CH13 – Recombinational DNA Repair & Homologous Recombination

Overview & Key Learning Goals

• Chapter 13 focuses on how cells repair double-stranded DNA breaks (DSBs) and other replication-blocking lesions by homologous recombination (HR) or, when HR is not possible, by non-homologous end joining (NHEJ).
• Major sections
– Recombination as a repair process (concepts & replication-fork problems)
– Bacterial enzyme machines (RecA, RecBCD, Ruv proteins, RecFOR)
– Homologous recombination in eukaryotes (mitosis, meiosis, programmed gene conversion)
– NHEJ pathway

Types of DNA Lesions Encountered During Replication

• Lesions (blue) are encountered by the replication fork (parental DNA = blue; nascent DNA = orange).
• Four fates once a polymerase meets a lesion:

  1. Trans-lesion synthesis
    – Special low-fidelity DNA polymerases insert across the lesion, leaving damage in the template and introducing mutations in the daughter strand.
  2. Lesion removal before fork arrival (BER or NER)
    – Creates a transient single-strand break; if the fork reaches it, the fork collapses, generating a one-ended DSB.
  3. Fork stalling & regression (fork backs up, forming a “chicken-foot” intermediate) so the lesion can be repaired or bypassed.
  4. Lagging-strand lesion bypass (gap left opposite lesion)
    – Polymerase re-primes downstream; a post-replicative ssDNA gap is left behind.
    • Recombination-based repair pathways have solutions for each of the above scenarios.

General Homologous Recombination (HR) Mechanism (DSB Model)

• Step 1 – End processing
– Helicase unwinds DNA; nuclease resects 5′→3′ to create 3′3' ssDNA overhangs.
• Step 2 – Strand invasion
– 3′3' tail coated with a recombinase (RecA in bacteria, Rad51/Dmc1 in eukaryotes) searches for homology in an intact duplex; invades to form a displacement loop (D-loop).
• Step 3 – DNA synthesis
– The invading 3′3' OH primes DNA polymerase; new DNA (red) extends the D-loop.
• Two principal resolution routes:

  1. Synthesis-Dependent Strand Annealing (SDSA)
    – The extended strand dissociates and re-anneals to the other processed end; ligase seals nicks.  Non-crossover; fast & favored in mitosis.
  2. Double-Strand-Break Repair (DSBR)
    – Extended strands remain paired; second end captured; ligation generates two Holliday junctions.
    – Resolution by specialized endonucleases (Holliday junction resolvases) can yield:
    • Non-crossover products (both cuts in X or both in Y plane)
    • Crossover products (one X + one Y cut)  reciprocal exchange of chromosomal arms.

Recombination-Based Repair of Replication-Fork Problems

1. Collapsed Forks at Nicks (One-Ended DSB)

• Nick in template causes leading-strand polymerase to detach; fork collapses.
• Processed end invades sister duplex; branch migration + resolution (usually non-crossover) recreate a functional fork.

2. Fork Regression at Lesions

• Stalled fork reverses, annealing parental template strands & forming a four-way junction; nascent strands pair with each other.
• Two outcomes:
– Lesion is excised (BER/NER) while fork is regressed; nascent strands degraded; fork resets & restarts.
– Nascent 3′3' end extended past lesion using the other nascent strand as template; branch migrates forward, bypassing lesion (damage left for later repair).

3. Lagging-Strand Gap Repair (Post-replicative)

• Gap opposite lesion is “healed” by invasion of the gapped strand into sister duplex, forming two Holliday junctions.
• Junctions migrate to bridge gap; polymerase fills; biased resolvase activity (both X or both Y) gives non-crossover outcome. Lesion itself remains for later excision.

Bacterial Enzymatic Machinery for HR

RecBCD Pathway (DSBs)

• RecBCD = RecB (helicase + nuclease), RecC (chi recognition), RecD (helicase).
• Binds blunt DSB end, unwinds & degrades both strands until an 8-nt chi (χ) site (5′-GCTGGTGG-3′5'\text{-GCTGGTGG-}3') is encountered.
• At χ, degradation of the 3′3' strand stops, 5′5' strand digestion continues → long 3′3' overhang.
• RecBCD loads RecA onto the 3′3' tail (5′→3′ direction) to begin homology search.

RecFOR Pathway (ssDNA Gaps)

• RecF, RecO, RecR replace SSB on a post-replication gap and load RecA.

Ruv Proteins – Branch Migration & Resolution

• RuvA + RuvB assemble on Holliday junctions; act as a rotary motor to drive branch migration.
• RuvC (resolvase) dimer binds opposite arms of junction and cleaves either X or Y pair of strands.

Regulation of RecA

• Induced transcriptionally by the SOS response (LexA repression relieved upon DNA damage).
• Requires RecBCD or RecFOR loaders; cannot bind random ssDNA  prevents unwanted recombination between repeated sequences (e.g., rRNA genes).

Homologous Recombination in Eukaryotes

Roles Beyond Repair
  1. DSB repair during S/G2 of mitosis (similar to bacterial pathway).
  2. Meiosis – HR deliberately creates crossovers that
    – Shuffle alleles between homologs (genetic diversity).
    – Establish physical links (chiasmata) that generate the tension required for bipolar attachment & proper segregation in meiosis I.
Meiotic Recombination Machinery (Saccharomyces cerevisiae prototype)

• DSB induction: Spo11 dimer cleaves both strands; each subunit becomes covalently attached (phosphotyrosyl bond).
• End processing: MRX complex (Mre11–Rad50–Xrs2) nicks 3′3' to Spo11 and releases Spo11-oligonucleotide; further resection by helicase Sgs1 and nucleases Dna2 / Exo1 / Sae2 → long 3′3' tails.
• Strand invasion: 3′3' ssDNA coated by Rad51 and meiosis-specific recombinase Dmc1.
• Outcomes
– SDSA (non-crossover) with potential gene conversion due to heteroduplex mismatches.
– DSBR with two junctions → non-crossover or crossover depending on resolution.

Gene Conversion Concept

• When heteroduplex DNA contains sequence mismatches, mismatch-repair enzymes may copy information from either strand.
• Example: If orange strand pairs with blue template, repair could overwrite orange sequence with blue, producing non-Mendelian inheritance (gene conversion).

Mitotic HR

• Predominantly repairs replication DSBs in S/G2; a DNA-damage checkpoint blocks mitosis until repair is complete.
• Only one RecA homolog active: Rad51 (no Dmc1).
• Loader protein Rad52 (human homolog = BRCA2) promotes Rad51 filament formation; BRCA2 defects ⇒ chromosome instability & breast cancer predisposition.
• SDSA strongly favored; crossovers rare to avoid loss of heterozygosity.

Programmed Gene Conversion – Yeast Mating-Type Switching

• S. cerevisiae haploids exist as MATa or MATα; opposite types fuse to form diploids.
• To ensure mating availability, cells can switch type via recombination.
• Chromosome III loci
– MAT (expressed): either MATaMAT_a or MATαMAT_\alpha.
– HMLα & HMRa (silent donors).
• Process

  1. Endonuclease HO creates site-specific DSB at MAT.
  2. 5′ ends resected → 3′3' tails.
  3. Rad51-mediated SDSA uses opposite silent cassette as template (HMLα when switching to α, HMRa when switching to a).
  4. No Holliday junctions, no crossovers; precise replacement of MAT sequence.

Non-Homologous End Joining (NHEJ)

• Backup pathway when no homologous template is available (G1 phase, terminally differentiated cells, heterochromatic regions).
• More prevalent in large-genome organisms (humans) because small indels are often tolerated.

Core Factors & Sequence of Events
  1. Ku70–Ku80 heterodimer binds each DSB end; protects DNA & recruits enzymes.
  2. DNA-PKcs binds Ku → forms active DNA-PK holoenzyme; juxtaposes ends.
  3. DNA-PKcs autophosphorylates and phosphorylates Artemis ⇒ Artemis endonuclease trims mismatched or ragged termini.
  4. Short microhomologies (1–4 nt) facilitate alignment of ends.
  5. Gap filling by specialized polymerases (Pol μ, Pol λ).
  6. Ligation by complex XRCC4–Ligase IV–XLF.
    • Consequences: small deletions, insertions, or base substitutions (mutagenic) but prevents lethal chromosome fragmentation.
Ethical / Medical Connections & Real-World Relevance

• HR defects (e.g., BRCA1/BRCA2, Rad51 paralogs) cause genome instability → cancer predisposition (breast, ovarian, Fanconi anemia).
• NHEJ is exploited in V(D)J recombination for antibody diversity and by genome-editing tools (CRISPR/Cas9) to create targeted indels.
• Understanding recombination informs chemotherapy design (PARP inhibitors target HR-deficient tumors) and gene-therapy safety.