6.2 DNA Damage & Repair Mechanisms

Cellular genomes are subject to constant chemical insult from endogenous metabolic byproducts and environmental mutagens. To preserve genetic fidelity across generations, organisms employ specialized DNA repair pathways.

DNA Replication Proofreading

During S phase, replicative DNA polymerases (prokaryotic DNA Pol III/I; eukaryotic DNA Pol $\delta$ and Pol $\epsilon$) achieve high fidelity through $3' \rightarrow 5'$ exonuclease proofreading activity.

When an incorrect nucleotide is incorporated, the mispaired base distorts the geometry of the polymerase catalytic site. This conformational distortion pauses $5' \rightarrow 3'$ synthesis and triggers shifting of the growing $3'$ terminus to the enzyme's intrinsic $3' \rightarrow 5'$ exonuclease domain. The exonuclease hydrolyzes the phosphodiester bond of the mismatched nucleotide, releasing the incorrect dNMP. The $3'$ end then swings back to the polymerase domain, and $5' \rightarrow 3'$ synthesis resumes. Proofreading reduces the baseline error rate of DNA polymerases from $10^{-5}$ ($1\text{ error in } 100,000\text{ bp}$) to $10^{-7}$ ($1\text{ error in } 10\text{ million bp}$).

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Post-Replicative Mismatch Repair (MMR)

Mismatch Repair (MMR) operates immediately after DNA replication to correct base-base mismatches and small insertion/deletion loops (indels) that escape polymerase proofreading, reducing the final genome error rate to $10^{-9}$ ($1\text{ in a billion bp}$).

Parent vs. Daughter Strand Discrimination

To avoid mutating the correct template, MMR must distinguish the original parental strand from the newly synthesized daughter strand containing the error:

• Prokaryotes (E. coli): The protein MutS binds the mismatch, and MutL forms a repair complex. The enzyme Dam methylase methylates adenine residues in $5'-GATC-3'$ sequences. Immediately after replication, the parental strand is fully methylated while the daughter strand is temporarily unmethylated (hemimethylated state). MutH endonucleolytically cleaves the unmethylated daughter strand at the nearest GATC site, followed by exonuclease digestion of the error, polymerisation by DNA Pol III, and sealing by ligase.
• Eukaryotes: Human homologs MSH2/MSH6 (MutS equivalent) and MLH1/PMS2 (MutL equivalent) recognize mismatches. Eukaryotes identify the daughter strand by single-strand nicks (unsealed gaps between Okazaki fragments on the lagging strand or terminal ends on the leading strand), independent of DNA methylation.

Clinical Correlation: Lynch Syndrome (HNPCC)

Germline loss-of-function mutations in human MMR genes (primarily MSH2 or MLH1) cause Hereditary Nonpolyposis Colorectal Cancer (HNPCC / Lynch Syndrome), an autosomal dominant disorder. Inactivation of MMR leads to Microsatellite Instability (MSI)—extreme hypermutability in short tandem repeat sequences—predisposing patients to early-onset colorectal, endometrial, and ovarian carcinomas.

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Base Excision Repair (BER)

Base Excision Repair (BER) removes non-bulky, non-helix-distorting damaged bases caused by spontaneous reactions, alkylation, or oxidation:

• Cytosine deamination to Uracil.
• Adenine deamination to Hypoxanthine.
• Guanine oxidation to 8-oxoguanine.
• Spontaneous depuration creating an abasic site.

Enzymatic Stepwise Cascade

1. DNA Glycosylase: Specific DNA glycosylases (e.g., Uracil DNA Glycosylase) recognize damaged bases and cleave the $N$-glycosidic bond connecting the base to the 2'-deoxyribose sugar, leaving an Apurinic/Apyrimidinic (AP / abasic) site.
2. AP Endonuclease: Recognizes the AP site and cleaves the $5'$ phosphodiester backbone immediately adjacent to the abasic sugar.
3. AP Lyase / Phosphodiesterase: Removes the remaining deoxyribose-phosphate sugar residue.
4. DNA Polymerase & Ligase: DNA Polymerase $\beta$ (in eukaryotes) or DNA Pol I (in prokaryotes) inserts the correct nucleotide, and DNA Ligase seals the phosphodiester nick.

Damaged Base ---> [DNA Glycosylase] ---> AP Site ---> [AP Endonuclease] 
             ---> Cleaved Backbone ---> [DNA Pol β & Ligase] ---> Repaired DNA

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Nucleotide Excision Repair (NER)

Nucleotide Excision Repair (NER) removes bulky, helix-distorting lesions that physically impede transcription and replication machinery. The classic trigger is ultraviolet (UV) radiation, which induces covalent crosslinking between adjacent pyrimidine bases to form cyclobutane pyrimidine dimers (CPDs / thymine dimers) or 6-4 photoproducts. NER also repairs bulky chemical adducts (e.g., benzo[a]pyrene from tobacco smoke).

Enzymatic Stepwise Cascade

1. Lesion Recognition: Multi-protein complexes (e.g., XPC or RNA Polymerase II stalled during transcription) detect structural helix distortion.
2. Excinuclease Cleavage: An endonuclease complex (Excinuclease; XPG at $3'$ side and XPF-ERCC1 at $5'$ side in humans; UvrABC in E. coli) makes dual nicks in the lesion-containing strand, cleaving several nucleotides upstream and downstream.
3. Oligonucleotide Excision: A $24\text{--}32\text{ nucleotide}$ single-stranded fragment containing the bulky lesion is excised (~29 nt in humans).
4. Resynthesis & Ligation: DNA Polymerase $\delta$ / $\epsilon$ fills the gap using the intact complementary strand as a template, and DNA Ligase seals the backbone.

Clinical Correlation: Xeroderma Pigmentosum (XP)

Autosomal recessive mutations in any of the XP genes (XPA through XPG) cause Xeroderma Pigmentosum (XP). Patients cannot repair UV-induced pyrimidine dimers. Clinical manifestations include extreme photosensitivity, severe cutaneous burning upon minimal sun exposure, poikiloderma, corneal opacities, and a $>2,000$-fold increased incidence of skin cancers (basal cell carcinoma, squamous cell carcinoma, malignant melanoma).

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Double-Strand Break (DSB) Repair Pathways

Double-strand breaks (DSBs) represent the most cytotoxic DNA lesion. Unrepaired DSBs trigger chromosomal fragmentation, translocations, or apoptosis. Cells utilize two primary pathways for DSB repair:

1. Non-Homologous End Joining (NHEJ)

• Mechanism: Error-prone, mutagenic pathway operating throughout all cell cycle phases (dominant in G0 and G1 phase when sister chromatids are absent).
• Steps: The Ku70/Ku80 heterodimer binds broken DNA ends and recruits the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). Nucleases (such as Artemis) trim single-stranded overhangs or fill gaps, and the ends are directly joined by DNA Ligase IV in complex with XRCC4.
• Consequence: End-processing lacks a template, frequently resulting in small insertions or deletions (indels) that disrupt open reading frames.

2. Homologous Recombination (HR)

• Mechanism: High-fidelity, error-free pathway restricted to late S phase and G2 phase, requiring an identical sister chromatid as a repair template.
• Steps: Nucleolytic 5' resection by the MRN complex (Mre11-Rad50-Nbs1) generates long $3'$ single-stranded DNA overhangs. Overhangs are coated by RPA, which is replaced by Rad51 recombinase facilitated by BRCA1 and BRCA2. Rad51 mediates strand invasion of the homologous sister chromatid duplex to form a Displacement loop (D-loop). DNA polymerase synthesizes across the break, followed by branch migration and resolution of Holliday junctions.

Clinical & Therapeutic Correlation: BRCA Mutations & PARP Inhibitors

Germline mutations in BRCA1 or BRCA2 disable HR repair, forcing cells to rely on error-prone NHEJ. This leads to genomic instability and dramatically elevated risks of hereditary breast and ovarian cancer.

Synthetic Lethality: Poly(ADP-ribose) Polymerase (PARP) enzymes repair single-strand DNA breaks via BER. PARP inhibitors (e.g., olaparib) trap PARP on single-strand nicks. During S phase, advancing replication forks collapse at trapped PARP lesions, generating double-strand breaks. In normal cells, these DSBs are accurately repaired by HR. In BRCA1/2-deficient tumor cells lacking HR, the accumulation of double-strand breaks causes catastrophic genomic destruction and selective apoptosis—a classic application of synthetic lethality.