Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins
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Ondrej Belan Consuelo Barroso Artur Kaczmarczyk Roopesh Anand Stefania Federico Nicola O'Reilly Matt Newton Erik Maeots Radoslav Enchev Enrique Martinez-Perez David S Rueda Simon BoultonAbstract
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.
Journal details
Journal Molecular Cell
Volume 81
Issue number 5
Pages 1058-1073.e7
Available online
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Publisher website (DOI) 10.1016/j.molcel.2020.12.020
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Europe PubMed Central 33421363
Pubmed 33421363
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