TY - JOUR T1 - Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins JF - bioRxiv DO - 10.1101/2020.11.05.369629 SP - 2020.11.05.369629 AU - Ondrej Belan AU - Consuelo Barroso AU - Artur Kaczmarczyk AU - Roopesh Anand AU - Stefania Federico AU - Nicola O’Reilly AU - Matthew D. Newton AU - Erik Maeots AU - Radoslav I. Enchev AU - Enrique Martinez-Perez AU - David S. Rueda AU - Simon J. Boulton Y1 - 2020/01/01 UR - http://biorxiv.org/content/early/2020/11/05/2020.11.05.369629.abstract N2 - 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 StatementThe authors have declared no competing interest. ER -