Unit 4: DNA Repair Mechanisms and Homologous Recombination

I. Orientation: Genome Maintenance and the Logic of Repair

DNA damage arises constantly from spontaneous chemistry, replication error, and environmental agents; cells counter it with pathways that either reverse the lesion or excise and resynthesise the affected strand. Recombination provides both an error-free route to repair double-strand breaks and a mechanism for genetic exchange.

• Two lesion classes: Base modifications and mismatches affect one strand and can be corrected using the intact complementary strand as template; double-strand breaks (DSBs) destroy both strands and require an external template or direct joining.
• Repair strategy hierarchy: Direct reversal (no excision) → excision repair (removes and resynthesises a patch) → recombinational repair (uses a homologous duplex).
• Core enzymatic activities: Glycosylases (base removal), endo/exonucleases (backbone cleavage and resection), helicases (strand separation), polymerases (gap filling), and ligases (nick sealing).
• Template requirement: Single-strand damage keeps sequence information on the partner strand; DSB repair by recombination needs a sister chromatid or homolog.

II. Direct Repair

Enzymatic reversal of a lesion without breaking the phosphodiester backbone.

• Purpose and principle: The chemical modification is undone in situ, restoring the original base with no excision and no template.

A. Molecular characterization of repair enzymes in direct repair

• Photolyase (photoreactivation): Reverses UV-induced cyclobutane pyrimidine dimers and 6-4 photoproducts using light energy (300–500 nm) captured by two chromophores.
  • Cofactors: FADH⁻ (catalytic) and a light-harvesting pterin (MTHF) or deazaflavin.
  • Mechanism: Absorbed photon energizes FADH⁻, which injects an electron into the dimer, splitting the cyclobutane ring and regenerating two monomeric pyrimidines.
• O⁶-methylguanine-DNA methyltransferase (MGMT/Ada): Removes alkyl groups from O⁶-methylguanine.
  • Suicide mechanism: Transfers the methyl group to an internal cysteine residue, inactivating the enzyme irreversibly — a stoichiometric, not catalytic, repair.
• AlkB dioxygenase: Oxidatively demethylates 1-methyladenine and 3-methylcytosine using Fe(II) and α-ketoglutarate, releasing the methyl group as formaldehyde.

III. Single Strand Damage Repair

Excision pathways that cut out damaged bases or nucleotides and resynthesise using the intact strand.

• Common principle: Recognise → excise → gap-fill by polymerase → ligate.

A. Molecular characterization of repair enzymes in single strand damage repair

• Base Excision Repair (BER): Corrects small, non-helix-distorting lesions (uracil, oxidised or alkylated bases).
  • DNA glycosylase: Flips the damaged base out and cleaves the N-glycosidic bond, creating an abasic (AP) site (e.g., uracil-DNA glycosylase, UNG).
  • AP endonuclease (APE1): Nicks the backbone 5′ to the AP site.
  • DNA polymerase β / Pol I: Fills the single-nucleotide gap; DNA ligase III/I seals the nick.
• Nucleotide Excision Repair (NER): Removes bulky, helix-distorting adducts such as pyrimidine dimers.
  • In E. coli (UvrABC): UvrA₂B scans and detects distortion; UvrB verifies and recruits UvrC, which incises ~7 nt on the 3′ side and ~4 nt on the 5′ side. UvrD (helicase II) releases the ~12–13 nt oligomer; Pol I and ligase complete repair.
  • In eukaryotes: XP proteins (XPA–XPG) and TFIIH perform equivalent recognition, dual incision, and resynthesis.
• Mismatch Repair (MMR): Corrects replication mismatches and small insertion/deletion loops.
  • Strand discrimination: In E. coli, MutH nicks the unmethylated (nascent) strand at hemimethylated GATC sites; MutS binds the mismatch, MutL couples the events, and UvrD unwinds for excision.

IV. Repair of Double Strand DNA Breaks

Rejoining or reconstructing a chromosome severed on both strands.

• Stakes: An unrepaired DSB causes chromosome loss or translocation; two pathways compete.

A. Molecular characterization of repair enzymes in double strand break repair

1. Homologous Recombination (HR): Error-free; uses a sister chromatid as template.
   • Resection: MRN complex (Mre11–Rad50–Nbs1) and exonucleases generate 3′ single-stranded overhangs.
   • Strand invasion: Rad51 (eukaryotic RecA homolog) coats the ssDNA and searches for homology.
2. Non-Homologous End Joining (NHEJ): Error-prone; ligates ends directly without a template.
   • Ku70/Ku80 binds the broken ends; DNA-PKcs tethers them; DNA ligase IV–XRCC4 seals the junction, often with small deletions.

V. Homologous and Site-Specific Recombination

Two mechanisms of DNA rearrangement distinguished by sequence-homology requirements.

A. Homologous recombination

• Definition: Exchange between DNA molecules sharing extensive sequence identity, requiring RecA/Rad51-mediated strand pairing.
• Features: Occurs anywhere identity exists; underlies meiotic crossing over and DSB repair; produces heteroduplex DNA and can yield crossover products.

B. Site-specific recombination

• Definition: Exchange at short, defined recognition sequences catalysed by a dedicated recombinase, needing little or no homology beyond those sites.
• Features: Precise, reciprocal, and RecA-independent; governs phage integration, gene inversion, and plasmid resolution.

VI. Models for Homologous Recombination — The Holliday Model

The classic scheme explaining heteroduplex formation and crossover geometry.

A. The Holliday Model

• Purpose: Proposed by Robin Holliday (1964) to account for gene conversion and reciprocal exchange via a crossed-strand intermediate.
• Steps:
  • Alignment: Two homologous duplexes align in register.
  • Nicking: Single-strand nicks at equivalent positions on strands of the same polarity.
  • Strand exchange: Nicked strands cross over and pair with the partner duplex, forming heteroduplex regions; ligation creates the Holliday junction (a four-way crossed structure).
  • Branch migration: The crossover point slides, extending heteroduplex DNA.
  • Resolution: The junction is cleaved by resolvases. Cutting the crossed strands gives non-crossover (patch) products; cutting the non-crossed strands gives crossover (splice) products.
• Explanatory power: Symmetric heteroduplex on both duplexes accounts for gene conversion; two resolution modes explain the ~50:50 crossover ratio.

VII. RecBCD Pathway

The E. coli enzyme that initiates recombinational DSB repair from a broken end.

A. RecBCD pathway

• Enzyme: RecBCD is a heterotrimer with helicase and nuclease activities that acts on double-stranded ends.
  • RecB: 3′→5′ helicase and nuclease.
  • RecD: 5′→3′ helicase (faster motor).
  • RecC: Recognises the Chi sequence.
• Chi (crossover hotspot instigator) site: The 8-nt sequence

5'-GCTGGTGG-3'

• Before Chi: RecBCD unwinds and degrades both strands, favouring the 3′-ended strand.
• At Chi: Recognition pauses the enzyme, attenuates 3′→5′ nuclease activity, and switches degradation so a 3′ single-stranded tail with Chi at its end is produced.
  • RecA loading: RecBCD loads RecA onto the 3′ tail; the RecA nucleoprotein filament promotes strand invasion of a homologous duplex, generating a D-loop that feeds into Holliday-junction processing (resolved by RuvABC).

VIII. Conserved Site-Specific Recombination

Recombinase families that exchange DNA through covalent protein–DNA intermediates.

A. Conserved site-specific recombination

• Principle: A recombinase binds a specific site, cleaves the backbone forming a transient covalent enzyme–DNA bond, exchanges strands, and reseals — conserving every phosphodiester bond (no synthesis or ligase needed).
• Two conserved families:
  1. Tyrosine recombinases (Int/Cre family): Use an active-site tyrosine to form a 3′-phosphotyrosine link; exchange one pair of strands at a time via a Holliday intermediate. Example: λ integrase acting at attP/attB.
  2. Serine recombinases (resolvase/invertase family): Use an active-site serine forming a 5′-phosphoserine link; cut all four strands, then rotate 180° and rejoin. Example: γδ resolvase, Hin invertase.
• Reaction outcomes: Sites in inverted orientation → inversion; sites in direct repeat on one molecule → deletion/excision; sites on separate molecules → integration.

IX. Cre/LoxP Recombination

A tyrosine-recombinase system widely used for controlled genome engineering.

A. Cre/LoxP recombination

• Components: Cre recombinase (from bacteriophage P1) and the 34-bp loxP site.
  • loxP structure: Two 13-bp palindromic arms flanking an 8-bp asymmetric spacer:

ATAACTTCGTATA - ATGTATGC - TATACGAAGTTAT
   13-bp arm      spacer       13-bp arm

• Spacer asymmetry: Sets loxP directionality, dictating whether the outcome is excision or inversion.
  • Mechanism: Four Cre monomers bind two loxP sites forming a synaptic complex; the active-site tyrosine cleaves the spacer boundaries, strands exchange through a Holliday junction, and isomerisation followed by a second exchange completes recombination.
  • Orientation-dependent outcomes:
    1. Direct repeat loxP: Intervening DNA is excised as a circle, leaving one loxP site — the basis of conditional gene knockout.
    2. Inverted loxP: Intervening segment is inverted rather than removed.
  • Utility: Combined with tissue-specific or inducible promoters (e.g., CreER activated by tamoxifen), it enables spatial and temporal control of gene deletion in model organisms, requiring no host cofactors or added energy.