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Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins.

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posted on 31.03.2021, 08:23 by Ondrej Belan, Consuelo Barroso, Artur Kaczmarczyk, Roopesh Anand, Stefania Federico, Nicola O'Reilly, Matthew D Newton, Erik Maeots, Radoslav I Enchev, Enrique Martinez-Perez, David S Rueda, Simon J Boulton
Homologous recombination (HR) is an essential DNA double-strand break (DSB) repair mechanism, which is frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated, resected DNA and catalyzes strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employed single-molecule imaging to investigate the mechanism of nematode RAD-51 filament growth in the presence of BRC-2 (BRCA2) and RAD-51 paralogs, RFS-1/RIP-1. BRC-2 nucleates RAD-51 on RPA-coated DNA, whereas RFS-1/RIP-1 acts as a "chaperone" to promote 3' to 5' filament growth via highly dynamic engagement with 5' filament ends. Inhibiting ATPase or mutation in the RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. The rfs-1 Walker box mutants display sensitivity to DNA damage and accumulate RAD-51 complexes non-functional for HR in vivo. Our work reveals the mechanism of RAD-51 nucleation and filament growth in the presence of recombination mediators.

Funding

Crick (Grant ID: 10013, Grant title: STP Peptide Chemistry) Crick (Grant ID: 10597, Grant title: Enchev FC001597) Crick (Grant ID: 10048, Grant title: Boulton FC001048) European Research Council (Grant ID: 742437 - TelMetab, Grant title: ERC 742437 - TelMetab)

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