The tumor suppressor complex BRCA1-BARD1 functions in DNA double-strand break repair by homologous recombination. Therein, BRCA1-BARD1 facilitates the nucleolytic resection of DNA ends to generate a single-stranded template for the recruitment of another tumor suppressor complex BRCA2-PALB2 and the recombinase RAD51. By examining purified BRCA1-BARD1 and mutants, we show that BRCA1 and BARD1 both bind DNA and interact with RAD51, and that BRCA1-BARD1 enhances the recombinase activity of RAD51. Mechanistically, BRCA1-BARD1 promotes the assembly of the synaptic complex, an essential intermediate in RAD51-mediated DNA joint formation. Evidence is provided that BRCA1 and BARD1 are both indispensable for RAD51 stimulation. Importantly, BRCA1-BARD1 mutants weakened for RAD51 interaction are compromised for DNA joint formation and for the mediation of homologous recombination and DNA repair in cells. Our results identify a late role of BRCA1-BARD1 in homologous recombination, a novel attribute of the tumor suppressor complex that could be targeted in cancer therapy.

In response to genotoxic stress, a transient arrest in cell cycle progression enforced by the DNA damage checkpoint (DDC) signaling pathway positively contributes to genome maintenance1. Because hyperactivated DDC can lead to a persistent and detrimental cell cycle arrest2,3, cells must tightly regulate the activity of DDC kinases. Despite their importance, the mechanisms for monitoring and modulating DDC signaling are not fully understood. Here we show that DNA repair scaffolding proteins Slx4 and Rtt107 prevent lesions generated during DNA replication from aberrantly hyperactivating DDC signaling in Saccharomyces cerevisiae. Upon replication stress, cells lacking Slx4 or Rtt107 exhibit hyperactivation of the downstream DDC kinase Rad53 while activation of the upstream DDC kinase Mec1 remains normal. An Slx4-Rtt107 complex counteracts the checkpoint adaptor Rad9 by physically interacting with Dpb11 and phospho-H2A, two positive regulators of Rad9-dependent Rad53 activation. Reduction of DDC signaling by hypomorphic mutations in RAD53 and H2A rescue the hyper-sensitivity of slx4Δ or rtt107Δ cells to replication stress. We propose that the Slx4-Rtt107 complex modulates Rad53 activation via a competition-based mechanism that balances the engagement of Rad9 at replication-induced lesions. Our findings reveal that DDC signaling is monitored and modulated through the direct action of DNA repair factors.

Homologous recombination (HR) is a crucial pathway for double-stranded DNA break (DSB) repair. During the early stages of HR, the newly generated DSB ends are processed to yield long single-stranded DNA (ssDNA) overhangs, which are quickly bound by replication protein A (RPA). RPA is then replaced by the DNA recombinase Rad51, which forms extended helical filaments on the ssDNA. The resulting nucleoprotein filament, known as the presynaptic complex, is responsible for pairing the ssDNA with homologous double-stranded DNA (dsDNA), which serves as the template to guide DSB repair. Here, we use single-molecule imaging to visualize the interplay between human RPA (hRPA) and human RAD51 during presynaptic complex assembly and disassembly. We demonstrate that ssDNA-bound hRPA can undergo facilitated exchange, enabling hRPA to undergo rapid exchange between free and ssDNA-bound states only when free hRPA is present in solution. Our results also indicate that the presence of free hRPA inhibits RAD51 filament nucleation, but has a lesser impact upon filament elongation. This finding suggests that hRPA exerts important regulatory influence over RAD51 and may in turn affect the properties of the assembled RAD51 filament. These experiments provide an important basis for further investigations into the regulation of human presynaptic complex assembly.

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.

Rad52 is a highly conserved protein involved in the repair of DNA damage. Human RAD52 has been shown to mediate single-stranded DNA (ssDNA) and is synthetic lethal with mutations in other key recombination proteins. For this study, we used single-molecule imaging and ssDNA curtains to examine the binding interactions of human RAD52 with replication protein A (RPA)-coated ssDNA, and we monitored the fate of RAD52 during assembly of the presynaptic complex. We show that RAD52 binds tightly to the RPA-ssDNA complex and imparts an inhibitory effect on RPA turnover. We also found that during presynaptic complex assembly, most of the RPA and RAD52 was displaced from the ssDNA, but some RAD52-RPA-ssDNA complexes persisted as interspersed clusters surrounded by RAD51 filaments. Once assembled, the presence of RAD51 restricted formation of new RAD52-binding events, but additional RAD52 could bind once RAD51 dissociated from the ssDNA. Together, these results provide new insights into the behavior and dynamics of human RAD52 during presynaptic complex assembly and disassembly.