrimidine nucleotide to another pyrimidine (C ↔ T). Transversion refers to the substitution of a purine for a pyrimidine or vice versa. Sometimes lesions may cause bases to be skipped during replication or cause extra nucleotides to be inserted into the backbone. DNA polymerases can also slip during the replication of regions of DNA that contain repeated sequences or large stretches of a single base. Larger lesions or cross-links in the DNA during replication can lead to more catastrophic DNA damage, including DNA strand breaks. Mutations may also occur during mitosis and meiosis when sister chromatids and/or homologous chromosomes are separated.

In nature, mutagenesis, or the process of generating DNA mutations, can lead to changes that are harmful, beneficial, or have no effect. Harmful mutations can lead to cancer and various heritable diseases, but beneficial mutations are the driving force of evolution. In 1927, Hermann Müller first demonstrated the effects of mutations with observable changes in chromosomes. He induced mutagenesis in fruit flies by exposing them to X-ray irradiation.

When a mutation is caused by an environmental factor or a chemical agent, that agent is referred to as a mutagen. Typical mutagens include chemicals, such as those inhaled when smoking, as well as radiation, including X-rays, ultraviolet light, and nuclear radiation. Different mutagens have distinct modes of DNA damage and are discussed further in the next section. It is important to note that DNA damage, in and of itself, does not necessarily lead to the formation of a mutation in the DNA. There are elaborate DNA repair processes that recognize and repair various types of DNA lesions. Fewer than 1 in 1,000 DNA lesions will result in a DNA mutation. The methods of DNA damage recognition and repair are the focus of later sections within this chapter.

Types of Mutations

There are various types of mutations. Two major categories of mutations are germline mutations and somatic mutations.

• Germline mutations occur in gametes, the sex cells, such as eggs and sperm. These mutations are especially significant because they can be transmitted to offspring, and every cell in the offspring will carry them.
• Somatic mutations occur in other cells of the body. These mutations may have little effect on the organism because they are confined to just one cell and its daughter cells. Somatic mutations also cannot be passed on to offspring.

Mutations also differ in how the genetic material is changed. Mutations may change an entire chromosome or just one or a few nucleotides.

Chromosomal alterations are mutations that alter the structure or number of chromosomes. They occur when a section of a chromosome breaks off and rejoins incorrectly or does not rejoin at all. Possible ways these mutations can occur are illustrated in the figure below. Chromosomal alterations are very serious. They often result in the death of the cell or organism in which they occur. If the organism survives, it may be affected in multiple ways. An example of a human chromosomal alteration is the mutation that causes Down Syndrome. It is a duplication mutation that leads to developmental delays and other abnormalities. It occurs when the individual inherits an extra copy of chromosome 21. It is also known as trisomy 21.

Thus, large-scale mutations in the chromosomal structure include (1) Amplifications (including gene duplications) where repetition of a chromosomal segment or presence of an extra piece of a chromosome broken piece of a chromosome may become attached to a homologous or non-homologous chromosome so that some of the genes are present in more than two doses leading to multiple copies of all chromosomal regions, increasing the dosage of the genes located within them, (2) Deletions of large chromosomal regions, leading to loss of the genes within those regions, and (3) Chromosomal Rearrangements such as translocations (which interchange of genetic parts from nonhomologous chromosomes), insertions (which insert segments of one chromosome into another nonhomologous chromosome), and inversions (which invert or flip a section of a chromosome into the opposite orientation), as shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): Chromosomal Alterations. Chromosomal alterations are major changes in the genetic material. Image modified from Dietzel65

There are also smaller mutations that can occur that only alter a single nucleotide or a small number of nucleotides within a localized region of the DNA. These are classified by how the DNA molecule is altered. One type, a point mutation, affects a single base and most commonly occurs when one base is substituted or replaced by another. Mutations also result from the addition of one or more bases, known as an insertion, or the removal of one or more bases, known as a deletion.

Figure \(\PageIndex{2}\): The Potential Effects of Point Mutations on Protein Coding Regions. The image shows various types of point mutations (silent, missense, and nonsense) that may alter protein structure. Figure from: Jonsta247

Point mutations (Table \(\PageIndex{1}\) and Figure \(\PageIndex{2}\)) may have a wide range of effects on protein function. As a consequence of the degeneracy of the genetic code, a point mutation will commonly result in the same amino acid being incorporated into the resulting polypeptide despite the sequence change. This change would not affect the protein’s structure and is thus referred to as a silent mutation. A missense mutation results in the incorporation of a different amino acid into the resulting polypeptide. The effect of a missense mutation depends on how chemically different the new amino acid is from the wild-type amino acid.

The location of the changed amino acid within the protein is also important. For example, suppose the changed amino acid is part of the enzyme’s active site or significantly affects the shape of the enzyme. In that case, the effect of the missense mutation may be significant. Many missense mutations result in proteins that are still functional, at least to some degree.

Sometimes, the impact of missense mutations may only be apparent under specific environmental conditions; such mutations are referred to as conditional mutations. Rarely, a missense mutation may be beneficial. Under appropriate environmental conditions, this type of mutation may confer a selective advantage on the organism that harbors it. Yet a different kind of point mutation, called a nonsense mutation, converts a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon). Nonsense mutations result in the synthesis of proteins that are shorter than the wild-type and typically nonfunctional.

Table \(\PageIndex{1}\): Types of Point Mutations

Type	Description	Example	Effect
Silent	mutated codon codes for the same amino acid	CAA (glutamine) → CAG (glutamine)	none
Missense	mutated codon codes for a different amino acid	CAA (glutamine) → CCA (proline)	variable
Nonsense	mutated codon is a premature stop codon	CAA (glutamine) → UAA (stop) usually	serious

Smaller-scale deletions and insertions also cause various effects. Because codons are triplets of nucleotides, insertions or deletions in groups of three nucleotides may lead to the insertion or deletion of one or more amino acids and may not cause significant effects on the resulting protein’s functionality. However, frameshift mutations, caused by insertions or deletions of a number of nucleotides that are not a multiple of three, are extremely problematic because a shift in the reading frame results, as shown in Figure \(\PageIndex{3}\). Because ribosomes read mRNA in triplet codons, frameshift mutations can change every amino acid after the mutation. The new reading frame may also include a stop codon before the end of the coding sequence. Consequently, proteins made from genes containing frameshift mutations are nearly always nonfunctional.

Figure \(\PageIndex{3}\): Summary of Small Scale Mutations on Protein Coding Regions. DNA alterations that change the protein sequence encoded by the DNA can include point mutations, insertions, and deletions. Figure from: Parker, et al (2019) Microbiology from Openstax

The majority of mutations have neither negative nor positive effects on the organism in which they occur. These mutations are called neutral mutations. Examples include silent point mutations, which are neutral because they do not change the amino acid sequence of the proteins they encode.

Some mutations have a positive effect on the organism in which they occur. They are referred to as beneficial mutations. If they appear in germline cells (such as eggs or sperm), these traits can be heritable and passed from one generation to the next. Beneficial mutations generally encode new protein variants that help organisms adapt to their environment. If mutations increase an organism’s chances of surviving or reproducing, they are likely to become more common within a population over time. There are several well-known examples of beneficial mutations. Here are just two:

1. Mutations have occurred in bacteria that enable them to survive in the presence of antibiotic drugs. The mutations have led to the evolution of antibiotic-resistant bacterial strains.
2. A unique mutation has been found in people from a small town in Italy. The mutation protects them from developing atherosclerosis, a dangerous buildup of fatty materials in blood vessels. The individual in whom the mutation first appeared has even been identified.

Harmful mutations can also occur. Imagine making a random change to a complex machine, such as a car engine. The likelihood that the random change would improve the car's functioning is very small. The change is far more likely to result in a vehicle that does not run well or perhaps does not run at all. Any random change in a gene's DNA is more likely to result in the production of a protein that does not function normally or may not function at all than in a mutation that improves the function. Such mutations are likely to be harmful. Harmful mutations may cause genetic disorders or cancer.

• A genetic disorder is a disease, syndrome, or other abnormal condition caused by a mutation in one or more genes or by a chromosomal alteration. An example of a genetic disorder is cystic fibrosis. A mutation in a single gene causes the body to produce thick, sticky mucus that clogs the lungs and blocks ducts in digestive organs. Genetic disorders are usually caused by gene mutations that occur within germline cells and are heritable.
• Illnesses caused by mutations that occur within an individual but are not passed on to their offspring are due to somatic cell mutations. Cancer is a disease caused by an accumulation of mutations within somatic cells. It results in cells that grow out of control and form abnormal masses of cells, known as tumors. It is generally caused by mutations in genes that regulate the cell cycle, DNA repair, angiogenesis, and other genes that favor cell growth and survival. Due to mutations, cells with mutated DNA have evolved to divide without restriction, evade the immune system, and develop drug resistance.

Types of DNA Damage

DNA damage, resulting from environmental factors and normal metabolic processes within the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day. While this constitutes only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs), if left unrepaired, it can cause mutations in critical genes (such as tumor suppressor genes), which can impede a cell's ability to carry out its function and appreciably increase the likelihood of tumor formation and disease states such as cancer.

The vast majority of DNA damage affects the primary structure of the double helix; that is, the bases themselves are chemically modified. These modifications can, in turn, disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix. Unlike proteins and RNA, DNA typically lacks a tertiary structure; therefore, damage or disturbance does not occur at that level. DNA is, however, supercoiled and wound around "packaging" proteins called histones (in eukaryotes), and both superstructures are vulnerable to DNA damage.

Several types of DNA damage can occur due to either normal cellular processes or environmental exposure of cells to DNA-damaging agents. DNA bases can be damaged by: (1) oxidative processes, (2) alkylation of bases, (3) base loss caused by the hydrolysis of bases, (4) bulky adduct formation, (5) DNA crosslinking, and (6) DNA strand breaks, including single and double-stranded breaks. An overview of these types of damage is described below.

Oxidative Damage

Reactive oxygen species (ROS) can cause significant cellular stress and damage, including oxidative DNA damage. Hydroxyl radicals (^•OH) are one of the most reactive and electrophilic of the ROS. They can be produced by ultraviolet and ionizing radiations or from other radicals arising from enzymatic reactions. The ^•OH can cause the formation of 8-oxo-7,8-dihydroguanine (8-oxoG) from guanine residues, among other oxidative products, as shown in Figure \(\PageIndex{4}\). Guanine is the most easily oxidized of the nucleic acid bases because it has the lowest ionization potential among the DNA bases. 8-oxo-dG is one of the most prevalent DNA lesions and is recognized as a biomarker of oxidative stress. It has been estimated that up to 100,000 8-oxo-dG lesions can occur per cell per day in DNA. The reduction potential of 8-oxo-dG is even lower (0.74 V vs. NHE) than that of guanosine (1.29 V vs NHE). Therefore, it can be further oxidized, creating a variety of secondary oxidation products.

Figure \(\PageIndex{4}\): Reactive Oxygen Species and DNA Damage. Under oxidative stress, 8-oxoG is formed when reactive oxygen species (ROS) modify guanine. Figure from: Poetsch, A.R. (2020) Comp & Struct Biotech J. 18:207-219.

As mentioned previously, increased levels of 8-oxo-dG in tissue can serve as a biomarker of oxidative stress. Furthermore, increased levels of 8-oxo-dG are frequently found associated with carcinogenesis and other disease states, as shown in Figure \(\PageIndex{5}\). During the replication of DNA that contains 8-oxo-dG, adenine is most often incorporated across from the lesion. Following replication, the 8-oxo-dG is excised during the repair process, and thymine is incorporated in its place. Thus, 8-oxo-dG mutations typically result in a G-to-T transversion.

Figure \(\PageIndex{5}\): Oxidative Stress and Human Health. DNA damage caused by oxidative stress has been linked to various human disease states. Figure from: Galano, A., Tan, D-X., and Reiter, R.J. (2018) Molecules 23(3)530

Alkylation of Bases

Alkylating agents are widespread in the environment and are also produced endogenously, as by-products of cellular metabolism. They introduce lesions into DNA or RNA bases that can be cytotoxic, mutagenic, or neutral to the cell. Figure \(\PageIndex{6}\) depicts the major reactive sites on the DNA bases that are susceptible to alkylation. Cytotoxic lesions block replication, interrupt transcription, or signal the activation of apoptosis, whereas mutagenic ones are miscoding and cause mutations in newly synthesized DNA. The most common type of alkylation is methylation, with the major products including N7-methylguanine (7meG), N3-methyladenine (3meA), and O6-methylguanine (O6meG). Smaller amounts of methylation also occur on other DNA bases, and include the formation of N1-methyladenine (1meA), N3-methylcytosine (3meC), O4-methylthymine (O4meT), and methyl phosphotriesters (MPT).

Figure \(\PageIndex{6}\): Major Sites of Alkylation in DNA. The diagram shows the Watson-Crick base pairs of DNA with principal damage sites modified by small alkylating agents (methyl and ethyl). (A) Sites of base modification by S_N1 and S_N2 alkylating agents. The orange indicates major damage sites, and the green indicates minor damage sites. (B) Phosphotriester formation is indicated by the presence of methyl groups, along with the Rp and Sp isomers. Figure from: Ahmad, A., Nay, S.L. and O'Conner, T.R. (2015). Chapter 4 Direct Reversal Repair in Mammalian Cells

Alkylating agents can cause damage to all exocyclic nitrogens and oxygens in DNA and RNA, as well as at ring nitrogens (Figure 25.2.6A). However, the percentage of each base site modified depends on the alkylating agent, the position in DNA or RNA, and whether the nucleic acid is single- or double-stranded. Interestingly, O-alkylations are more mutagenic and harmful than N-alkylations, which may be more cytotoxic but less mutagenic.

As we will explore in Chapter 13, DNA methylation also serves as a crucial mechanism for regulating gene expression.

Base Loss

An AP site (apurinic/apyrimidinic site), also known as an abasic site, is a location in DNA (also in RNA but much less likely) that has neither a purine nor a pyrimidine base, either spontaneously or due to DNA damage, as shown in Figure \(\PageIndex{7}\). It has been estimated that, under physiological conditions, 10,000 apurinic sites and 500 apyrimidinic sites may be generated per cell per day.

Figure \(\PageIndex{7}\): Abasic Sites. Apurinic and Apyrimidinic (AP) sites occur due to unstable hydrolysis. Figure from Mayhew

AP sites can be formed by spontaneous depurination, but also occur as intermediates in base excision repair, the repair process described in section 25.2.5. If left unrepaired, AP sites can lead to mutation during semiconservative replication. They can cause replication fork stalling and are often bypassed by translesion synthesis, a process discussed in greater detail in Section 12.8. In E. coli, adenine is preferentially inserted opposite AP sites, a pattern known as the "A rule". The situation becomes more complex in higher eukaryotes, where different nucleotides exhibit preferences that vary depending on the organism and environmental conditions.

Bulky Adduct Formation

Some chemicals are biologically reactive and will form covalent linkages with biological molecules such as DNA and proteins, creating large, bulky adducts or appendages that branch off from the main molecule. We will use the mutagen/carcinogen benzo[a]pyrene as an example of this process.

Benzo[a]pyrene is a polycyclic aromatic hydrocarbon that forms during the incomplete combustion of organic matter at temperatures between 300°C (572°F) and 600°C (1,112°F). The ubiquitous compound can be found in coal tar, tobacco smoke, and many foods, especially grilled meats. Benzo[a]pyrene is a procarcinogen that needs to be biologically activated by metabolism before it forms a reactive metabolite, as in Figure \(\PageIndex{8}\). Usually, when the body is exposed to foreign molecules, it initiates a metabolic process that renders them more hydrophilic, making them easier to remove as waste products. Unfortunately, in the case of benzo[a]pyrene, the resulting metabolite is a highly reactive epoxide that forms a bulky adduct preferentially with guanine residues in DNA. If left unrepaired, during DNA replication, an adenine will usually be inserted opposite the lesion in the daughter molecule. Subsequent repair of the adduct will result in the replacement of the damaged guanine base with thymine, causing a G --> T transversion mutation.

Figure \(\PageIndex{8}\): Benzo[a]pyrene Activation and Adduct Formation. (A) Benzo[a]pyrene is biologically activated to a dihydrodiol epoxide during normal metabolic processes. (B) Cigarette smoke is a source of benzo[a]pyrene. (C) The activated (+)benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide can form a DNA adduct with guanine residues. (D) The benzo[a]pyrene adduct forms a bulky lesion that distorts the DNA backbone. Figures from: (A) Eleska, (B) Elizabeth Aguilera (C) DI93biochemist, and (D) Zephryis

DNA Crosslinking

Crosslinking of DNA occurs when various exogenous or endogenous agents react with two nucleotides of DNA, forming a covalent linkage between them. This crosslink can occur within the same strand (intrastrand) or between opposite strands of double-stranded DNA (interstrand), as shown in Figure \(\PageIndex{9}\). These adducts interfere with cellular processes, including DNA replication and transcription, thereby triggering cell death.

Figure \(\PageIndex{9}\): f Intrastrand and Interstrand DNA Crosslinks. Lopez and Martinez, Cell. Mol. Life Sci. (2016) 73:3097–3114
DOI 10.1007/s00018-016-2218-x. Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/),

UV light can cause molecular crosslinks to form between two pyrimidine residues, commonly two thymine residues, that are positioned consecutively within a strand of DNA, as shown in Figure \(\PageIndex{10}\). Two common UV products are cyclobutane pyrimidine dimers (CPDs) and 6–4 photoproducts. These premutagenic lesions alter the structure and possibly the base pairing. Up to 50–100 such reactions per second might occur in a skin cell during exposure to sunlight, but are usually corrected within seconds by photolyase reactivation or nucleotide excision repair. Uncorrected lesions can inhibit polymerases, cause misreading during transcription or replication, or lead to replication arrest. Pyrimidine dimers are the primary cause of melanomas in humans.

Figure \(\PageIndex{10}\): Double-Stranded DNA breaks and Thymine Dimer Formation (a) Ionizing radiation, such as X-rays and γ-rays, contains enough energy to cause single and double-stranded breaks in the DNA backbone. (b) Energy from non-ionizing radiation, such as UV light, is directly absorbed by DNA, causing adjacent thymine residues within a single DNA strand to become cross-linked. (c) a 6,4-dimer that forms a single covalent bond and (d) thymine-thymine cyclobutane dimer that forms two covalent bonds between the thymine residues. Figures from (a and b) Parker, et al (2019) Microbiology from Openstax and (c and d) Smokefoot

DNA Strand Breaks

Ionizing radiation, such as that created by radioactive decay or cosmic rays, causes breaks in DNA strands (see Figure above). Low-level ionizing radiation may induce irreparable DNA damage, leading to replication and transcription errors that are necessary for neoplasia, or may trigger viral interactions, resulting in premature aging and cancer. Chemical agents that form crosslinks within the DNA, especially interstrand crosslinks, can also lead to DNA strand breaks if the damaged DNA undergoes DNA replication. Crosslinked DNA can cause topoisomerase enzymes to stall in the transition state when the DNA backbone is cleaved. Instead of relieving supercoiling and resealing the backbone, the stalled topoisomerase remains covalently linked to the DNA in a process called abortive catalysis. This leads to the formation of a single-stranded break with Top1 enzymes or a double-stranded break with Top2 enzymes. DNA double-strand breaks can also occur due to topoisomerase stalling during DNA transcription, as shown in Figure \(\PageIndex{11}\). Abortive catalysis and the formation of DNA strand breaks during transcriptional events may serve as a damage sensor within the cell, helping to instigate DNA damage response signaling pathways that initiate DNA repair processes.

Figure \(\PageIndex{11}\): Model of Topoisomerase IIB (TOP2B)-mediated DNA Double Strand Breaks During Transcription. Morimoto, S., et al (2019) Genes 10:868

Panel (1) shows that in the uninduced state of transcription, Pol II is paused between +25 and +100 from the transcription start site. The pausing is attributed to different elements, including pausing-stabilizing transcription factors, the +1 nucleosome, and DNA structure and torsion. Positive supercoiling ahead of Pol II may require TOP2B function.

Panel (2) shows transcription activation induced by various stimuli activates TOP2B to resolve DNA torsion in the promoter and gene body.

Panel (3) shows that, in this process, double-strand breaks can be formed via abortive TOP2B catalysis, which occurs frequently in certain genes. This may be responsible for the DNA damage response signaling observed in several stimulus-inducible genes in humans. 

Cellular Stress and DNA Damage Response

Genetic damage, whether produced by exogenous or endogenous mechanisms, represents an ongoing threat to the cell. To preserve genome integrity, eukaryotic cells have evolved repair mechanisms specific to different types of DNA Damage. Regardless of the type of damage, a sophisticated surveillance mechanism that elicits DNA damage checkpoints detects and signals it to the DNA repair machinery. DNA damage checkpoints have been functionally conserved throughout eukaryotic evolution, with most of the relevant players in the checkpoint response highly conserved from yeast to humans. Checkpoints are induced to delay cell cycle progression and to allow cells time to repair damaged DNA before DNA replication, as shown in Figure \(\PageIndex{12}\). Once the damaged DNA is repaired, the checkpoint machinery triggers signals that will resume cell cycle progression. Within cells, multiple pathways contribute to DNA repair.  Independent of the specific repair pathway involved, three phases of checkpoint activation are traditionally identified: (1) Sensing of damage, (2) activating the signaling cascade, and (3) switching on downstream effectors. The sensor phase recognizes the damage and activates the signal transduction phase to block cell cycle progression and select the appropriate repair pathway.

Figure \(\PageIndex{12}\): Cell Fates Following DNA Damage. Cell cycle checkpoints are induced by DNA damage, shown in red. Cyclin-Dependent Kinase Inhibitors (CDKIs), shown in purple, block cell cycle progression across all phases (G1, S, G2, or M) following DNA damage by inhibiting cyclin-dependent kinase complexes, shown in green. Signaling cascades activated in response to DNA damage also elicit DNA repair pathways. If the DNA damage is severe, programmed cell death (Apoptosis) will be activated. Image from: Ding, L. et al. (2020) Int. J. Mol. Sci. 21(6):1960

In addition to blocking cell cycle progression, DNA damage sensors also activate DNA repair mechanisms that are specific to the type of damage present. For example, single-stranded DNA breaks are repaired primarily by Base Excision Repair, bulky DNA adducts and crosslinks are repaired by Nucleotide Excision Repair, and smaller nucleotide mutations, such as alkylation, are repaired by Mismatch Repair. Cells also have two primary mechanisms for repairing Double-Strand breaks (DSBs). They include Non-Homologous End-Joining (NHEJ) and Homologous Recombination (HR). If damage is too extensive to be repaired, apoptotic pathways will be elicited. In the following sections, details about the major DNA repair pathways will be given.

In multicellular organisms, the response to DNA damage can result in two major physiological consequences: (1) Cells can undergo cell cycle arrest, repair the damage, and re-enter the cell cycle, or (2) cells can be targeted for cell death (apoptosis) and removed from the population. The cell cycle process is highly conserved and precisely controlled to govern genome duplication and the separation of the genome into the daughter cells. The cell cycle consists of four distinct and ordered phases, termed G0/G1 (gap 1), S (DNA synthesis), G2 (gap 2), and M (mitosis).

Multiple checkpoints exist within each stage of the cell cycle to ensure faithful DNA replication in the S phase and the precise separation of chromosomes into daughter cells. The G1 and G2 phases are critical regulatory checkpoints, where the restriction point between the G1 and S phases determines whether cells enter the S phase or exit the cell cycle to halt at the G0 phase. The cell cycle progression requires the activity of cyclin-dependent kinases (CDKs), a group of serine/threonine kinases. CDKs are activated when they form complexes with cyclin regulatory proteins, which are expressed at specific stages of the cell cycle. Cyclins bind to and stabilize CDKs in their active conformation. The formation of cyclin/CDK complexes controls cell-cycle progression by phosphorylating target genes, such as the tumor suppressor protein retinoblastoma (Rb).

During DNA damage, the cell cycle is arrested by cyclin-dependent kinase inhibitors. As noted in Figure 12.12, this is a complex signal transduction cascade with numerous downstream effects. A primary function of cell cycle arrest is that CDK inhibition allows time for DNA repair before cell cycle progression into the S phase or mitosis. As shown in Figure 25.2.12, two major cell-cycle checkpoints respond to DNA damage: one occurring pre-DNA synthesis in the G1 phase and the other post-DNA synthesis in the G2 phase, both affecting the activity of specific CDK complexes.

The checkpoint kinases phosphatidylinositol 3-kinase (PI3K)-like protein kinases (PI3KKs), ataxia telangiectasia and Rad3-related (ATR) or ataxia telangiectasia mutated (ATM) protein, and the transducer checkpoint kinases CHK1 (encoded by the CHEK1 gene) and CHK2 (encoded by the CHEK2 gene) are key regulators of DNA damage signaling. DNA damage signaling is detected by the ATM/ATR complex, which then phosphorylates and activates CHK2 and CHK1, respectively. Activated CHK2 promotes p53 activation, leading to p53-dependent early-phase G1 arrest, allowing time for DNA repair. The activation of p53 induces the expression of the Cyclin-Dependent Kinase Inhibitor (CKI) p21^CIP1 gene, leading to the inhibition of cyclin E/CDK2 complexes and subsequent upregulation of DNA repair machinery.

If DNA repair cannot be completed successfully, or the cells cannot program to respond to the stresses of viable cell-cycle arrest, the cells face the fate of p53-induced apoptosis. The activated CHK1 mediates a temporary S-phase arrest through phosphorylation of CDC25A, thereby inactivating CDC25A and promoting its ubiquitination and proteolysis. Moreover, the activated CHK1 phosphorylates and inactivates CDC25C, leading to cell-cycle arrest in the G2 phase. The active CHK1 also directly stimulates the phosphorylation of WEE1, thereby enhancing the inhibitory Tyr15 phosphorylation of CDK2 and CDK1 and subsequently blocking the cell cycle in the G2 phase. The activity of WEE1 can also be stimulated by low CDK activity during the G2 cell-cycle phase. The SAC, also known as the mitotic checkpoint, functions as the monitor of the correct attachment of the chromosomes to the mitotic spindle in metaphase, which is regulated by the TTK protein kinase (TTK, also known as monopolar spindle 1 (MPS1)).

The activation of SAC transiently induces cell-cycle arrest by inhibiting APC/C activation. To establish and maintain the mitotic checkpoint, the TTK recruits numerous checkpoint proteins to kinetochores during mitosis by phosphorylating its substrates, ensuring adequate chromosome segregation and genomic integrity. In this way, the genomic instability from chromosome segregation defects is protected by SAC. Once the SAC is passed, the APC/C E3 ligase complex stimulates and tags cyclin B and securin for ubiquitin-mediated degradation, thereby initiating mitosis. In a word, the checkpoints offer a failsafe mechanism to ensure the genomic integrity from the parental cell to the daughter cell. The signal transduction cascade of checkpoint activation ultimately converges on CDK inhibition, indicating that CDK function is a key driver of cell-cycle progression.

Mismatch Repair

DNA mismatch repair (MMR) is a highly conserved DNA repair system that significantly contributes to maintaining genome stability by correcting mismatched base pairs and small modifications of DNA bases, such as alkylation. The primary source of mismatched base pairs is replication error, although it can also arise from other biological processes. Thus, the MMR machinery must have a mechanism to determine which DNA strand is the template and which has been newly synthesized. In E. coli, DNA methylation is a common post-replicative modification. Thus, in newly synthesized DNA, the unmethylated strand is recognized as the new strand, and the methylated strand is used as the template to repair mismatches. In E. coli, MMR increases the accuracy of DNA replication by 20–400-fold. Mutations and epigenetic silencing in MMR genes have been implicated in up to 90% of human hereditary nonpolyposis colon cancers, indicating the significance of this repair system in maintaining genomic stability. Post-replicative MMR is carried out by the long-patch MMR mechanism, which involves multiple proteins and excises a relatively long tract of the oligonucleotide during repair. In contrast, particular kinds of mismatched base pairs are repaired through very short-patch MMR, in which a short oligonucleotide tract is excised to remove the lesion. Table\(\PageIndex{2}\) below shows mismatch repair enzymes in bacteria, yeast, and humans.

Table\(\PageIndex{2}\): Mismatch repair enzymes in bacteria, yeast, and humans

MMR in eukaryotes and most bacteria directs the repair to the error-containing strand of the mismatched duplex by recognizing the strand discontinuities. On the other hand, E. coli MMR interprets the absence of methylation as a strand-discrimination signal. The MutS protein recognizes mismatches. In both MMR systems, strand discrimination is conducted by nicking endonucleases. MutL homologs from eukaryotes and most bacteria incise the discontinuous strand to introduce the entry or termination point for the excision reaction. In E. coli,  MutH nicks the unmethylated strand of the duplex to generate the entry point for excision. Figure \(\PageIndex{13}\) shows different MMR pathway models.

Figure \(\PageIndex{13}\): A schematic representation of MMR pathway models. Fukui, K. (2010) J. Nuc. Acids 260512. Creative Commons Attribution License

Vertical panel (a): Eukaryotic MMR. The misincorporation of a base during DNA replication generates a DNA mismatch. MutSα recognizes base-base mismatches and MutLα nicks the 3'- or 5'-side of the mismatched base on the discontinuous strand. The resulting DNA segment is excised by the EXO1 exonuclease, in cooperation with the single-stranded DNA-binding protein RPA. The DNA strand is resynthesized by DNA polymerase δ and DNA ligase 1.

Vertical panel (b): MMR in mutH-less bacteria. MutS recognizes mismatched bases. After the incision of the discontinuous strand by MutL, the error-containing DNA strand is removed by the cooperative functions of DNA helicases, such as UvrD, the exonucleases RecJ and ExoI, and the single-stranded DNA-binding protein SSB. DNA polymerase III and DNA ligase fill the gap to complete the repair.

Vertical panel (c): E. coli MMR. MutS recognizes mismatched bases, and MutL interacts with and stabilizes the complex. Then, the MutH endonuclease is activated to incise the unmethylated GATC site, creating an entry point for the excision reaction. DNA helicase, a single-stranded DNA-binding protein, and several exonucleases are involved in the excision reaction. PDB IDs of crystal structures in this figure are 2O8B (human MutSα), 1H7S (human MutLα), 1L1O (human RPA), 3IAY (human DNA polymerase δ), 1X9N (human DNA ligase 1), 1E3M (bacterial MutS), 1B63 (bacterial MutL), 2AZO (E. coli MutH), 2ISI (bacterial UvrD), 2ZXO (bacterial RecJ), 3C95 (bacterial ExoI), 2CWA (bacterial SSB), 2HQA (bacterial DNA polymerase III), and 2OWO (bacterial DNA ligase).

Figure \(\PageIndex{14}\) shows an interactive iCn3D model of the E. Coli DNA Mismatch Repair Protein Muts Binding to a G-T Mismatch (1E3M).

Figure \(\PageIndex{14}\): E. Coli DNA Mismatch Repair Protein MutS Binding to a G-T Mismatch (1E3M). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/icn3d/share.html?YHB4ptjaS1mt7gp3A

The MutS monomers are colored gray and cyan. The mismatched G9-T22 base pair is labeled. ADP is shown in spacefill. Phe 36 from the gray monomer is shown in magenta.

The monomers' conformations differ, resulting in pseudo-symmetry in the dimer. Both subunits contribute to DNA binding, but only one (gray) binds both ADP and the actual mismatched GT base pair, the latter through minor-groove interactions that kink the DNA. The general major-groove interaction clamps the DNA. Note how far away the ADP binds. Phenylalanine 36 in the gray subunit (which binds the mismatch) inserts adjacent to the mismatch.

ATP is bound and hydrolyzed to ADP by the MutS protein on binding the mismatch. Next, a MutL dimer binds in a process that also requires ATP. MutH, a nuclease, also binds to MutL. The bound DNA is scanned until a "signal" is detected. In E. coli, the signal is a GATC sequence that is methylated on only one strand and nicked by the MutH protein on the unmethylated GATC. Helicase II binds to and unwinds the DNA at the mismatch site. Exonucleases (3' to 5' or 5' to 3') remove the sequence on the mismatched strand. PolII and DNA ligase then repair the DNA.

MutS is yet another fascinating enzyme, as it must scan millions of DNA bases without initiating repair until it localizes a mismatch. A series of conformational changes must occur to enable specific recognition of the mismatch.

Fernandez-Leiro et al. have determined the structure of MutS at various stages along the repair pathway. Figure \(\PageIndex{15}\) shows interactive iCn3D models of the E. Coli MutS scanning form (EMD-11791, PDB 7AI5) and the more progressed MutS:MutL kink-clamped form (EMD-11795, PDB 7AIC)

E. Coli MutS scanning form (EMD-11791, PDB 7AI5)	E. Coli MutS with MutL in kink-clamped form (EMD-11795, PDB 7AIC)
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...pdHkXxzXsZnqu6	Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...hQ6PeKbQeCw916

Figure \(\PageIndex{15}\): E. Coli MutS scanning form (EMD-11791, PDB 7AI5) (left) and the more progressed MutS:MutL kink-clamped form (EMD-11795, PDB 7AIC) (right)

In the scanning form, the two monomers have been color-coded as follows: monomer 1, the N-terminal part interacting with DNA in magenta, with the rest of the protein in red; monomer 2, the N-terminal part interacting with DNA in cyan, and the rest of the chain is blue. In the kink-clamped state (right), the N-terminal magenta and N-terminal cyan sections were not present in the resolved structure. ATP is shown in spacefill.

These structures suggest that during scanning by the homoduplex of normal DNA, the conformational change necessary for MutS to morph to the kink-clamped state can not occur due to a steric block. Kinking of the DNA at the mismatch removes the steric block.

Click the links to download videos that feature animations illustrating the role of MutS in mismatch repair. (Fernandez-Leiro, R., Bhairosing-Kok, D., Kunetsky, V. et al. The selection process of licensing a DNA mismatch for repair. Nat Struct Mol Biol 28, 373–381 (2021). https://doi.org/10.1038/s41594-021-00577-7, with permission)

Video 1: Molecular mechanism of DNA mismatch repair initiation. Front and side views of MutS passing through the first four stages of the repair cascade: DNA scanning, mismatch recognition, intermediate state, and MutL recruitment. The movements show a computational morphing between the four cryo-EM structures. MutS monomer A is shown in a pale-green color, monomer B in pale blue, DNA in dark gray, and MutL^LN40 in yellow.

Video 2: Mismatch repair licensing at a mismatch. Top and side views of MutS as it transforms from the DNA-scanning state to the mismatch-bound state. The initial part of the movie depicts the movement of monomer B relative to monomer A during the scanning state, as determined by the principal component analysis of the multibody refinement. Note that the MutS dimer explores multiple conformations, attempting to distort the DNA, without crossing over the opposite monomer. When a mismatch is present in the DNA, it allows MutS to deform and kink the DNA, and the two MutS monomers to cross over in a clockwise manner. Movements show a computational morphing between the different states. MutS monomer A is shown in a pale-green color, monomer B in pale blue, and DNA in gray. The DNA mismatch is highlighted in pink.

Video 3: Multiple conformational changes of mismatch and connector domains tracking DNA. Front and side views of MutS as it goes from the mismatch-bound state to the MutL^LN40-bound clamp state via the intermediate state. MutS monomer A is shown in a pale green color, monomer B in pale blue, and DNA in dark gray. DNA mismatch is highlighted in pink. The mismatch domain is shown in dark green, and the connector domain is shown in light green. The ends of a central helix in the connector domain are colored in red and blue for clarity. Movements show a computational morphing between the different states.

Base Excision Repair

Most oxidized bases are removed from DNA by enzymes operating within the Base Excision Repair (BER) pathway. Single-stranded DNA breaks can also be repaired through this process. Removal of oxidized bases in DNA is pretty rapid. For example, 8-oxo-dG was increased 10-fold in the livers of mice subjected to ionizing radiation, but the excess 8-oxo-dG was removed with a half-life of 11 minutes. 8-oxoG is excised by 8-oxoguanine DNA glycosylase (OGG1), leaving an apurinic site (AP site), as shown in Figure \(\PageIndex{16}\). AP sites are then processed further into single-strand breaks via backbone incision of AP-endonuclease 1 (APE1). In long-patch base excision repair, the base and some additional nucleotides are replaced, depending on the activity of polymerase delta (Polδ) and epsilon (Polε), together with proliferating cell nuclear antigen (PCNA). The old strand is removed by Flap-endonuclease 1 (FEN1), before Ligase I (LigI) ligates the backbone back together. Short patch base excision repair consists of polymerase beta (Polβ) replacing the single missing base, ligase III (LigIII) ligating the DNA backbone, and X-ray repair cross-complementing protein 1 (XRCC1) aiding the process and serving as a scaffold for additional factors.

Figure (\PageIndex{16}\): Base Excision Repair. Poetsch, A.R. (2020) Comp & Struct Biotech J. 18:207-219.

Base excision repair (BER) of 8-oxo-7,8-dihydroguanine (8-oxoG). Oxidative DNA damage is repaired via several repair intermediates by base excision repair (BER). Through the removal of the oxidized base, a reactive apurinic site (AP site) is formed. Incision of the strand creates a single-strand break, and the damaged site is then repaired through either short or long patch BER.

25.2.6 Nucleotide Excision Repair

Bulky DNA adducts and DNA crosslinks, such as those caused by UV light, are repaired using Nucleotide Excision Repair (NER) pathways. In higher eukaryotic cells, NER excises 24-32 nucleotide DNA fragments containing the damaged lesion with extreme accuracy. Reparative synthesis, using the undamaged strand as a template, followed by ligation of the single-strand break that results from the damage, is the final stage of DNA repair. The process involves the coordinated action of approximately 30 proteins that sequentially form complexes of variable composition on DNA. NER consists of two pathways that differ in their initial damage recognition. Global genome nucleotide excision repair (GG-NER) detects and eliminates bulky DNA damage throughout the entire genome, including untranscribed regions and silent chromatin.

In contrast, transcription-coupled nucleotide excision repair (TC-NER) operates when damage to a transcribed DNA strand impairs transcription. TC-NER is activated by the stalling of RNA polymerase II at the damaged sites of a transcribed strand, while GG-NER is controlled by the protein XPC, a specialized protein factor that reveals the damage. A schematic GG-NER process is presented in Figure (\PageIndex{17}\) below.

Figure (\PageIndex{17}\): Schematic of Global Genome Nucleotide Excision Repair. Figure from: Petruseva, I.O., et al (2014) 6(1):23-34.

Genetic mutations in NER pathway genes can lead to UV-sensitive and high-carcinogenic pathologies, such as xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD), as well as specific neurodegenerative manifestations.

Understanding Xeroderma pigmentosum has identified several genes involved in nucleotide excision repair (NER). Mutations in XP genes and loss of proper nucleotide excision repair (NER) function cause the symptoms associated with the disease. People with XP have an impaired ability to repair bulky DNA adducts and crosslinks, such as thymine dimers, induced by UV light exposure. People suffering from XP have extreme photosensitivity, skin atrophy, hyperpigmentation, and a high rate of sunlight-induced skin cancer. The risk of internal tumors in XP patients is also 1,000-fold higher. Moreover, the disease is often associated with neurologic disorders. Currently, there is no effective treatment for this disorder.

The detection of bulky DNA lesions during NER is particularly challenging for cells and can be solved only through highly sensitive recognition that requires multiple protein components. In contrast to BER, where a damaged base is simultaneously recognized and eliminated by a single specialized glycosylase, specialized groups of proteins are responsible for the recognition and excision of the lesion in NER. In eukaryotic NER, universal sensor proteins perform the initial recognition of the total range of bulky damages. In the case of TC-NER, it occurs when RNA polymerase II, the transcribing enzyme, is stalled by damage. In GG-NER, these are complexes of the XPC factor and the DDB1-DDB2 heterodimer (XPE factor) that enhance UV damage repair. In general, NER recognition of damage is a multistep process involving several proteins that form near-damaged complexes of variable compositions. The process is completed by the formation of a preincision complex ready to eliminate a damaged DNA fragment by specialized NER endonucleases.

In a eukaryotic cell, after the stable XPC/DNA complex forms during the initial recognition of damage, NER is carried out by a repairasome, a complex of variable composition and architecture comprising many subunits. Individual subunits of the complex have insufficient affinity and selectivity to the substrate (DNA containing bulky damage). The situation changes when specific protein complexes are established at the damage site. A total of 18 polypeptides must be accurately positioned within two or three DNA turns when a stable structure, ready for damage removal, is formed and excision starts. The structure of NER-associated proteins enables them to interact with the DNA substrate and facilitates dynamic, specific protein-protein interactions. Changes in interactions mediated by the same protein are among the mechanisms that regulate the repair process and fine-tune complexes, thereby providing high-precision nucleotide excision repair.

25.2.7 Repair of Double-Stranded DNA Breaks

Cells have evolved two main pathways to repair double-strand breaks within the DNA: the non-homologous end-joining (NHEJ) pathway, which ensures direct resealing of DNA ends; and the homologous recombination (HR) pathway that relies on the presence of homologous DNA sequences for DSB repair, as shown in Figure (\PageIndex{18}\) below.

NHEJ repair is the simplest and most widely used mechanism for repairing DSBs that occur in DNA. Repair by NHEJ involves direct resealing of the two broken ends independently of sequence homology. Although being active throughout the cell cycle, NHEJ is relatively more critical during the G1 phase. Proteins required for NHEJ include, but are not restricted to, the highly conserved Ku70/Ku80 heterodimeric complex, DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and DNA Ligase IV (LIG4) in complex with XRCC4. By directly binding to DNA ends, Ku70/Ku80 protects them against exonucleases and thus acts as an inhibitor of homologous recombination (HR). Very short sequence homologies are likely to aid in DNA end alignment before NHEJ-dependent repair; however, they are not strictly required. NHEJ protects genetic integrity by rejoining broken DNA strands that might otherwise be lost during DNA replication and cell regeneration. However, during the process of NHEJ, insertions or deletions within the joined regions may occur (Fig 25.2.17).

Figure (\PageIndex{18}\): Main Pathways of DNA Double-Stranded Break Repair. Diccotignies, A. (2013) Frontiers in Genetics 4(48):48

Non-homologous end-joining (NHEJ) and homologous recombination (HR) pathways act competitively to repair DNA double-strand breaks (DSBs). Key players of NHEJ and HR are depicted. The MRE11/RAD50/XRS2 (MRX) complex is recruited very early at DNA ends and appears to play important roles for both NHEJ and HR. The Ku70/Ku80 heterodimer is required for NHEJ and, by inhibiting DNA end resection (5′–3′ exonuclease activity), acts as a repressor of HR. The fidelity of NHEJ-dependent DSB repair is low and, most of the time, associated with nucleotide deletions and/or insertions at repair junctions. The common early step of HR-dependent mechanisms is the formation of ssDNA, which is then coated by replication protein A (RPA). Single-strand annealing (SSA) mechanism requires the presence of direct repeats (shown in orange) on both sides of the break. SSA does not imply any strand invasion process and is therefore not dependent on the RAD51 protein. Strand invasion and D-loop formation are, however, common steps of synthesis-dependent strand annealing (SDSA) and double Holliday junction (HJ) dissolution mechanisms. In the latter case, double Holliday junctions are resolved with or without crossing over.

In contrast to NHEJ, homologous recombination (HR) requires a homologous DNA sequence to serve as a template for DNA synthesis-dependent repair and involves extensive DNA end processing. As expected, HR is highly accurate, leading to precise repair of the damaged locus using DNA sequences homologous to the broken ends. HR predominantly uses the sister chromatid as a template for double-strand break (DSB) repair, rather than the homologous chromosome. Correspondingly, HR is largely inhibited while cells are in the G1 phase of the cell cycle when the sister chromatid has not yet been replicated, as shown in panel (A) of Figure (\PageIndex{19}\) below. HR repair mechanisms play a bigger role in DSB repair that occurs after S-phase DNA replication (S-phase, G2, and M).

Repair through HR is not defined by a single mechanism but involves various mechanistically distinct DSB repair processes, including synthesis-dependent strand annealing (SDSA), double Holliday junction resolution, and single-strand annealing (SSA). A common step in HR-dependent DSB repair mechanisms is the initial formation of single-stranded DNA (ssDNA) to pair with homologous DNA template sequences. For this to occur, the 5' DNA strand at the DSB is processed by multiple nucleases and accessory proteins to generate a 3' single-stranded DNA (ssDNA) end that can serve as a template for recombination (see Figure 18 above).

Panel B of Figure (\PageIndex{19}\) below provides a more detailed look at the HR process. During the highly regulated HR process, three main phases can be distinguished. Firstly, 3′-single-stranded DNA (ssDNA) ends are generated by nucleolytic degradation of the 5′-strands. This first step is catalyzed by endonucleases, including the MRN complex, which consists of Mre11, Rad50, and Nbs1. In the second step, the ssDNA ends are coated by replication protein A (RPA) filaments. In the third step, RPA is replaced by Rad51 in a BRCA1- and BRCA2-dependent process, ultimately performing the recombinase reaction using a homologous DNA template.

Importantly, HR is not only employed to repair DNA lesions induced by DNA-damaging agents but is also essential for proper chromosome segregation during meiosis. The relevance of HR in these physiological processes is illustrated by its strict requirement during development. Mice lacking key HR genes, such as Brca1, Brca2, or Rad51, exhibit extensive genetic alterations that result in early embryonic lethality. Whereas homozygous inactivation of HR genes is usually embryonic lethal, heterozygous inactivation of BRCA1 and BRCA2 does not interfere with cellular viability but rather predisposes individuals to cancer, including breast and ovarian cancer. The tumors that develop in individuals with heterozygous BRCA1/2 mutations invariably lose their second BRCA1/2 allele, indicating that in certain cancers, the absence of BRCA1/2 is compatible with cellular proliferation. How exactly such tumors cope with their HR defect is currently not fully understood.

Figure (\PageIndex{19}\): DNA double-strand break (DSBs) repair. Krajewska, M., Fehrmann, R.S.N., de Vries, E.G.E., and van Vugt, A.A.T.M. (2015) Front. Genet. 6:96

Panel (A) illustrates the DNA double-strand break (DSB) repair pathways within the context of cell cycle regulation. Non-homologous end joining (NHEJ) can occur throughout the cell cycle and is indicated by the red line. Homologous recombination (HR) can only be employed in S/G2 phases of the cell cycle and is indicated in green.

Pane (B) shows key steps in the HR repair pathway. After DSB recognition, 5′–3′ end resection is initiated by the MRN (Mre11, Rad50, Nbs1) complex and CtIP. Subsequently, further resection by the Exo1, DNA2, and Sgs1 proteins is conducted to ensure ‘maintained’ resection. Then, resected DNA ends are bound by replication protein A (RPA). The actual recombination step in HR repair, termed strand exchange, is carried out by the recombinase Rad51. Rad51 replaces RPA to assemble helical nucleoprotein filaments called ‘presynaptic filaments.’ Other HR components, including BRCA1 and BRCA2, facilitate this process. The final step of junction resolution is carried out by helicases, including the RecQ-like (BLM) helicase.

Error-Prone Bypass and Translesion Synthesis

If DNA is not repaired before DNA replication, the cell must employ an alternative strategy to replicate it, even in the presence of a DNA lesion. This is crucial to prevent causing double-stranded DNA breaks, which can occur when a replisome stalls at the replication fork. Under these circumstances, another strategy cells use to respond to DNA damage is to bypass lesions encountered during DNA replication and continue replication. DNA damage bypass can occur through recombination mechanisms or via a novel mechanism known as translesion synthesis. Translesion synthesis employs an alternative DNA polymerase that can substitute for one that has stalled at the replication fork due to DNA damage. Specialized DNA polymerases, which are active in regions of DNA damage, have active sites that accommodate fluctuations in DNA topology, enabling them to bypass lesions and continue replication.

The evolution of DNA polymerases that can tolerate distorted DNA lesions and continue replication is evident at all levels of life, from prokaryotic, single-celled organisms to eukaryotic, multicellular organisms, including humans. In fact, within vertebrates, there has been a significant expansion of DNA polymerases that play a role in DNA damage bypass mechanisms, highlighting the importance of these processes in damage tolerance and cell survival, as shown in Table (\PageIndex{3}\) below.

	Polymerase
B-Family	Pol ζ (Pol zeta)   (Rev3 / Rev7 / Pol31 / Pol32)	Eukaryotes (Yeast, Humans)	Responsible for nearly all damage-induced mutagenesis. Uniquely extends mismatched or distorted primer termini after an initial nucleotide insertion opposite a lesion (Northam et al., 2013).
Y-Family	Pol ι (Pol iota)	Humans	The most error-prone Y-family member. Highly mutagenic when inserting opposite undamaged G or T templates, but can act in a protective or provocative bypass manner depending on major-groove adducts (Kim et al., 2014).
	Pol η (Pol eta)   (RAD30 / XPV)	Eukaryotes (Yeast, Humans)	Error-free when bypassing UV-induced Thymine-Thymine (TT) dimers. However, it displays error-prone lesion bypass when encountering chemical adducts like benzo[a]pyrene-derived lesions (Zhang, 2000).
	Pol κ (Pol kappa)	Eukaryotes (Humans)	Highly accurate at bypassing specific bulky minor-groove $N^2$-guanine adducts, but can be highly error-prone on other non-bulky DNA distortions.
	REV1	Eukaryotes (Yeast, Humans)	A specialized deoxycytidyl transferase that specifically inserts a 'C' opposite abasic sites or damaged guanines. Crucially acts as a structural scaffold to recruit Pol ζ (Northam et al., 2013; Goodman & Woodgate, 2013).
	Pol V (UmuD'₂C)	Prokaryotes (E. coli)	Strictly regulated by the SOS response; highly error-prone and responsible for the majority of bacterial damage-induced mutagenesis.
	Pol IV (DinB)	Prokaryotes (E. coli)	Prone to generating -1 frameshift mutations during translesion synthesis.

Table (\PageIndex{3}\): DNA Polymerases involved in Error-Prone Bypass

The activity of error-prone DNA polymerases is tightly regulated to avoid the rampant introduction of mutations within the DNA sequence. One of the main mechanisms employed within a replisome stalled at the replication fork due to DNA damage involves the monoubiquitination of PCNA. Recall from Chapter 9 that PCNA is the sliding clamp that enables the DNA polymerase to bind tightly enough to the DNA during replication to mediate efficient DNA synthesis. Monoubiquitination of PCNA enables the recruitment of translesion DNA polymerases and the bypass of the damaged lesions during DNA synthesis.

During translesion synthesis, the polymerase must insert a dNTP opposite the lesion. None of the dNTP bases is likely to form stable hydrogen-bond interactions with the damaged lesion. Thus, the nucleotide that causes the least distortion or repulsion will usually be added across from the lesion. This can cause transition or transversion mutations to occur at the site of the lesion. Alternatively, translesion polymerases are prone to slippage, which can cause an insertion or deletion mutation near the DNA lesion. These slippages can lead to frameshift mutations if they occur within gene coding regions. Thus, over a lifetime, translesion synthesis in multicellular organisms can lead to the accumulation of mutations in somatic cells, causing tumor formation and cancer development.

Evolution by natural selection is also possible due to random mutations that occur within germ cells. Occasionally, germline mutations may lead to a beneficial mutation that enhances an individual's survival within a population. If this gene proves to enhance population survival, it will be selected over time and drive the evolution of that species. An example of a beneficial mutation is the case of a population of people that shows resistance to HIV infection. Since the first case of infection with human immunodeficiency virus (HIV) was reported in 1981, nearly 40 million people have died from HIV infection, the virus that causes acquired immune deficiency syndrome (AIDS). The virus targets helper T cells, which play a crucial role in bridging innate and adaptive immune responses, infecting and killing cells that are normally involved in the body’s response to infection. There is no cure for HIV infection, but many drugs have been developed to slow or block the progression of the virus. Although individuals around the world may be infected, the highest prevalence is among people 15–49 years old in sub-Saharan Africa, where nearly one person in 20 is infected, accounting for more than 70% of the infections worldwide, as shown in Figure (\PageIndex{20}\) below. Unfortunately, this is also a part of the world where prevention strategies and drugs to treat the infection are the most lacking.

Figure (\PageIndex{20}\): HIV is highly prevalent in sub-Saharan Africa, but its prevalence is quite low in some other parts of the world. Figure from: Parker, et al (2019) Microbiology from Openstax

In recent years, scientific interest has been piqued by the discovery of a few individuals from northern Europe who are resistant to HIV infection. In 1998, American geneticist Stephen J. O’Brien at the National Institutes of Health (NIH) and colleagues published the results of their genetic analysis of more than 4,000 individuals. These findings indicate that many individuals of Eurasian descent (up to 14% in some ethnic groups) carry a deletion mutation, known as CCR5-delta 32, in the gene encoding CCR5. CCR5 is a coreceptor on the surface of T-cells that is necessary for many strains of the virus to enter host cells. The mutation leads to the production of a receptor to which HIV cannot effectively bind, thereby blocking viral entry. People homozygous for this mutation have greatly reduced susceptibility to HIV infection, and those who are heterozygous have some protection from infection as well.

It is not clear why people of northern European descent, specifically, carry this mutation. Still, its prevalence seems to be highest in northern Europe and steadily decreases in populations as one moves south. Research indicates that the mutation has been present since before HIV appeared and may have been selected for in European populations as a result of exposure to the plague or smallpox. This mutation may protect individuals from the plague, caused by the bacterium Yersinia pestis, and smallpox, caused by the variola virus, as this receptor may also be involved in the pathogenesis of these diseases. The age of this mutation is a matter of debate, but estimates suggest it appeared between 1875 and 225 years ago, and may have been spread from Northern Europe through Viking invasions.

This exciting finding has led to new avenues in HIV research, including looking for drugs to block CCR5 binding to HIV in individuals who lack the mutation. Although DNA testing to determine which individuals carry the CCR5-delta 32 mutation is possible, there are documented cases of individuals homozygous for the mutation contracting HIV. For this reason, DNA testing for the mutation is not widely recommended by public health officials, as it may encourage risky behavior among those who carry it. Nevertheless, inhibiting the binding of HIV to CCR5 continues to be a valid strategy for the development of drug therapies for those infected with HIV.

Summary

(Summary written by Claude, Sonnet 4.6, Anthropic)

DNA integrity is continuously challenged by both replication errors and exogenous and endogenous damaging agents, generating an estimated 1,000 to 1,000,000 molecular lesions per cell per day. Despite DNA polymerase error rates of 10⁻⁶ to 10⁻⁹ per base—maintained by intrinsic proofreading exonucleases—the sheer scale of the human genome means each replication cycle produces up to a few thousand uncorrected errors before repair mechanisms intervene. Mutations are classified by scale: chromosomal alterations (amplifications, deletions, translocations, inversions) affect large genomic regions and are usually catastrophic; point mutations alter single bases and may be silent (same amino acid due to codon degeneracy), missense (different amino acid; effect depends on chemical similarity and location in the protein), or nonsense (premature stop codon, typically inactivating the protein); and insertions or deletions not in multiples of three cause frameshifts that alter all downstream codons and are almost universally devastating to protein function.

Six mechanistically distinct classes of DNA damage arise from different chemical insults. Reactive oxygen species—particularly hydroxyl radicals generated by UV and ionizing radiation or normal cellular metabolism—oxidize guanine to 8-oxo-dG, the most prevalent oxidative lesion and a biomarker of oxidative stress, which mispairs with adenine to produce G→T transversions. Alkylating agents methylate or ethylate ring and exocyclic oxygens and nitrogens on all four bases; O⁶-methylguanine is particularly mutagenic as it mispairs with thymine. Spontaneous hydrolysis generates approximately 10,000 apurinic and 500 apyrimidinic (AP) sites per cell per day; if unrepaired, AP sites cause translesion polymerases to insert adenine across the abasic site ("A rule"), producing mutations. Metabolic activation of polycyclic aromatic hydrocarbons such as benzo[a]pyrene produces reactive epoxides that form bulky adducts with guanine, distorting the double helix and causing G→T transversions. UV radiation generates pyrimidine dimers—primarily cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts—between adjacent thymine residues, blocking replication and transcription; these are the primary cause of melanoma. Ionizing radiation and abortive topoisomerase catalysis generate single- and double-stranded breaks, with DSBs being the most cytotoxic form of DNA damage.

The DNA damage checkpoint response integrates lesion detection with cell cycle control through the ATM/ATR → CHK2/CHK1 → p53/CDC25 signaling cascade. ATM (activated by DSBs through the MRN sensor complex) and ATR (activated by RPA-coated ssDNA at stalled replication forks) phosphorylate CHK2 and CHK1, respectively. CHK2 activates p53, inducing p21^CIP1 expression that inhibits cyclin E/CDK2 to arrest cells in G1. CHK1 phosphorylates and inactivates CDC25A (causing S-phase arrest) and CDC25C (causing G2 arrest), while also activating WEE1 kinase to impose inhibitory Tyr15 phosphorylation on CDK1 and CDK2. If damage is irreparable, p53-dependent apoptosis is triggered rather than continued cell cycle progression.

Four major repair pathways address specific lesion types. Base excision repair (BER) handles small, non-helix-distorting base modifications: damage-specific DNA glycosylases (e.g., OGG1 for 8-oxo-dG) cleave the N-glycosidic bond to generate AP sites, AP endonuclease 1 (APE1) incises the backbone, and either Pol β (short-patch) or Pol δ/ε + FEN1 (long-patch) fill the gap before ligation. Nucleotide excision repair (NER) removes bulky helix-distorting lesions through either global genome recognition (XPC-RAD23B) or transcription-coupled recognition (stalled RNA Pol II), followed by TFIIH-mediated unwinding, dual incision removing a 24–32 nt oligonucleotide by XPF-ERCC1 and XPG, and gap-filling synthesis. NER gene mutations cause xeroderma pigmentosum, with >1000-fold elevated skin cancer risk. Mismatch repair (MMR) corrects replication errors through MutS/MutSα mismatch recognition (involving Phe36 insertion into the kinked DNA minor groove), strand discrimination (GATC methylation/MutH nicking in E. coli; strand discontinuities in eukaryotes), and exonuclease-mediated excision of the error-containing strand; MMR deficiency underlies ~90% of hereditary nonpolyposis colon cancers. Double-strand break repair uses two competing mechanisms: error-prone NHEJ (Ku70/Ku80 end protection → DNA-PKcs bridging → Lig IV/XRCC4 ligation), active throughout the cell cycle; and high-fidelity HR (MRN-mediated resection → RPA/RAD51 filament formation with BRCA1/BRCA2 assistance → strand invasion using the sister chromatid as template → Holliday junction resolution), restricted to S/G2/M. BRCA1/BRCA2 heterozygous loss-of-function mutations predispose to breast and ovarian cancer by compromising HR fidelity. When lesions are not repaired before the replication fork arrives, translesion synthesis polymerases—recruited by PCNA monoubiquitination—bypass lesions at the cost of mutagenic nucleotide insertion, contributing to somatic mutation accumulation and cancer over a lifetime, while rare germline translesion events occasionally produce beneficial mutations such as the CCR5-Δ32 deletion that confers resistance to HIV infection.

 

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Practice Problems

Multiple Choice

Which of the following is a change in the sequence that leads to the formation of a stop codon?

1. missense mutation
2. nonsense mutation
3. silent mutation
4. deletion mutation

[reveal-answer q=”745512″]Show Answer[/reveal-answer]
[hidden-answer a=”745512″]Answer b. A nonsense mutation is a change in the sequence that leads to formation of a stop codon.[/hidden-answer]

The formation of pyrimidine dimers results from which of the following?

1. spontaneous errors by DNA polymerase
2. exposure to gamma radiation
3. exposure to ultraviolet radiation
4. exposure to intercalating agents

[reveal-answer q=”709151″]Show Answer[/reveal-answer]
[hidden-answer a=”709151″]Answer c. The formation of pyrimidine dimers results from exposure to ultraviolet radiation.[/hidden-answer]

Which of the following is an example of a frameshift mutation?

1. a deletion of a codon
2. missense mutation
3. silent mutation
4. deletion of one nucleotide

[reveal-answer q=”688366″]Show Answer[/reveal-answer]
[hidden-answer a=”688366″]Answer a. The deletion of one nucleotide is an example of a frameshift mutation.[/hidden-answer]

Which of the following is the type of DNA repair in which thymine dimers are directly broken down by the enzyme photolyase?

1. direct repair
2. nucleotide excision repair
3. mismatch repair
4. proofreading

[reveal-answer q=”755583″]Show Answer[/reveal-answer]
[hidden-answer a=”755583″]Answer a. In a direct repair, thymine dimers are directly broken down by the enzyme photolyase.[/hidden-answer]

Which of the following regarding the Ames test is true?

1. It is used to identify newly formed auxotrophic mutants.
2. It is used to identify mutants with restored biosynthetic activity.
3. It is used to identify spontaneous mutants.
4. It is used to identify mutants lacking photoreactivation activity.

[reveal-answer q=”770537″]Show Answer[/reveal-answer]
[hidden-answer a=”770537″]Answer b. It is used to identify mutants with restored biosynthetic activity.[/hidden-answer]

Fill in the Blank

A chemical mutagen that is structurally similar to a nucleotide but has different base-pairing rules is called a ________.

[reveal-answer q=”702924″]Show Answer[/reveal-answer]
[hidden-answer a=”702924″]A chemical mutagen that is structurally similar to a nucleotide but has different base-pairing rules is called a nucleoside analog.[/hidden-answer]

The enzyme used in light repair to split thymine dimers is called ________.

[reveal-answer q=”939657″]Show Answer[/reveal-answer]
[hidden-answer a=”939657″]The enzyme used in light repair to split thymine dimers is called photolyase.[/hidden-answer]

The phenotype of an organism that is most commonly observed in nature is called the ________.

[reveal-answer q=”640686″]Show Answer[/reveal-answer]
[hidden-answer a=”640686″]The phenotype of an organism that is most commonly observed in nature is called the wild type.[/hidden-answer]

True/False

Carcinogens are typically mutagenic.

[reveal-answer q=”166576″]Show Answer[/reveal-answer]
[hidden-answer a=”166576″]True[/hidden-answer]

Think about It

Why is it more likely that insertions or deletions will be more detrimental to a cell than point mutations?

Critical Thinking

Below are several DNA sequences that are mutated compared with the wild-type sequence: 3′-T A C T G A C T G A C G A T C-5′. Envision that each is a section of a DNA molecule that has separated in preparation for transcription, so you are only seeing the template strand. Construct the complementary DNA sequences (indicating 5′ and 3′ ends) for each mutated DNA sequence, then transcribe (indicating 5′ and 3′ ends) the template strands, and translate the mRNA molecules using the genetic code, recording the resulting amino acid sequence (indicating the N and C termini). What type of mutation is each?

Mutated DNA Template Strand #1: 3′-T A C T G T C T G A C G A T C-5′
Complementary DNA sequence:	[practice-area rows=”1″][/practice-area]
mRNA sequence transcribed from template:	[practice-area rows=”1″][/practice-area]
Amino acid sequence of peptide:	[practice-area rows=”1″][/practice-area]
Type of mutation:	[practice-area rows=”1″][/practice-area]

Mutated DNA Template Strand #2: 3′-T A C G G A C T G A C G A T C-5′
Complementary DNA sequence:	[practice-area rows=”1″][/practice-area]
mRNA sequence transcribed from template:	[practice-area rows=”1″][/practice-area]
Amino acid sequence of peptide:	[practice-area rows=”1″][/practice-area]
Type of mutation:	[practice-area rows=”1″][/practice-area]

Mutated DNA Template Strand #3: 3′-T A C T G A C T G A C T A T C-5′
Complementary DNA sequence:	[practice-area rows=”1″][/practice-area]
mRNA sequence transcribed from template:	[practice-area rows=”1″][/practice-area]
Amino acid sequence of peptide:	[practice-area rows=”1″][/practice-area]
Type of mutation:	[practice-area rows=”1″][/practice-area]

Mutated DNA Template Strand #4: 3′-T A C G A C T G A C T A T C-5′
Complementary DNA sequence:	[practice-area rows=”1″][/practice-area]
mRNA sequence transcribed from template:	[practice-area rows=”1″][/practice-area]
Amino acid sequence of peptide:	[practice-area rows=”1″][/practice-area]
Type of mutation:	[practice-area rows=”1″][/practice-area]

<h212references">25.2.10 References

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