A Polar and Nucleotide-Dependent Mechanism of Action for RAD51 Paralogs in RAD51 Filament Remodeling

• Published in: Molecular cell 2016-12, Vol.64 (5), p.926-939
• Main Authors: Taylor, Martin R.G., Špírek, Mário, Jian Ma, Chu, Carzaniga, Raffaella, Takaki, Tohru, Collinson, Lucy M., Greene, Eric C., Krejci, Lumir, Boulton, Simon J.
• Format: Article
• Language: English
• Subjects: Caenorhabditis elegans Proteins - genetics; Caenorhabditis elegans Proteins - metabolism; Carrier Proteins - genetics; Carrier Proteins - metabolism; DNA repair; DNA, Single-Stranded - genetics; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; filaments; genome stability; homologous recombination; Intermediate Filaments - genetics; Intermediate Filaments - metabolism; Multiprotein Complexes - metabolism; Protein Binding; Rad51; Rad51 paralogs; Rad51 Recombinase - genetics; Rad51 Recombinase - metabolism; Recombinational DNA Repair
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2016.10.020

Central to homologous recombination in eukaryotes is the RAD51 recombinase, which forms helical nucleoprotein filaments on single-stranded DNA (ssDNA) and catalyzes strand invasion with homologous duplex DNA. Various regulatory proteins assist this reaction including the RAD51 paralogs. We recently discovered that a RAD51 paralog complex from C. elegans, RFS-1/RIP-1, functions predominantly downstream of filament assembly by binding and remodeling RAD-51-ssDNA filaments to a conformation more proficient for strand exchange. Here, we demonstrate that RFS-1/RIP-1 acts by shutting down RAD-51 dissociation from ssDNA. Using stopped-flow experiments, we show that RFS-1/RIP-1 confers this dramatic stabilization by capping the 5′ end of RAD-51-ssDNA filaments. Filament end capping propagates a stabilizing effect with a 5′→3′ polarity approximately 40 nucleotides along individual filaments. Finally, we discover that filament capping and stabilization are dependent on nucleotide binding, but not hydrolysis by RFS-1/RIP-1. These data define the mechanism of RAD51 filament remodeling by RAD51 paralogs. [Display omitted] •A nematode RAD51 paralog complex binds the 5′ end of RAD51 pre-synaptic filaments•Filament binding drives pre-synaptic complex remodeling with 5′→3′ polarity•Pre-synaptic complex remodeling propagates up to 40 nucleotides from the 5′ end•Filament binding is dependent on a nucleotide co-factor, but not ATP hydrolysis RAD51 paralogs promote homologous recombination by remodeling RAD51-ssDNA filaments. Here, Taylor et al. demonstrate that RAD51 paralogs mediate remodeling by specifically binding the 5′ end of RAD51 filaments in a nucleotide-dependent manner. This propagates a stabilizing effect with 5′→3′ polarity distal to their binding site, slowing RAD51 dissociation from DNA.