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

Martin R.G Taylor,Mário Špírek,Chu Jian Ma,Raffaella Carzaniga,Tohru Takaki,Lucy M Collinson,Eric C Greene,Lumir Krejci,Simon J Boulton

doi:10.1016/j.molcel.2016.10.020

Martin R.G Taylor, Mário Špírek + Show 7 more

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https://doi.org/10.1016/j.molcel.2016.10.020

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SummaryCentral 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.

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Homologous recombination (HR) is a highly conserved mechanism for the repair of DNA double-strand breaks and stalled replication forks
We present evidence that RFS-1/RIP-1 binds to the 50 end of individual RAD-51-single-stranded DNA (ssDNA) filaments and mediates remodeling in a 50/30 direction, in a manner dependent on ATP binding, but not hydrolysis by the complex
RFS-1/RIP-1 Shuts down RAD-51 Dissociation from ssDNA To investigate the mechanism by which RAD51 paralogs stabilize pre-synaptic filaments, we monitored the stability of RAD51 filaments assembled on long ssDNA curtains using the rebinding of fluorescently labeled yeast RPA to naked ssDNA (ScRPA-eGFP, hereafter abbreviated to RPA) (Figures 1A and 1B) (Gibb et al, 2014a; Qi et al, 2015)

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Homologous recombination (HR) is a highly conserved mechanism for the repair of DNA double-strand breaks and stalled replication forks. HR mediators, such as BRCA2, promote RAD51 filament assembly on ssDNA coated with the ssDNA binding protein RPA (Jensen et al, 2010; Liu et al, 2010; Shahid et al, 2014; Thorslund et al, 2010), while RAD54 promotes RAD51 dissociation from dsDNA after strand invasion (Solinger et al, 2002) These activities allow these proteins to stimulate strand exchange by RAD51 in vitro (Jensen et al, 2010; Solinger et al, 2002; Thorslund et al, 2010) and promote HR and DNA damage resistance in vivo (Johnson and Jasin, 2001)

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