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

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

doi:10.1016/j.molcel.2020.12.020

Ondrej Belan, Consuelo Barroso + Show 10 more

Open Access

https://doi.org/10.1016/j.molcel.2020.12.020

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Abstract

SummaryHomologous 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.

Highlights

Homologous recombination (HR) is a largely error-free mechanism of DNA double-strand break (DSB) repair
Through a combination of microfluidics, optical tweezers, and fluorescence microscopy, we show that BRC-2 acts primarily as a RAD-51 nucleation factor on replication protein A (RPA)-coated single-stranded DNA (ssDNA), whereas RFS-1/RIP-1 acts on nucleated RAD51-ssDNA complexes to stimulate filament growth
RAD-51 assembly and RPA-eGFP displacement was monitored by loss of eGFP fluorescence and simultaneous decrease in the force exerted on ssDNA that accompanies recombinase filament formation (Hegner et al, 1999)

Summary

Introduction

Homologous recombination (HR) is a largely error-free mechanism of DNA double-strand break (DSB) repair. Broken double-stranded DNA (dsDNA) ends are nucleolytically processed, yielding 2–4 kb of single-stranded DNA (ssDNA) coated by replication protein A (RPA). RPA is displaced by Rad, a eukaryotic recombinase that forms helical nucleoprotein filaments with single-stranded DNA (ssDNA). DNA is extended 1.5-fold over the dsDNA contour length, with a stoichiometry of 3 nt per RAD51 monomer and six RAD51 monomers per helical turn (Xu et al, 2017). Rad filaments search for a homologous DNA sequence within sister chromatids or homologous chromosomes, followed by strand invasion and displacement loop (D-loop) intermediate formation. The invading 30 DNA end is extended by polymerases and processed by multiple redundant pathways to complete the repair and to restore the broken DNA strand (Chapman et al, 2012)

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