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#homologous recombination

19 public questions tagged with this topic.

Recombination

RecA binds preferentially to

Single-stranded DNA preference of RecA ensures that recombinogenic ends not double-stranded regions are targeted. ssDNA generated after resection or RecBCD processing is immediately bound by SSB in bacteria or RPA in eukaryotes, which melts secondary structures. RecA mediator proteins such as RecFOR facilitate replacement of SSB with cooperative ATP-bound RecA nucleating into contiguous filament. Within filament ssDNA adopts extended conformation facilitating base-triplet scanning for homology on intact duplex. This selective binding avoids illegitimate association with undamaged chromosomes, channeling strand invasion specifically to broken resected termini requiring repair.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 12: RecA Preferential Binding to ssDNA

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Recombination

RecC subunit recognizes

Within RecBCD trimer, RecC subunit does not possess catalytic domains but functions as scanner for recombination hotspots. Crystal structures show tunnel where duplex DNA threaded before reaching helicase motors, containing specific pocket recognizing Chi octamer 5'-GCTGGTGG-3' in single-stranded form. Recognition occurs when RecBCD unwinds DNA, Chi strand exposed interacts with RecC residues. Binding triggers conformational rearrangement transmitted through flexible linker changing RecB and RecD motor speeds and attenuating nuclease, effectively transforming enzyme from destructive nuclease to RecA loading machinery, explaining RecC mutations abolishing Chi response without affecting helicase activity.

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 10: RecC Recognition of Chi Site Mechanism

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Recombination

In mating type switching, donor locus is

In budding yeast, chromosome III harbors two silent donor repositories: HML containing alpha information near left telomere and HMR containing a information near right telomere, each flanked by homology boxes X and Z1 shared with MAT. During switching, MAT is recipient receiving new genetic identity, while HML or HMR acts as template providing sequence without being altered itself, analogous to SDSA. Choice of donor regulated by recombination enhancer controlling accessibility; MATa cells preferentially recombine with HML alpha, MAT alpha cells with HMR a, ensuring productive switching rather than futile copying of identical information.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 21: Silent Loci as Donors for MAT Switching

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Recombination

Mating type switching occurs via which recombination pathway?

Mating type conversion involves copying information from silent donor into broken MAT locus. After HO induced break, resected MAT end invades HML or HMR sharing Z1 and W homology regions forming D-loop. Repair synthesis copies Ya or Yα sequence from donor, then nascent strand dissociates from donor and anneals back to second end of MAT, filling gap without establishing stable double Holliday junction or crossover between chromosome III regions. This synthesis-dependent strand annealing mechanism results in non-crossover gene conversion, preserving donor cassette unchanged while converting recipient MAT sequence, ensuring donor not lost and maintains switching plasticity.

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 18: Mating Type Switching via SDSA Mechanism

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Recombination

SDSA pathway mainly results in

Synthesis-dependent strand annealing represents predominant mitotic homologous recombination pathway designed to avoid crossovers. After limited invasion and extension of one 3' end using donor chromatid as template, polymerase delta synthesizes short tract, then disrupted D-loop releases nascent strand through action of Mph1, Srs2, RTEL1, or FANCM helicases, restoring donor duplex unchanged. Dissociated strand anneals to second broken end, allowing fill-in synthesis and ligation without formation of stable double Holliday junction. Genetic markers flanking break remain parental configuration, thereby generating only non-crossover recombinants, critical for genome stability.

Ref: NCBI Bookshelf, Biochemistry, SDSA Pathway Results in Non-Crossover Products

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Recombination

BRCA2 assists homologous recombination by regulating

BRCA2 is prominent tumor suppressor containing BRC repeats that directly interact with Rad51. Under normal conditions RPA rapidly coats ssDNA preventing spontaneous Rad51 nucleation. After DNA damage, BRCA2 is recruited via PALB2 and BRCA1 to break sites where it mediates nucleation of Rad51 filament, stabilizing ATP-bound active conformation, blocking ATP hydrolysis mediated disassembly, and delivering Rad51 to ssDNA replacing RPA. This loader function is essential for strand invasion, D-loop formation, and homology-directed repair. Pathogenic BRCA2 mutations cause defective Rad51 recruitment, reliance on error-prone NHEJ, and breast-ovarian cancer susceptibility.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 19: BRCA2 Regulation of Rad51 Loading in HR

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Recombination

Dmc1 protein functions in

Dmc1 is meiosis-specific RecA homolog expressed during prophase I together with Rad51. While Rad51 participates broadly in repair, Dmc1 is enriched at meiotic double-strand breaks and preferentially drives interhomolog rather than intersister recombination, ensuring at least one crossover per homolog pair. Dmc1 filaments are more resistant to dissociation and cooperate with auxiliary factors Mei5-Sae3 and Hop2-Mnd1 to stabilize heteroduplex and promote directed homology search toward homologous chromosome. Knockout mice show sterility with normal vegetative growth because Dmc1 dispensable for mitotic repair.

Ref: Watson et al., Molecular Biology of the Gene, 7th ed., Chapter 12: Dmc1 Meiosis-Specific Recombinase Role

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Recombination

Rad51 functions in

Rad51 is eukaryotic ortholog of bacterial RecA and central recombinase for homologous recombination. Expression is not restricted to meiotic tissue; it operates in mitotic somatic cells repairing collapsed forks and double-strand breaks using sister chromatid template, and in meiotic cells alongside Dmc1. Rad51 forms presynaptic filament that executes homology search, invasion, and D-loop formation. Its activity regulated by mediators BRCA2, Rad52, and Rad51 paralogs that facilitate RPA displacement. Loss causes hypersensitivity to ionizing radiation, embryonic lethality, and cancer predisposition in both mitotic and meiotic contexts.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: Rad51 Function in Mitosis and Meiosis

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Recombination

Primary function of MRX complex is

Efficient homologous recombination depends on conversion of blunt double-strand breaks into 3' single-stranded overhangs suitable for Rad51 filament formation. MRX and Sae2 initiate this resection by Mre11 introducing endonucleolytic nick distal to 5' blocked ends such as Spo11 adducts or Ku-bound ends. Nick creation allows bidirectional exonucleolytic degradation, primarily 5'→3' by Exo1 or Sgs1-Dna2 in extended resection. This step commits cell to homology-directed repair because long 3' tails incompatible with NHEJ. Thus MRX does not perform strand invasion or direct ligation but provides substrate for homology search.

Ref: Lodish et al., Molecular Cell Biology, 9th ed., Chapter 12: MRX Initiated 5' Strand Resection Mechanism

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Recombination

MRX complex is composed of

In Saccharomyces cerevisiae, initial DSB processing requires trimeric Mre11-Rad50-Xrs2 complex, whose mammalian counterpart is MRN containing Nbs1 instead of Xrs2. Mre11 is nuclease with both endonuclease and 3'→5' exonuclease activities, Rad50 is long coiled-coil SMC-like protein dimerizing via zinc-hook at apex and binding ATP to undergo conformational switching between open and closed states, Xrs2/Nbs1 mediates protein interactions, nuclear localization, and Tel1/ATM checkpoint signaling. Together they sense Spo11 blocks, tether DNA ends, activate checkpoint, and initiate resection together with Sae2/CtIP, bridging structural maintenance and enzymatic nucleolysis.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 5: MRX Complex Composition Mre11 Rad50 Xrs2

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Recombination

Spo11 forms a covalent complex with DNA via which amino acid residue?

Spo11 mechanism mirrors type II topoisomerase chemistry. Each monomer of Spo11 dimer contributes nucleophilic tyrosine hydroxyl that attacks scissile phosphate in DNA backbone. Conserved 5Y-CAP domain contains critical Tyr135 in Saccharomyces and Tyr138 in mouse, forming 5'-phosphotyrosyl covalent intermediate retaining break ends bound after cleavage. This linkage resembles topoisomerase-DNA adduct that prevents diffusion of ends and flags break for MRX-mediated removal as Spo11-oligonucleotide complexes. Mutation of catalytic tyrosine to phenylalanine eliminates DSB formation and recombination, causing meiotic arrest despite normal protein expression.

Ref: NCBI Bookshelf, Molecular Cell Biology, Spo11 Tyrosine-Mediated Covalent Complex Mechanism

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Recombination

Strand invasion during homologous recombination is mediated by

Resected 3' single-stranded overhangs are rapidly coated by RPA, which must be replaced by recombinase to become invasive. In bacteria RecA, and in eukaryotes Rad51 and meiosis-specific Dmc1, polymerize directionally on ssDNA into right-handed helical nucleoprotein filaments with ATP. This filament stretches DNA and performs homology search via base-triplet sampling, then catalyzes pairing and strand exchange with intact duplex donor. Invasion creates a displacement loop and provides 3'-OH primer for repair synthesis, establishing heteroduplex DNA without requiring ATP hydrolysis for pairing itself.

Ref: NCBI Bookshelf, Molecular Cell Biology, Homologous Recombination – RecA/Rad51 Mediated Strand Invasion

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