Abstract
SummaryRepair 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.
Highlights
Homologous recombination (HR) is an essential mechanism for the repair of DNA double strand breaks (DSBs) and damaged replication forks
We previously showed that rfs-1 mutant strains are sensitive to DNA damage, defective for RAD-51 focus formation at stalled replication forks, and exhibit meiotic defects when combined with helq-1 mutations (Ward et al, 2007, 2010)
Using a yeast two-hybrid screen (Boulton et al, 2002), we identified an orphan protein encoded by R01H10.5 (Uniprot ID code Q21621) that we named RIP-1 (RFS-1 interacting protein) (Figure 1A), which interacted with RFS-1 by yeast two-hybrid (Figure 1B), glutathione S-transferase (GST) pulldowns of C. elegans RFS-1 and RIP-1 expressed in human cells (Figure 1C), and FLAG co-immunoprecipitation from yeast cells (Figure 2A)
Summary
Homologous recombination (HR) is an essential mechanism for the repair of DNA double strand breaks (DSBs) and damaged replication forks. HR is initiated at single-stranded DNA (ssDNA) exposed at nucleolytically processed DSB ends or post-replicative ssDNA gaps by the exchange of the ssDNA binding protein RPA for the recombinase enzyme Rad, which forms helical nucleoprotein filaments on ssDNA. Unloading of Rad from double-stranded DNA (dsDNA) permits the initiation of repair DNA synthesis and the resulting joint molecules are processed by various enzymes to complete the repair reaction (Chapman et al, 2012; San Filippo et al, 2008). The molecular mechanism underlying the stimulation of HR by the Rad paralogs has remained elusive
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