DNA break repair goes live

Homologous recombination (HR) is used to repair DNA double-strand breaks (DSBs) that threaten the genomic stability of prokaryotic and eukaryotic cells. In bacteria, the RecBCD complex processes the ends of DSBs to generate 3′-terminal single-strand extensions for RecA recruitment. RecA binding nucleates assembly of a nucleoprotein filament that aligns with sequences of a homologous duplex and promotes strand invasion leading to exchange. Although RecA function has been extensively studied in vitro, its intracellular activities have not been directly observed, and it is not known whether HR repair is restricted to sister duplexes that remain in proximity after replication. Now, Sherratt and colleagues use a combination of super-resolution structured illumination microscopy (SIM) and wide-field time-lapse microscopy to monitor the recruitment of fluorescently labeled RecA proteins to RecBCD-processed DSBs induced by I-SceI endonuclease in living cells. Remarkably, they observe pairing and recombination between homologous sister loci that have segregated to opposite halves of the cell after replication. Time-lapse analysis of repair-competent Escherichia coli revealed that both ends of the DSB are processed simultaneously by RecBCD and recruit 3,000 RecA molecules to generate filaments termed 'RecA bundles' that extend along the long axis of the cell between the bulk DNA and the inner membrane. The cut ends then travel towards the uncut sister locus, resulting in rapid sister pairing and RecA-bundle disassembly. Repair-deficient RecA mutants unable to bind or hydrolyze ATP do not form bundles, consistent with the bundles being functionally relevant. Recombination factors that promote downstream events are recruited to the sites after bundle disassembly. The current findings suggest that RecA bundles facilitate the homology search by directionally 'channeling' the motion of cut ends over long distances, by an as-yet-uncharacterized mechanism, to permit efficient HR-mediated DNA repair after sister-chromosome segregation. (Nature doi:10.1038/nature12868, 22 December 2013)


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Moorefield, B. DNA break repair goes live. Nat Struct Mol Biol 21, 117 (2014). https://doi.org/10.1038/nsmb.2772

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