Belan, O., Barroso, C., Kaczmarczyk, A. orcid.org/0000-0002-2100-2780 et al. (9 more authors) (2021) Single-molecule analysis reveals cooperative stimulation of Rad51 filament nucleation and growth by mediator proteins. Molecular Cell, 81 (5). 1058-1073.e7. ISSN: 1097-2765
Abstract
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. Belan et al. exploit single-molecule approaches to reveal the mechanism of C. elegans Rad51 filament assembly in the presence of recombination mediators. BRCA2 primarily enhances Rad51 nucleation on ssDNA, whereas Rad51 paralogs dynamically engage with 5′ Rad51 filament ends and stimulate filament growth in a 3′–5′ direction.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 The Authors. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
Keywords: | BRCA2; DNA repair; Rad51 nucleoprotein filaments; Rad51 paralogs; homologous recombination; single molecule approaches; Animals; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carrier Proteins; DNA Breaks, Double-Stranded; DNA, Helminth; DNA-Binding Proteins; Gene Expression Regulation; Molecular Chaperones; Mutation; Protein Binding; Rad51 Recombinase; Recombinational DNA Repair; Replication Protein A; Signal Transduction; Single Molecule Imaging |
Dates: |
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Institution: | The University of Sheffield |
Academic Units: | The University of Sheffield > Faculty of Science (Sheffield) > School of Biosciences (Sheffield) |
Date Deposited: | 17 Oct 2025 13:51 |
Last Modified: | 17 Oct 2025 13:51 |
Status: | Published |
Publisher: | Elsevier BV |
Refereed: | Yes |
Identification Number: | 10.1016/j.molcel.2020.12.020 |
Related URLs: | |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:232914 |