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

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

Posted 05 Nov 2020
bioRxiv DOI: 10.1101/2020.11.05.369629

Homologous recombination (HR) is an essential DNA double-strand break (DSBs) repair mechanism frequently inactivated in cancer. During HR, RAD51 forms nucleoprotein filaments on RPA-coated resected DNA and catalyses strand invasion into homologous duplex DNA. How RAD51 displaces RPA and assembles into long HR-proficient filaments remains uncertain. Here, we employ 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, while 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 RFS-1 Walker box leads to RFS-1/RIP-1 retention on RAD-51 filaments and hinders growth. 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. ### Competing Interest Statement The authors have declared no competing interest.

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