DNA Repair

Core Explanation

Overview of Lesion Types and Repair Pathways

Mismatch Repair (MMR)

MMR corrects replication errors that escape the proofreading (3′→5′ exonuclease) activity of DNA polymerases. It improves replication fidelity by 100- to 1000-fold.

Recognition: In eukaryotes, MutSα (MSH2–MSH6 heterodimer) recognizes single base mismatches and small insertion-deletion loops (1–2 nt). MutSβ (MSH2–MSH3) recognizes larger insertion-deletion loops.

Mechanism:

1. MutSα binds the mismatch and undergoes an ATP-dependent conformational change, forming a sliding clamp that diffuses along DNA.
2. MutSα recruits MutLα (MLH1–PMS2 heterodimer), an endonuclease.
3. MutLα nicks the newly synthesized (daughter) strand. Strand discrimination in eukaryotes is thought to rely on the single-strand nicks present during lagging-strand synthesis, or from PCNA-oriented nicking.
4. Exonuclease I (EXO1) digests the daughter strand from the nick past the mismatch.
5. DNA Pol δ (or Pol ε) resynthesizes the excised region, and DNA ligase I seals the nick.

Clinical significance: Mutations in MSH2, MLH1, MSH6, or PMS2 cause Lynch syndrome (hereditary non-polyposis colorectal cancer, HNPCC). Tumors exhibit microsatellite instability (MSI) — contraction/expansion of repetitive sequences because MMR cannot correct polymerase slippage errors at microsatellites.

Base Excision Repair (BER)

BER handles small, non-helix-distorting base lesions — the high-volume repair pathway.

Recognition: DNA glycosylases scan DNA and flip damaged bases out of the helix for inspection. Each glycosylase recognizes a specific lesion class:

• UNG (uracil-DNA glycosylase): removes uracil from DNA (resulting from cytosine deamination or misincorporation)
• OGG1: removes 8-oxoguanine (oxidative damage)
• MPG (N-methylpurine DNA glycosylase): removes alkylated bases

Mechanism:

1. Glycosylase cleaves the N-glycosidic bond, releasing the damaged base and creating an abasic (AP) site.
2. APE1 (AP endonuclease 1) nicks the phosphodiester backbone 5′ to the AP site.
3. Short-patch BER: Pol β removes the 5′ deoxyribose phosphate (dRPase activity) and fills the single-nucleotide gap. Ligase III–XRCC1 seals.
4. Long-patch BER: If the AP site is oxidized/reduced and resistant to dRPase, Pol δ/ε extends 2–12 nt, displacing a flap removed by Fen1. Ligase I seals.

Nucleotide Excision Repair (NER)

NER removes bulky, helix-distorting lesions that block replication and transcription — the classic example is UV-induced cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts.

NER has two subpathways:

• Global genome NER (GG-NER): scans the entire genome for distortions. Recognition: XPC–RAD23B–CETN2 complex detects helical distortion, with UV-DDB (DDB1–DDB2/XPE) assisting at CPDs.
• Transcription-coupled NER (TC-NER): specifically repairs the transcribed strand of active genes. Recognition: stalled RNA Polymerase II is recognized by CSA, CSB, and UVSSA.

Both converge on a common mechanism:

1. TFIIH (containing XPB and XPD helicases) unwinds ~25–30 bp around the lesion.
2. XPA and RPA stabilize the open bubble and verify the lesion.
3. XPF–ERCC1 endonuclease nicks 5′ to the lesion; XPG nicks 3′.
4. The 24–32 nt oligonucleotide containing the lesion is released.
5. Pol δ/ε and ligase fill and seal.

Clinical significance: Mutations in NER genes cause xeroderma pigmentosum (XP) — extreme UV sensitivity, >10,000-fold increased skin cancer risk. Cockayne syndrome (CS) results from TC-NER defects and includes neurodegeneration and premature aging without elevated cancer risk. The contrast between XP (cancer-prone) and CS (not cancer-prone) reveals that unrepaired transcription-blocking lesions trigger apoptosis rather than mutagenesis.

Double-Strand Break Repair

Double-strand breaks (DSBs) are the most dangerous DNA lesions — one unrepaired DSB can cause cell death or, if misrepaired, chromosomal translocations. Two mechanistically distinct pathways exist:

Non-Homologous End Joining (NHEJ)

NHEJ directly ligates broken DNA ends with minimal or no processing. It is the dominant DSB repair pathway in G1 and G0 (quiescent) cells because it does not require a homologous template.

Mechanism:

1. Ku70/Ku80 heterodimer rapidly binds DNA ends with high affinity, protecting them from resection and recruiting DNA-PKcs.
2. DNA-PKcs (catalytic subunit of DNA-dependent protein kinase) is activated and phosphorylates downstream targets.
3. Ends may be processed by Artemis (endonuclease activity upon DNA-PKcs phosphorylation), polynucleotide kinase/phosphatase (PNKP), and DNA polymerases λ/μ (fill-in synthesis).
4. XRCC4–Ligase IV–XLF complex ligates the ends.

NHEJ is faster (minutes vs. hours for HR) and template-independent. It can be mutagenic at the repair junction (small insertions/deletions) if end processing occurs. This property is exploited in CRISPR knockout: NHEJ introduces frameshift indels that inactivate target genes.

Homologous Recombination (HR)

HR uses an intact homologous sequence (the sister chromatid, present only after S phase) as a template for accurate repair. It is restricted to S/G2 phases.

Mechanism:

1. End resection: The MRN complex (MRE11–RAD50–NBS1) and CtIP initiate 5′→3′ resection. Exo1 and Dna2/BLM helicase–nuclease extend resection, generating long 3′ ssDNA overhangs.
2. RPA coats ssDNA, then RAD51 displaces RPA (with BRCA2/PALB2 assistance) to form the RAD51 nucleoprotein filament.
3. Strand invasion: RAD51 filament searches for and invades the homologous sequence on the sister chromatid, forming a D-loop.
4. DNA synthesis: Pol δ extends the invading 3′ end, using the sister chromatid as template.
5. Resolution: Depending on D-loop processing (dissolution via BTR complex or resolution via resolvases), the outcome can be gene conversion (non-crossover predominantly, via synthesis-dependent strand annealing) or crossover.

HR is template-dependent and therefore inherently high-fidelity in principle, but requires the sister chromatid — hence its restriction to S/G2. BRCA1 and BRCA2 are essential HR factors; their mutation causes homologous recombination deficiency and is synthetically lethal with PARP inhibition (PARP inhibitors trap PARP1 on DNA at single-strand breaks, which are converted to DSBs during replication — HR-deficient cells cannot repair these, leading to cell death).

NHEJ vs. HR: Pathway Choice

Critical precision: Neither pathway is universally "good" or "bad." NHEJ is essential: Ku80 knockout mice are radiation-sensitive and immunodeficient (V(D)J recombination requires NHEJ). HR is essential for accurate repair in replicating cells but requires a homologous template. In G1 cells, NHEJ is the only option — even if error-prone, it is better than an unrepaired DSB. The pathway choice is regulated: 53BP1 promotes NHEJ by protecting ends from resection; BRCA1 promotes HR by removing 53BP1 and recruiting resection machinery.

Fanconi Anemia Pathway (Interstrand Crosslink Repair)

Interstrand crosslinks (ICLs) covalently link the two DNA strands, blocking both replication and transcription. The Fanconi anemia (FA) pathway coordinates ICL repair using components of NER, translesion synthesis, and HR:

1. Stalled replication fork at ICL → FANCM and FA core complex recognize and ubiquitinate FANCD2–FANCI.
2. Structure-specific nucleases (XPF–ERCC1, SLX4) unhook the crosslink.
3. Translesion synthesis polymerases bypass the unhooked adduct.
4. DSB intermediate is repaired by HR.

Experimental Evidence

• XP complementation groups: Cell fusion experiments in the 1960s–70s identified seven NER complementation groups (XPA–XPG), each corresponding to a distinct gene. This classic somatic cell genetics approach mapped the entire NER pathway before any genes were cloned.
• γ-H2AX foci: Phosphorylated H2AX (γ-H2AX) forms foci at DSB sites within minutes, serving as a quantitative readout of DSB induction and repair. Counting γ-H2AX foci by immunofluorescence is a standard assay for DSB repair kinetics.
• I-SceI reporter assays: A single I-SceI endonuclease cleavage site integrated into the genome allows tracking of NHEJ (re-ligation with indels) vs. HR (gene conversion using an engineered donor cassette). GFP reconstitution upon HR provides a direct fluorescence readout.
• Synthetic lethality: BRCA1/2-deficient cells are exquisitely sensitive to PARP inhibitors, providing the preclinical rationale for olaparib, niraparib, and rucaparib in BRCA-mutant breast and ovarian cancer.
• MSI testing in Lynch syndrome: PCR amplification of five standardized microsatellite markers (Bethesda panel, including BAT25, BAT26) reveals MSI-high status in Lynch-associated tumors, used clinically for diagnosis.

Disease and Clinical Connections

High-Yield Summary

• MMR: MutSα/MutLα → EXO1 → Pol δ → Ligase I. Strand-specific via PCNA-associated nicks. Lynch syndrome = MMR defect → MSI.
• BER: Glycosylase → APE1 → short-patch (Pol β, Ligase III–XRCC1) or long-patch (Pol δ/ε, Fen1, Ligase I).
• NER: Global genome (XPC) vs. transcription-coupled (CSA/CSB stalled Pol II) → common TFIIH unwinding → XPA, XPF-ERCC1, XPG → 24–32 nt excision. XP = NER defect.
• DSB repair: NHEJ (Ku70/80, DNA-PKcs, XRCC4–Ligase IV; fast, template-independent, potentially mutagenic; all cell-cycle phases). HR (MRN resection → RAD51 strand invasion; template-based, generally high-fidelity; S/G2 only).
• Pathway choice: 53BP1 favors NHEJ; BRCA1 favors HR. Neither pathway is universally superior — each is essential in its biological context.
• PARP inhibitors are synthetically lethal with BRCA1/2 mutations.
• CRISPR genome editing exploits NHEJ (indels → knockouts) and HR (donor template → knock-ins).

Practice Questions

1. A tumor with MLH1 promoter hypermethylation exhibits microsatellite instability. Would this tumor respond to a PARP inhibitor? Why or why not?

Answer: Not necessarily. MLH1 silencing causes mismatch repair deficiency, not homologous recombination deficiency. PARP inhibitors are synthetically lethal with HR defects (BRCA1/2 mutation), not MMR defects. MLH1-deficient tumors are instead vulnerable to immune checkpoint inhibitors because MMR deficiency produces a high mutational burden with abundant neoantigens (frameshift peptides from MSI), triggering anti-tumor immune responses. The FDA approval of pembrolizumab for MSI-high tumors, regardless of tissue of origin, reflects this distinct biology.

2. A cell is treated with ionizing radiation. γ-H2AX foci are counted at 1 hour and 24 hours post-irradiation. In wild-type cells, >90% of foci resolve by 24 hours. In a mutant line, >50% persist at 24 hours. What repair pathway is likely defective, and what additional experiment would distinguish an NHEJ defect from an HR defect?

Answer: Persisting γ-H2AX foci indicate a DSB repair defect — either NHEJ or HR, or a damage-signaling defect. To distinguish NHEJ from HR, irradiate cells synchronized in G1 (when HR is inactive) vs. late S/G2 (when both pathways are available). If the defect is in NHEJ, G1-irradiated cells will show persisting foci while S/G2 cells may still resolve them via HR. If the defect is in HR, G1-irradiated cells will resolve foci but S/G2 cells will not. Alternatively, examine RAD51 foci (marker of HR) — absence of RAD51 foci indicates an HR defect, while normal RAD51 foci with persisting γ-H2AX suggests NHEJ failure. An ATM inhibitor can also help distinguish signaling from repair defects.

3. Why do cisplatin-treated tumor cells that acquire carboplatin resistance often show upregulation of translesion synthesis polymerases (Pol η, Pol ζ, REV1) or increased expression of XPF–ERCC1?

Answer: Cisplatin forms intrastrand crosslinks and interstrand crosslinks (ICLs). ICL repair requires XPF–ERCC1 for unhooking, followed by translesion synthesis (TLS) polymerases to bypass the residual adduct before HR completes repair. Upregulation of XPF–ERCC1 increases ICL unhooking capacity; upregulation of TLS polymerases allows the cell to bypass DNA lesions that would otherwise stall replication forks. Both mechanisms enable the cell to survive cisplatin-induced damage — this is not "repair" in the traditional sense but rather damage tolerance. These resistant cells often accumulate mutations due to the error-prone nature of TLS polymerases, potentially driving further tumor evolution.

Common Misconceptions

"HR is good/high-fidelity; NHEJ is bad/error-prone." This is a misleading oversimplification. Both are essential and both can produce desirable or undesirable outcomes depending on context. NHEJ is critical for V(D)J recombination (generating antibody diversity via deliberate errors) and is the only DSB repair pathway available in G1. HR is unavailable in G1 and can produce loss of heterozygosity through crossover events. The cell carefully regulates pathway choice — neither is universally "good."

"DNA repair mutants always cause cancer." Not always. Cockayne syndrome involves NER (TC-NER) defects and causes neurodegeneration and premature aging without cancer predisposition, because cells with unrepaired transcription-blocking lesions undergo apoptosis rather than accumulating mutations. The specific molecular defect, not just "repair failure," determines phenotype.

"Mismatch repair is just for replication errors." MMR also plays a role in suppressing recombination between divergent (homeologous) sequences and in signaling DNA damage-induced apoptosis (the cytotoxic mechanism of some chemotherapeutics like 6-thioguanine).