Molecular Cell
Volume 81, Issue 5, 4 March 2021, Pages 1058-1073.e7
Journal home page for Molecular Cell

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

https://doi.org/10.1016/j.molcel.2020.12.020Get rights and content
Under a Creative Commons license
open access

Highlights

  • C. elegans BRCA2 and Rad51 paralogs synergistically promote Rad51 filament assembly

  • Rad51 paralogs dynamically engage with the 5′ Rad51 filament ends

  • Rad51 paralogs stimulate Rad51 filament growth in a 3′–5′ direction

  • Retention of Rad51 paralogs at filament ends hinders growth and blocks HR in vivo

Summary

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.

Keywords

DNA repair
homologous recombination
single molecule approaches
Rad51 nucleoprotein filaments
BRCA2
Rad51 paralogs

Cited by (0)

7

Lead contact