DNA annealing by Rad52 Protein is stimulated by specific interaction with the complex of replication protein A and single-stranded DNA

Abstract

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...

Sequential and Synergistic Modification of Human RPA Stimulates Chromosomal DNA Repair

“…For Thr(P) 21 , a peptide containing RPA2 residues 16 -26 was used to generate the anti-Thr(P) 21 antibody. 5 This is consistent with the fact that the S29A mutation did not affect formation of Thr(P) 21 , indicating that this mutation does not affect recognition of the epitope (Figs. 4C).…”

A Novel Function of Rad54 Protein

“…For instance, the Rad51 protein interacts with a number of other homologous recombination proteins, including Rad52 protein (8), Rad55 protein (40), and Rad54 protein (41,42). In addition, Rad55 protein forms a stable heterodimer with Rad57 protein (21); Rad50 protein, Mre11 protein, and Xrs2 protein form a heterotrimer (43); and Rad52 protein interacts with RPA (44,45). These multiple interactions permit formation of a variety of complexes, whose dynamic properties permit great flexibility in responding to various endogenous or exogenous DNA damages.…”

DNA-Pairing and Annealing Processes in Homologous Recombination and Homology-Directed Repair

“…This activity is important for the second-end capture phases of dHJ and SDSA pathways, as described below. The mechanism of Rad52-promoted annealing of RPA-ssDNA complexes involves the formation of a ternary Rad52-RPA-ssDNA complex (Shinohara et al 1998;Sugiyama et al 1998). This mechanism appears to be conserved as both RecO and UvsY show similar interactions with cognate SSB-ssDNA complexes (Sweezy and Morrical 1999;Ryzhikov et al 2011;Ryzhikov and Korolev 2012).…”

“…The ability to anneal two complementary strands of ssDNA constitutes a separate and distinct function of certain proteins with recombination mediator activity including S. cerevisiae Rad52, E. coli RecO, and T4 UvsY (Yonesaki and Minagawa 1989;Mortensen et al 1996;Reddy et al 1997;Kantake et al 2002). A remarkable and conserved activity of these proteins is their ability to anneal complementary ssDNA strands that are complexed with cognate ssDNA-binding protein (RPA, SSB, or Gp32, respectively) (Shinohara et al 1998;Sugiyama et al 1998;Kantake et al 2002). This activity is important for the second-end capture phases of dHJ and SDSA pathways, as described below.…”