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bioRxiv · 10.1101/2020.11.05.369629

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

Belan, O.·Barroso, C.·Kaczmarczyk, A.·Anand, R.·Federico, S.·O'Reilly, N.·Newton, M. D.·Maeots, E.·Enchev, R. I.·Martinez-Perez, E.·Rueda, D. S.·Boulton, S. J.
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Abstract

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.

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Belan, O., Barroso, C., Kaczmarczyk, A., Anand, R., Federico, S., O'Reilly, N., Newton, M. D., Maeots, E., Enchev, R. I., Martinez-Perez, E., Rueda, D. S., Boulton, S. J.. 2020-11-05. Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins. https://doi.org/10.1101/2020.11.05.369629

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