Key DNA Repair Mechanisms

Why This Matters

Your cells face an onslaught of DNA damage every single day. Estimates suggest 10,000 to 100,000 lesions per cell daily from replication errors, reactive oxygen species, UV radiation, and chemical exposure. The repair mechanisms covered here aren't just molecular housekeeping; they're the reason your genome doesn't collapse into chaos. When these systems fail, the consequences are severe: cancer predisposition syndromes, premature aging, neurodegeneration, and immunodeficiency.

You're being tested on more than pathway names and protein players. Exams will ask you to match damage types to appropriate repair mechanisms, explain why certain pathways are error-prone while others maintain fidelity, and predict what happens when specific repair proteins are mutated. Don't just memorize the steps. Know when each pathway activates, what triggers it, and why cells sometimes choose a less accurate repair route over a precise one.

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Excision-Based Repair: Cut It Out and Start Fresh

These pathways share a common logic: recognize the damage, excise it, and use the undamaged strand as a template for accurate repair. The key difference lies in the size of what gets removed.

Base Excision Repair (BER)

BER handles the most common everyday damage your DNA encounters, particularly lesions caused by reactive oxygen species and spontaneous hydrolysis.

• Targets small, non-helix-distorting lesions: oxidized bases (like 8-oxoguanine), deaminated cytosines (which become uracil), and alkylated bases that don't dramatically warp the DNA structure
• DNA glycosylases initiate repair by flipping the damaged base out of the helix and cleaving the N-glycosidic bond, creating an abasic (AP) site. There are multiple glycosylases, each recognizing a different type of damaged base.
• AP endonuclease then cleaves the phosphodiester backbone at the AP site, and DNA polymerase β fills in the single-nucleotide gap (short-patch repair). DNA ligase III/XRCC1 seals the nick.

Nucleotide Excision Repair (NER)

NER tackles damage that BER can't: lesions large enough to physically distort the double helix.

• Removes bulky, helix-distorting adducts: UV-induced cyclobutane pyrimidine dimers (CPDs), 6-4 photoproducts, chemical crosslinks, and large covalent modifications that bend or unwind the DNA
• Excises a 24-32 nucleotide segment in eukaryotes (about 12-13 nt in prokaryotes). A multi-protein complex including XPA (damage verification), XPC (global genome damage recognition), and the TFIIH helicase (which unwinds DNA around the lesion) coordinates dual incisions by XPF-ERCC1 and XPG endonucleases. This is why it's called "nucleotide" excision: it removes a whole oligonucleotide chunk, not just a single base.
• NER has two sub-pathways: global genome NER (GG-NER) scans the entire genome, while transcription-coupled NER (TC-NER) specifically repairs lesions on the transcribed strand of active genes, triggered when RNA polymerase II stalls at damage.
• Defects cause xeroderma pigmentosum (XP): patients are extremely UV-sensitive and develop skin cancers at very young ages, making NER a classic example of repair-disease connections. Cockayne syndrome results from defects specifically in TC-NER.

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Replication Quality Control: Catching Mistakes Before They Stick

Mismatch repair operates during and shortly after DNA replication to correct errors that escaped polymerase proofreading. The central challenge: distinguishing the newly synthesized (error-containing) strand from the parental template strand.

Mismatch Repair (MMR)

• Corrects base-pair mismatches and small insertion/deletion loops (IDLs) that occur during replication. These errors don't distort the helix enough for NER recognition, and they aren't chemically damaged bases, so BER won't catch them either.
• MutS homologs recognize the mismatch (MSH2-MSH6 for base mismatches; MSH2-MSH3 for larger IDLs in eukaryotes), and MutL homologs (MLH1-PMS2) coordinate the downstream response. In E. coli, MutH nicks the unmethylated (newly synthesized) strand at hemimethylated GATC sites. Eukaryotes lack MutH and instead use strand discontinuities (Okazaki fragment nicks on the lagging strand, or the 3' terminus on the leading strand) for strand discrimination.
• Defective MMR causes Lynch syndrome (hereditary nonpolyposis colorectal cancer, HNPCC) with characteristic microsatellite instability (MSI). Microsatellites are short tandem repeats that are especially prone to slippage during replication; without MMR to correct the resulting IDLs, these repeats expand or contract, producing a distinctive molecular signature.

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Double-Strand Break Repair: The Most Dangerous Lesion

Double-strand breaks (DSBs) are catastrophic because there's no intact complementary strand to guide repair. Cells have two main options: accurate but slow (homologous recombination) or fast but error-prone (non-homologous end joining). The choice depends largely on cell cycle phase and whether a sister chromatid template is available.

Homologous Recombination (HR)

HR is the high-fidelity option for DSB repair, but it comes with a strict requirement.

• Uses a homologous sequence (the sister chromatid) as a template, making HR essentially error-free. This restricts HR to S and G2 phases, when a sister chromatid is available after replication.
• The process begins with 5' to 3' end resection by the MRN complex (Mre11-Rad50-Nbs1) and nucleases like CtIP and Exo1, generating 3' single-stranded DNA overhangs. RPA coats the ssDNA to prevent secondary structures, then BRCA2 loads Rad51 onto the ssDNA, displacing RPA. Rad51 nucleoprotein filaments catalyze strand invasion into the homologous duplex, forming a D-loop.
• BRCA1/BRCA2 mutations impair HR and cause hereditary breast and ovarian cancer. These tumors become dependent on error-prone repair pathways like NHEJ and alternative end joining, which is why PARP inhibitors are effective therapeutics: PARP inhibition causes replication-associated breaks that normally require HR, creating synthetic lethality in BRCA-deficient cells.

Non-Homologous End Joining (NHEJ)

NHEJ prioritizes speed over accuracy. It's the cell's way of quickly sealing a dangerous break, even at the cost of losing or altering a few nucleotides.

• Directly ligates broken DNA ends without a homologous template, which often causes small insertions or deletions at the junction site
• The Ku70/Ku80 heterodimer rapidly binds and protects broken ends, then recruits DNA-PKcs (DNA-dependent protein kinase catalytic subunit). End processing by Artemis trims incompatible overhangs, and DNA ligase IV (with XRCC4) seals the break.
• Active throughout the cell cycle and is the dominant DSB repair pathway in G1 (when no sister chromatid exists). NHEJ is also essential for V(D)J recombination in developing lymphocytes, where controlled imprecision at the junctions generates the antibody and T-cell receptor diversity needed for adaptive immunity.

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Direct Reversal: No Excision Required

Some damage types can be fixed by simply reversing the chemical modification. These pathways are fast and don't require DNA synthesis or strand breakage, but they're limited to specific lesion types.

O6-Methylguanine Repair by MGMT

• O6-methylguanine-DNA methyltransferase (MGMT) transfers the methyl group from $O^6$-methylguanine directly to a cysteine residue in its own active site. This is a suicide reaction: the transfer permanently inactivates the enzyme, meaning each MGMT molecule can only repair one lesion.
• Alkylation damage from environmental carcinogens and chemotherapy drugs (like temozolomide) is the primary target. $O^6$-methylguanine is dangerous because it mispairs with thymine during replication, causing G:C to A:T transition mutations. High MGMT expression in tumors causes resistance to alkylating chemotherapy.
• No template needed, no strand breaks created: this is the fastest possible repair, but the one-and-done nature of the enzyme means cells must continuously synthesize new MGMT to maintain protection.

Photoreactivation

• Photolyase directly cleaves UV-induced cyclobutane pyrimidine dimers using energy absorbed from visible/near-UV light (300-500 nm), a light-dependent mechanism
• The enzyme contains FAD and a pterin or deazaflavin cofactor that absorb photons and transfer electrons to break the cyclobutane ring of the dimer, restoring the two bases to their original monomeric form
• Present in bacteria, plants, and many animals but absent in placental mammals: humans rely entirely on NER for UV damage repair

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Damage Tolerance: When You Can't Fix It, Work Around It

Sometimes repair must wait. Replication forks can't stall indefinitely without risking fork collapse and DSBs. These mechanisms allow DNA synthesis to continue past unrepaired lesions, prioritizing genome duplication over perfect repair.

Translesion Synthesis (TLS)

• Specialized Y-family polymerases (Pol η, Pol ι, Pol κ, Rev1) have open, flexible active sites that can accommodate distorted template bases but lack $3' \to 5'$ proofreading exonuclease activity
• The switch from replicative to TLS polymerase is triggered by monoubiquitination of PCNA (the sliding clamp) by the Rad6/Rad18 complex at stalled forks. This modified PCNA recruits TLS polymerases to the damage site.
• Pol η accurately bypasses thymine dimers by inserting AA opposite the T-T dimer. Mutations in the POLH gene (encoding Pol η) cause the XP variant form (XP-V), where patients are UV-sensitive despite having fully functional NER. This shows that even with NER, some dimers persist to S phase and require TLS for bypass.
• Inherently mutagenic overall because TLS polymerases have low fidelity on undamaged DNA. This is a last resort mechanism that trades accuracy for survival.

Interstrand Crosslink Repair

Interstrand crosslinks (ICLs) are among the most toxic lesions because they affect both strands simultaneously.

• Covalent links between complementary strands block both replication and transcription. Neither strand can serve as a clean template, so no single repair pathway can handle ICLs alone.
• Requires coordination of multiple pathways: the Fanconi anemia (FA) pathway acts as the central coordinator. During replication, converging forks stall at the ICL. The FA core complex monoubiquitinates FANCD2-FANCI, which recruits nucleases for "unhooking" (incisions flanking the crosslink on one strand). TLS polymerases then bypass the unhooked adduct, and HR restores the DSB generated during unhooking.
• Fanconi anemia mutations (in any of over 20 FA genes) cause bone marrow failure, developmental abnormalities, and cancer predisposition. These patients are hypersensitive to crosslinking agents like cisplatin and mitomycin C.

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Quick Reference Table

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Self-Check Questions

1. A patient presents with extreme UV sensitivity and early-onset skin cancers but has normal NER function. Which repair pathway is most likely defective, and what is this condition called?

2. Compare and contrast how cells distinguish the error-containing strand in mismatch repair between E. coli (using MutH and hemimethylation) and eukaryotes (using strand discontinuities).

3. Why is homologous recombination restricted to S and G2 phases of the cell cycle, while NHEJ can operate in G1? What molecular requirement explains this difference?

4. Both BER and NER use excision-based strategies. If a cell is exposed to cigarette smoke containing bulky polycyclic aromatic hydrocarbons, which pathway responds, and why can't the other pathway handle this damage?

5. A tumor shows high MGMT expression and is resistant to temozolomide chemotherapy. Explain the molecular basis of this resistance and why MGMT is called a "suicide enzyme."