Repair of DNA double strand breaks by homologous recombination (HR) is initiated by Rad51 filament nucleation on single-stranded DNA (ssDNA), which catalyzes strand exchange with homologous duplex DNA. BRCA2 and the Rad51 paralogs are tumor suppressors and critical mediators of Rad51. To gain insight into Rad51 paralog function, we investigated a heterodimeric Rad51 paralog complex, RFS-1/RIP-1, and uncovered the molecular basis by which Rad51 paralogs promote HR. Unlike BRCA2, which nucleates RAD-51-ssDNA filaments, RFS-1/RIP-1 binds and remodels pre-synaptic filaments to a stabilized, "open," and flexible conformation, in which the ssDNA is more accessible to nuclease digestion and RAD-51 dissociation rate is reduced. Walker box mutations in RFS-1, which abolish filament remodeling, fail to stimulate RAD-51 strand exchange activity, demonstrating that remodeling is essential for RFS-1/RIP-1 function. We propose that Rad51 paralogs stimulate HR by remodeling the Rad51 filament, priming it for strand exchange with the template duplex.

Department of Biochemistry and Molecular Genetics University of Virginia School of Medicine Charlottesville VA 22908 USA

Department of Biology Masaryk University 62500 Brno Czech Republic; International Clinical Research Center St Anne's University Hospital in Brno 62500 Brno Czech Republic

Department of Biology Masaryk University 62500 Brno Czech Republic; International Clinical Research Center St Anne's University Hospital in Brno 62500 Brno Czech Republic; National Centre for Biomolecular Research Masaryk University 62500 Brno Czech Republic

DNA Damage Response Laboratory Clare Hall Laboratory The Francis Crick Institute South Mimms EN6 3LD UK

DNA Damage Response Laboratory Clare Hall Laboratory The Francis Crick Institute South Mimms EN6 3LD UK; UCSF Mission Bay Genentech Hall S574 San Francisco CA 94158 USA

Electron Microscopy Science Technology Platform Lincoln's Inn Fields Laboratory The Francis Crick Institute London WC2A 3LY UK

Section of Virology Single Molecule Imaging Group and MRC Clinical Sciences Centre Department of Medicine Imperial College London London W12 0NN UK

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Mechanism of BCDX2-mediated RAD51 nucleation on short ssDNA stretches and fork DNA

Nucleotide proofreading functions by nematode RAD51 paralogs facilitate optimal RAD51 filament function

Antibiotic-induced DNA damage results in a controlled loss of pH homeostasis and genome instability

DSS1 interacts with and stimulates RAD52 to promote the repair of DSBs

Human RAD51 rapidly forms intrinsically dynamic nucleoprotein filaments modulated by nucleotide binding state

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