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Simon Boulton : Mechanics of the homologous recombination reaction

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Homologous recombination (HR) is an essential mechanism for the repair of DNA double strand breaks (DSBs) and ssDNA gaps arising at damaged replication forks. HR is a complex multistep process initiated by the loading of the RAD51 recombinase onto single stranded DNA (ssDNA) to form a helical nucleoprotein filament. Once RAD51 has loaded, the resulting RAD51-ssDNA filament structure is primed to commence homology search and strand invasion into an intact homologous duplex DNA molecule located elsewhere in the genome. Following strand invasion, DNA synthesis can initiate from the broken 3’ DNA end allowing the correct sequence information to be copied from the homologous sequence into the break site. To complete the repair reaction, the resulting joint molecules can be disentangled by dissolution using branch migration and topoisomerase activities or resolved by structure specific nucleases.

Homologous Recombination

Eukaryotic RAD51 is an inefficient enzyme requiring numerous accessory factors to facilitate: i) targeting to and loading onto ssDNA, ii) the homolog search, iii) stimulation of strand exchange, and iv) displacement (D)-loop stabilization. In general, RAD51 accessory factors have been identified by genetic means as being essential for HR in vivo. By integrating data from biochemical and biophysical approaches, we recently uncovered the mechanism of action of the Rad51 paralogs in modulating RAD51 to promote HR. We showed that the Rad51 paralogs preferentially bind to and remodel the pre-synaptic filament, converting it to a stable conformation primed for strand exchange (Taylor et al., Cell 2015). In a follow-up study, we employed stopped flow and immuno-gold EM to show that Rad51 paralogs specifically bind to the 5’ end of RAD-51-ssDNA filaments and mediate remodeling in a 5’→3’ direction, in a manner dependent on ATP binding but not ATP hydrolysis. Using DNA curtain technology pioneered by the Greene lab, we showed that the end capping and remodeling activity of Rad51 paralogs acts to block the turnover of RAD51 from ssDNA, thus stabilizing the pre-synaptic filament in an active state (Taylor et al., Molecular Cell 2016).

We are currently applying cutting-edge biophysical and structural approaches to understand HR in unprecedented detail. We are studying multiple different HR regulators (BRCA2, Rad51 paralogs, Rad51AP1, SHU complex, RECQ-5 and HELQ) to gain insights into how they work individually and how they act cooperatively during the HR reaction.