The generation of a double-strand break in the Saccharomyces cerevisiae genome is a potentially catastrophic event that can induce cell-cycle arrest or ultimately result in loss of cell viability. The repair of such lesions is strongly dependent on proteins encoded by the RAD52 epistasis group of genes (RAD50-55, RAD57, MRE11, XRS2), as well as the RFA1 and RAD59 genes. rad52 mutants exhibit the most severe phenotypic defects in double-strand break repair, but almost nothing is known about the biochemical role of Rad52 protein. Rad51 protein promotes DNA strand exchange and acts similarly to RecA protein. Yeast Rad52 protein interacts with Rad51 protein, binds single-stranded DNA and stimulates annealing of complementary single-stranded DNA. We find that Rad52 protein stimulates DNA strand exchange by targeting Rad51 protein to a complex of replication protein A (RPA) with single-stranded DNA. Rad52 protein affects an early step in the reaction, presynaptic filament formation, by overcoming the inhibitory effects of the competitor, RPA. Furthermore, stimulation is dependent on the concerted action of both Rad51 protein and RPA, implying that specific protein-protein interactions between Rad52 protein, Rad51 protein and RPA are required.

Homologous recombination in Saccharomyces cerevisiae depends critically on RAD52 function. In vitro, Rad52 protein preferentially binds single-stranded DNA (ssDNA), mediates annealing of complementary ssDNA, and stimulates Rad51 protein-mediated DNA strand exchange. Replication protein A (RPA) is a ssDNA-binding protein that is also crucial to the recombination process. Herein we report that Rad52 protein effects the annealing of RPA-ssDNA complexes, complexes that are otherwise unable to anneal. The ability of Rad52 protein to promote annealing depends on both the type of ssDNA substrate and ssDNA binding protein. RPA allows, but slows, Rad52 protein-mediated annealing of oligonucleotides. In contrast, RPA is almost essential for annealing of longer plasmid-sized DNA but has little effect on the annealing of poly(dT) and poly(dA), which are relatively long DNA molecules free of secondary structure. These results suggest that one role of RPA in Rad52 protein-mediated annealing is the elimination of DNA secondary structure. However, neither Escherichia coli ssDNA binding protein nor human RPA can substitute in this reaction, indicating that RPA has a second role in this process, a role that requires specific RPA-Rad52 protein interactions. This idea is confirmed by the finding that RPA, which is complexed with nonhomologous ssDNA, inhibits annealing but the human RPA-ssDNA complex does not. Finally, we present a model for the early steps of the repair of double-strand DNA breaks in yeast.In the yeast Saccharomyces cerevisiae, homologous recombination typically initiates at double-strand DNA breaks (DSBs). At an early step of meiosis, DSBs are introduced at specific loci in the chromosome; these breaks serves as sites for initiation of recombination, and loci with a high frequency of DSBs are recombination hot spots (1-4). In addition to its intimate association with the meiotic process, homologous recombination is responsible for the repair of DSBs that are introduced by DNA alkylating reagents, ionizing-radiation, and specific endonucleases (5, 6). A group of genes, referred to as the RAD52 epistasis group, are involved in both homologous recombination and the repair of DSBs (7,8). Analyses of mutants within this group of genes showed that the RAD52 gene is critically important for both recombination and resistance to x-rays (9-11). Although the RAD52 gene does not show any obvious homology to the known recombination proteins of Escherichia coli, it is conserved in yeast, human, and mice (12, 13), suggesting that the Rad52 protein is unique to eukaryotic organisms. Purified Rad52 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) and can anneal complementary ssDNA oligonucleotides (14, 15). More recent reports show that Rad52 protein stimulates DNA strand exchange mediated by Rad51 protein, which is a homologue of the E. coli RecA protein (16)(17)(18)(19). In the yeast system, stimulation is due to Rad52 protein interaction with, and alleviation of an inhibitory consequence of...

Rad52 protein plays a central role in double strand break repair and homologous recombination in Saccharomyces cerevisiae. We have identified a new mechanism by which Rad52 protein stimulates Rad51 protein-promoted DNA strand exchange. This function of Rad52 protein is revealed when subsaturating amounts (relative to the single-stranded DNA concentration) of replication protein-A (RPA) are used. Under these conditions, Rad52 protein is needed for extensive DNA strand exchange. Interestingly, in this new role, Rad52 protein neither acts simply as a single strand DNA-binding protein per se nor, in contrast to its previously identified stimulatory roles, does it require physical interaction with RPA because it can be substituted by the Escherichia coli single strand DNA-binding protein. We propose that Rad52 protein acts by stabilizing the Rad51 presynaptic filament.Homologous recombination and double strand break (DSB) 1 repair in the yeast Saccharomyces cerevisiae are under the control of the RAD52 epistasis group of genes, which includes RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, RDH54/ TID1, RAD50, MRE11, and XRS2 (1,2). For these processes, there exist multiple pathways that can be distinguished according to their need for Rad51 protein, the structural and functional homologue of the Escherichia coli DNA strand exchange protein, RecA. Both RAD51-dependent and RAD51-independent pathways exist, and they share a common requirement for RAD52 (1). Several proteins of the RAD52 epistasis group interact directly with one or more other proteins of the group. Rad52 protein physically interacts with Rad51 protein (3) and with replication protein-A (RPA) (4), the yeast functional homologue of the E. coli single strand DNA-binding protein SSB.Rad51 protein promotes DNA strand exchange in vitro (5). Using the single strand circular and homologous linear duplex DNA substrate system, complete heteroduplex formation by Rad51 protein was strongly dependent on the presence of RPA. RPA maximizes heteroduplex product yield by increasing the amount of ssDNA that can be incorporated into the active complex, which is composed of Rad51 protein and ssDNA and known as the presynaptic filament. In this respect, the RecA protein-and Rad51 protein-promoted reactions are different. Although SSB stimulates the activity of RecA protein, RecA protein can promote a significant level of DNA strand exchange in the absence of SSB (6). The most effective level of RPA was found to be that which could completely saturate the ssDNA (7), and complete product formation was extremely sensitive to the order of protein addition to ssDNA (5). When Rad51 protein is introduced to the ssDNA before RPA, DNA strand exchange occurs efficiently. However, RPA and Rad51 protein compete for binding to the same ssDNA binding sites. Because RPA binds more rapidly to ssDNA, when RPA is bound to ssDNA prior to or simultaneous with Rad51 protein, formation of the Rad51 nucleoprotein filament is inhibited and DNA strand exchange is blocked. Consequently, while RPA is an e...

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