Rad52 Protein Has a Second Stimulatory Role in DNA Strand Exchange That Complements Replication Protein-A Function

Abstract

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...

Heteroduplex Joint Formation by a Stoichiometric Complex of Rad51 and Rad52 of Saccharomyces cerevisiae

“…1E), unlike the observations reported by Sugiyama et al (34). It was reported that Rad51 required 30 -60 mM KCl for heteroduplex joint formation between circular single-stranded DNA and linear double-stranded DNA (43), and that heteroduplex joint formation by Rad52 was stimulated by a lower concentration of KCl, but inhibited by KCl at 50 mM (33). The formation of D-loops by Rad51-Rad52 was decreased 1-3) or RecA (lanes 4 -6) were cleaved by NheI outside the sequence homologous to the pNS11 single-stranded DNA fragments and were incubated at 37°C.…”

“…In this reaction, Rad52 facilitates the binding of Rad51 to RPA-coated singlestranded DNA and displacement of the RPA (34), and stabilizes the binding of Rad51 to single-stranded DNA (33). In this study, by use of a short single-stranded DNA fragment as a substrate, the requirement of RPA for the reaction was eliminated.…”

“…RPA is required for this reaction, but the binding of Rad51 to single-stranded DNA is inhibited by previously bound RPA on the single-stranded DNA. Thus, it is assumed that Rad52 promotes the loading of Rad51 onto the RPA-coated circular single-stranded DNA and stabilizes Rad51 binding to the single-stranded DNA (29,33,34). By the use of a short single-stranded DNA that bypasses the requirement of RPA for heteroduplex joint formation, we now show that Rad52 plays an additional important function in the Rad51-promoted formation of D-loops.…”

“…The molar concentration of Rad52 in fraction V was determined from the extinction coefficient reported by New et al (31). The purified Rad52 exhibited annealing activity between complementary single-stranded DNAs (data not shown) and stimulated (data not shown) the strand exchange by Rad51 and RPA, at the same stoichiometry of Rad52 as described by New and Kowalczykowski (33).…”

“…This requirement is fulfilled by RPA, which unfolds secondary structures in single-stranded DNA, but the preceding binding of RPA prevents the binding of Rad51. Rad52 is required for loading of Rad51 onto RPA-coated single-stranded DNA (29,33,34), and for the stabilization of the Rad51-single-stranded DNA complex (called the presynaptic filament (33)). The cooperative nature of Rad51 binding to single-stranded DNA could explain why only a small amount of Rad52 is required for these functions.…”