4.4 DNA damage and repair mechanisms

DNA Damage Types and Causes

Every cell in your body accumulates tens of thousands of DNA lesions per day. These come from normal metabolism and from the environment, and if left unrepaired, they drive mutations, cancer, and aging. The repair systems that counteract this damage are just as important to genomic integrity as accurate replication itself.

Endogenous and Exogenous Sources

DNA damage arises from two broad categories of sources:

• Endogenous sources stem from normal cellular processes. Reactive oxygen species (ROS) generated during oxidative metabolism are a major culprit, along with spontaneous hydrolysis of the glycosidic bond (depurination/depyrimidination) and spontaneous deamination of bases.
• Exogenous sources come from the environment: ultraviolet (UV) radiation, ionizing radiation (X-rays, gamma rays), and chemical mutagens such as those found in cigarette smoke or industrial chemicals.

Chemical Modifications and Structural Alterations

Base modifications change the chemical structure of individual nucleotides, which can alter their base-pairing behavior:

• Oxidation converts guanine to 8-oxoguanine, which mispairs with adenine instead of cytosine during replication.
• Alkylation adds methyl or ethyl groups to bases. A classic example is $O^6$-methylguanine, which mispairs with thymine.
• Deamination removes an amino group. Cytosine deaminates to uracil, so what was a C:G pair now codes as a U:A pair if unrepaired.

Strand breaks disrupt the sugar-phosphate backbone:

• Single-strand breaks (SSBs) often result from oxidative damage or aborted topoisomerase I activity. Because one strand remains intact, these are generally easier to repair.
• Double-strand breaks (DSBs) are far more dangerous. Ionizing radiation and certain chemotherapeutic agents (e.g., etoposide, a topoisomerase II poison) can cause DSBs. Both strands are severed, so there's no intact complementary strand to use as a simple template.

Pyrimidine dimers are UV-induced lesions that covalently link adjacent pyrimidines on the same strand, distorting the helix:

• Cyclobutane pyrimidine dimers (CPDs) form a four-membered ring between adjacent thymines (or other pyrimidines).
• 6-4 photoproducts involve a covalent bond between the C6 and C4 positions of adjacent pyrimidines.
• Both types block DNA and RNA polymerase progression, stalling replication and transcription.

Complex DNA Lesions and Replication Errors

Some lesions are especially difficult to repair because they affect both strands or involve non-DNA molecules:

• DNA-protein crosslinks (DPCs) occur when proteins become covalently attached to DNA. Agents like cisplatin and formaldehyde can induce these. DPCs physically obstruct the replication and transcription machinery and can cause replication fork collapse.
• Interstrand crosslinks (ICLs) form covalent bonds between bases on opposite strands. Bifunctional alkylating agents (nitrogen mustards) and psoralen plus UVA light cause ICLs. Because they prevent strand separation entirely, ICLs are among the most cytotoxic lesions a cell can encounter.
• Replication errors occur when DNA polymerase incorporates the wrong nucleotide. Even with proofreading, polymerase errors happen at a rate of roughly $10^{-7}$ per base pair per replication. Damaged template bases increase this error rate further. Unrepaired mismatches become permanent point mutations after the next round of replication.

DNA Repair Mechanisms

Excision Repair Pathways

These pathways work by cutting out the damaged section and resynthesizing it using the undamaged strand as a template.

Base Excision Repair (BER) handles small, non-helix-distorting base lesions (oxidized bases, deaminated bases, alkylated bases):

1. A DNA glycosylase specific to the type of damage recognizes and cleaves the glycosidic bond, removing the damaged base and leaving an abasic (AP) site. For example, uracil-DNA glycosylase removes uracil that arose from cytosine deamination.
2. An AP endonuclease (APE1) nicks the backbone at the AP site.
3. The repair is completed by one of two sub-pathways:
   • Short-patch BER: DNA polymerase $\beta$ fills in a single nucleotide, and DNA ligase III/XRCC1 seals the nick.
   • Long-patch BER: DNA polymerase $\delta$ or $\varepsilon$ synthesizes 2–10 nucleotides, displacing a flap that is removed by FEN1, followed by ligation.

Nucleotide Excision Repair (NER) handles bulky, helix-distorting lesions like pyrimidine dimers and large chemical adducts. NER comes in two flavors:

• Global genomic NER (GG-NER) scans the entire genome. The XPC-RAD23B complex detects helix distortion.
• Transcription-coupled NER (TC-NER) is triggered when RNA polymerase II stalls at a lesion in an actively transcribed gene. CSA and CSB proteins recruit the repair machinery.

Both sub-pathways converge on the same core steps:

1. Damage verification by TFIIH (which includes the XPB and XPD helicases) unwinds DNA around the lesion.
2. Dual incision: XPF-ERCC1 cuts on the 5' side and XPG cuts on the 3' side, excising a 24–32 nucleotide oligonucleotide containing the damage.
3. Gap filling by DNA polymerase $\delta$ or $\varepsilon$ using the undamaged strand as template.
4. Ligation by DNA ligase I or III seals the remaining nick.

Double-Strand Break Repair

DSBs are the most dangerous lesions because they can lead to chromosomal rearrangements. Cells have two main strategies:

Homologous Recombination (HR) is high-fidelity but requires a homologous template, so it operates primarily during S and G2 phases when a sister chromatid is available.

1. End resection: The MRN complex (Mre11-Rad50-Nbs1) and nucleases like CtIP generate 3' single-stranded overhangs.
2. Strand invasion: RPA coats the ssDNA, then RAD51 (with help from BRCA2) forms a nucleoprotein filament that invades the homologous duplex.
3. DNA synthesis: The invading strand primes new synthesis using the sister chromatid as template.
4. Resolution: The resulting Holliday junctions are resolved (or dissolved) to restore two intact duplexes.

Non-Homologous End Joining (NHEJ) is faster and works throughout the cell cycle, but it's error-prone because it ligates broken ends directly without a template.

1. Ku70/Ku80 heterodimer binds the broken ends and recruits DNA-PKcs.
2. End processing enzymes trim incompatible overhangs if needed.
3. DNA ligase IV (with XRCC4) ligates the ends.

NHEJ can introduce small insertions or deletions at the junction site, which is why HR is preferred when a template is available.

Specialized Repair Mechanisms

Direct reversal repairs specific lesions without excising any nucleotides:

• Photolyase uses blue light energy to directly split pyrimidine dimers. This enzyme is found in bacteria, plants, and many animals, but not in placental mammals (including humans), which rely on NER instead.
• $O^6$-methylguanine-DNA methyltransferase (MGMT) transfers the alkyl group from $O^6$-methylguanine onto a cysteine residue in its own active site. This is a "suicide" reaction: each MGMT molecule can only be used once.

Translesion synthesis (TLS) is a damage tolerance mechanism rather than a true repair pathway. When the replicative polymerase stalls at a lesion, specialized TLS polymerases (Pol $\eta$, Pol $\kappa$, Pol $\iota$, Rev1) are recruited via ubiquitination of PCNA. These polymerases have larger, more flexible active sites that can accommodate damaged templates, but they lack proofreading and have lower fidelity. TLS doesn't fix the damage; it allows replication to continue past it, preventing fork collapse at the cost of potentially introducing mutations.

Mismatch Repair for DNA Integrity

Mismatch Recognition and Strand Discrimination

Mismatch repair (MMR) corrects base-base mismatches and small insertion/deletion loops (IDLs) that escape polymerase proofreading. The central challenge of MMR is strand discrimination: the system must identify and correct the newly synthesized strand (which contains the error), not the template strand.

Recognition is handled by MutS homologs:

• MutS$\alpha$ (MSH2-MSH6) recognizes single base-base mismatches and small (1–2 nucleotide) IDLs.
• MutS$\beta$ (MSH2-MSH3) recognizes larger IDLs.

After mismatch recognition, MutL homologs are recruited:

• MutL$\alpha$ (MLH1-PMS2) is the primary MutL complex in human cells and has endonuclease activity.

Strand discrimination differs between prokaryotes and eukaryotes:

• In E. coli, the Dam methylase methylates adenine in GATC sequences. Immediately after replication, the template strand is methylated but the new strand is not yet methylated. MutH nicks the unmethylated (new) strand, directing repair.
• In eukaryotes, the mechanism is less clear-cut but likely involves strand discontinuities (Okazaki fragment nicks on the lagging strand, or the 3' terminus on the leading strand) and the orientation of the PCNA sliding clamp, which is loaded asymmetrically during replication.

MMR Mechanism and Significance

The eukaryotic MMR process proceeds through these steps:

1. MutS$\alpha$ or MutS$\beta$ recognizes the mismatch and undergoes an ATP-dependent conformational change.
2. MutL$\alpha$ is recruited and nicks the daughter strand (guided by strand discrimination signals).
3. Exonuclease 1 (EXO1) degrades the error-containing strand, removing up to 1–2 kb of DNA.
4. RPA stabilizes the resulting single-stranded gap.
5. DNA polymerase $\delta$ resynthesizes the correct sequence.
6. DNA ligase I seals the nick.

MMR reduces the overall replication error rate by 50- to 1000-fold, bringing the final mutation rate to approximately $10^{-9}$ to $10^{-10}$ per base pair per cell division.

Defects in MMR genes (especially MLH1 and MSH2) cause microsatellite instability (MSI), where short tandem repeat sequences expand or contract due to unrepaired IDLs. This is the molecular hallmark of Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC), which carries a significantly elevated risk of colorectal, endometrial, and other cancers.

Additional Roles of MMR

MMR has functions beyond correcting replication errors:

• DNA damage signaling: MMR proteins can recognize certain lesions (e.g., $O^6$-methylguanine paired with thymine) and trigger cell cycle arrest or apoptosis through ATR/Chk1 signaling. This is why MMR-deficient tumors can be resistant to certain alkylating agents.
• Antibody diversification: MMR participates in somatic hypermutation and class switch recombination in B cells, contributing to the generation of antibody diversity.
• Triplet repeat expansion: MMR processing of slipped DNA structures at trinucleotide repeats can paradoxically promote repeat expansion, which is relevant to diseases like Huntington's disease and myotonic dystrophy.

Consequences of Unrepaired DNA Damage

Genomic Instability and Cellular Dysfunction

When DNA damage accumulates faster than repair systems can handle it, the result is genomic instability:

• Point mutations can activate oncogenes (e.g., a single base change in RAS) or inactivate tumor suppressors (e.g., TP53).
• Chromosomal aberrations such as translocations, deletions, and amplifications disrupt gene dosage and regulation. The Philadelphia chromosome translocation $t(9;22)$ in chronic myeloid leukemia is a well-known example.

Cells have checkpoint mechanisms to detect damage before it becomes permanent:

• ATM responds primarily to DSBs; ATR responds to replication stress and ssDNA.
• Both kinases activate downstream effectors (Chk1, Chk2) that stabilize p53, which induces p21 expression, causing G1/S arrest.
• If damage is too severe to repair, prolonged checkpoint activation leads to cellular senescence (permanent growth arrest) or apoptosis.

Impaired Cellular Processes and Tissue Function

• Unrepaired lesions in the template strand block RNA polymerase, reducing transcription of affected genes. This can disrupt protein production across multiple pathways.
• Accumulated transcription errors from damaged templates can produce aberrant proteins that overwhelm the proteasome and protein quality control systems.
• DNA damage in stem cells is particularly consequential because it impairs tissue regeneration. A shrinking stem cell pool and reduced differentiation capacity contribute to age-related decline in the hematopoietic system, skin, and intestinal epithelium.

Genetic Disorders and Cancer Susceptibility

Inherited defects in specific repair pathways cause well-characterized syndromes:

More broadly, accumulated mutations from impaired repair can drive cells toward the hallmarks of cancer: sustained proliferative signaling, evasion of growth suppressors, and resistance to cell death.

Neurodegeneration and Developmental Abnormalities

• Neurons are post-mitotic and have limited replacement capacity in the adult brain, making them especially vulnerable to accumulated DNA damage. Oxidative DNA damage has been implicated in Alzheimer's disease and Parkinson's disease, where elevated levels of 8-oxoguanine are found in affected brain regions.
• During development, exposure to DNA-damaging agents combined with inadequate repair can cause birth defects. Ethanol metabolism generates acetaldehyde, which forms DNA adducts and contributes to fetal alcohol spectrum disorders. Maternal smoking increases the risk of both birth defects and childhood cancers through direct DNA damage from tobacco carcinogens.