No abstract

Rad51, Rad52, and RPA play central roles in homologous DNA recombination. Rad51 mediates DNA strand exchange, a key reaction in DNA recombination. Rad52 has two distinct activities: to recruit Rad51 onto single-strand (ss)DNA that is complexed with the ssDNA-binding protein, RPA, and to anneal complementary ssDNA complexed with RPA. Here, we report that Rad52 promotes annealing of the ssDNA strand that is displaced by DNA strand exchange by Rad51 and RPA, to a second ssDNA strand. An RPA that is recombination-deficient (RPA(rfa1-t11)) failed to support annealing, explaining its in vivo phenotype. Escherichia coli RecO and SSB proteins, which are functional homologues of Rad52 and RPA, also facilitated the same reaction, demonstrating its conserved nature. We also demonstrate that the two activities of Rad52, recruiting Rad51 and annealing DNA, are coordinated in DNA strand exchange and second ssDNA capture.

Replication protein-A (RPA) is involved in many processes of DNA metabolism, including DNA replication, repair, and recombination. Cells carrying a mutation in the largest subunit of RPA (rfa1-t11: K45E) have defects in meiotic recombination, mating-type switching, and survival after DNA damage caused by UV and methyl methanesulfonate, as well as increased genome instability; however, this mutant has no significant defect in DNA replication. We purified the RPA heterotrimer containing the rfa1-t11 substitution (RPA(rfa1-t11)). This mutant RPA binds single-stranded DNA (ssDNA) with the same site size, and the RPA(rfa1-t11)⅐ssDNA complex shows a similar sensitivity to disruption by salt as the wild-type RPA⅐ssDNA complex. RPA(rfa1-t11) stimulates DNA strand exchange, provided that the Rad51 protein⅐ssDNA nucleoprotein complex is assembled prior to introduction of the mutant RPA. However, RPA(rfa1-t11) is displaced from ssDNA by Rad51 protein more slowly than wild-type RPA and, as a consequence, Rad51 protein-mediated DNA strand exchange is inhibited when the ssDNA is in a complex with RPA(rfa1-t11). Rad52 protein can stimulate displacement of RPA(rfa1-t11) from ssDNA by Rad51 protein, but the rate of displacement remains slow compared with wild-type RPA. These in vitro results suggest that, in vivo, RPA is bound to ssDNA prior to Rad51 protein and that RPA displacement by Rad51 protein is a critical step in homologous recombination, which is impaired in the rfa1-t11 mutation.

In Saccharomyces cerevisiae, Rad52 protein plays an essential role in the repair of DNA double-stranded breaks (DSBs). Rad52 and its orthologs possess the unique capacity to anneal single-stranded DNA (ssDNA) complexed with its cognate ssDNA-binding protein, RPA. This annealing activity is used in multiple mechanisms of DSB repair: single-stranded annealing, synthesis-dependent strand annealing, and crossover formation. Here we report that the S. cerevisiae DNA strand exchange protein, Rad51, prevents Rad52-mediated annealing of complementary ssDNA. Efficient inhibition is ATP-dependent and involves a specific interaction between Rad51 and Rad52. Free Rad51 can limit DNA annealing by Rad52, but the Rad51 nucleoprotein filament is even more effective. We also discovered that the budding yeast Rad52 paralog, Rad59 protein, partially restores Rad52-dependent DNA annealing in the presence of Rad51, suggesting that Rad52 and Rad59 function coordinately to enhance recombinational DNA repair either by directing the processed DSBs to repair by DNA strand annealing or by promoting second end capture to form a double Holliday junction. This regulation of Rad52-mediated annealing suggests a control function for Rad51 in deciding the recombination path taken for a processed DNA break; the ssDNA can be directed to either Rad51-mediated DNA strand invasion or to Rad52-mediated DNA annealing. This channeling determines the nature of the subsequent repair process and is consistent with the observed competition between these pathways in vivo.In Saccharomyces cerevisiae, the repair of DNA doublestranded breaks (DSBs) 3 is accomplished primarily by homologous recombination. Genes from the RAD52 epistasis group, including RAD50, RAD51, RAD52, RAD54, RAD55-57, RAD59, MRE11, XRS2, and RFA1, are responsible for this recombination-dependent DSB repair (1, 2). To repair a DSB, the DNA end is first processed to produce a 3Ј single-stranded tailed duplex DNA. The ssDNA is then channeled into one of the many recombinational pathways, which can be further categorized into RAD51-dependent and -independent pathways.RAD51-dependent recombination requires functions of RAD51, RAD52, RAD54, RAD55, RAD57, and RFA1 for efficient DNA repair (1). The central step of this pathway involves DNA strand invasion of homologous duplex DNA by the processed DSB complexed with Rad51 protein (3). DNA replication from the invading 3Ј-end replaces the genetic information missing from the broken dsDNA, and subsequent DNA pairing and resolution steps restore DNA integrity.RAD51-independent recombination requires RAD52 (4) and is enhanced by RAD59 (5-7). In addition to the recombination genes MRE11, RAD50, XRS2, RAD52, and RAD59 (6,8), this pathway also depends on MSH2, MSH3 (6, 9), RAD1, and RAD10 (10, 11). This RAD51-independent recombination is most easily assayed as DSB repair occurring between directly repeated sequences, by a mechanism termed single-stranded annealing (SSA) (8, 12). As implied, the central step of SSA is annealing between complementary singl...