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

• Published in: Molecular cell Vol. 81; no. 5; pp. 1058 - 1073.e7
• Main Authors: Belan, Ondrej, Barroso, Consuelo, Kaczmarczyk, Artur, Anand, Roopesh, Federico, Stefania, O’Reilly, Nicola, Newton, Matthew D., Maeots, Erik, Enchev, Radoslav I., Martinez-Perez, Enrique, Rueda, David S., Boulton, Simon J.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04-03-2021; Cell Press
• Subjects: Animals; BRCA2; Caenorhabditis elegans - genetics; Caenorhabditis elegans - metabolism; Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; DNA Breaks, Double-Stranded; DNA repair; DNA, Helminth - genetics; DNA, Helminth - metabolism; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; Gene Expression Regulation; homologous recombination; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Mutation; Protein Binding; Rad51 nucleoprotein filaments; Rad51 paralogs; Rad51 Recombinase - genetics; Rad51 Recombinase - metabolism; Recombinational DNA Repair; Replication Protein A - genetics; Replication Protein A - metabolism; Signal Transduction; single molecule approaches; Single Molecule Imaging; single molecule approaches; Rad51 paralogs; homologous recombination; BRCA2; DNA repair; Rad51 nucleoprotein filaments
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2020.12.020

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.
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•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
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.