Studies of the interaction between Rad52 protein and the yeast single-stranded DNA binding protein RPA.

The yeast single-stranded DNA binding protein replication protein A (RPA) is a multisubunit complex containing three polypeptides of 70, 30, and 14 kDa. This heterotrimeric structure is conserved across all eukaryotic species where the protein is found. In Saccharomyces cerevisiae, deletion of any one of the three subunits is lethal (3), a reflection of the critical role the protein complex plays in DNA replication. RPA’s role in yeast is not limited to replication, however; it participates in repair and recombination as well. RPA’s involvement in yeast recombination is revealed in biochemical studies of Rad51, which show that RPA is required for the Rad51-catalyzed formation of both joint molecules and fully exchanged products from single-stranded circular DNA and linear double-stranded DNA with an overhanging complementary end (31, 38, 39). Genetic analysis of yeast has underscored the importance of RPA in recombination, as mutations in the gene RFA1, encoding the large (70-kDa) subunit, affect recombination ability (8, 24, 37). One of these mutations, the rfa1-44 allele, results in a 390-fold reduction in a recombination assay that is based on the recombinational repair of HO-endonuclease-induced double-stranded breaks (DSBs) (8). In fact, the rfa1-44 mutant strain is approximately as deficient in its ability to repair a DSB—whether induced by the action of HO endonuclease or by exposure to X rays—as the rad55 and rad57 mutants tested (8, 13, 14). A further indication of the reduced recombinational activity of the rfa1-44 mutant is the 25-fold reduction in the sporulation efficiency and spore viability of the strain compared to those of the wild type (8). The rfa1-44 mutant shows moderate UV radiation sensitivity as well (8). Interestingly, this mutation does not appear to have an effect on DNA replication, as the [3H]uracil incorporation and growth rates of the rfa1-44 mutant and wild-type strains are not substantially different (data not shown). Accordingly, we have previously suggested that the rfa1-44 mutation is a separation-of-function allele (8). Earlier evidence stemming from the experiments on the recombination and repair defects of the rfa1-44 mutant indicated the possibility of an interaction between RFA1 and RAD52. First, the rfa1-44 mutant’s recombination and repair defects are suppressible by dose-dependent overexpression of RAD52 (8), indicative of a genetic interaction between RAD52 and RFA1. In addition, an allele-specific genetic interaction between rfa1-44 and one of the rad52 mutants in our collection was observed. Briefly, a diploid strain carrying both the rfa1-44 and rad52-34 mutations (as well as the corresponding wild-type copies of the genes) is only partially complemented with respect to X-ray survival (8). Diploids carrying both the rfa1-44 mutation and any of the other rad52 mutations in our collection show the expected full complementation (7, 8). Given these genetic interactions, we surmised that the RPA and Rad52 proteins might interact physically as well. Such a direct interaction between the mammalian forms of Rad52 and RPA has been demonstrated (32). A number of proteins involved in yeast DSB repair appear to function as a complex (4, 14, 17, 18, 25, 27, 36) that has been dubbed a “recombinosome” (8). Relying on earlier successes in which interactions between Rad52 and Rad51 were detected by using the yeast two-hybrid system (6), we used the same approach to search for interactions between Rad52 and the individual subunits of RPA. Additionally, we used the same assay to examine interactions between two Rad52 mutant proteins, Rad52-34 and Rad52-38 (8), each of whose mutations reside in the region believed to be responsible for Rad52’s DNA binding activity (28). Our results indicate that these mutations affect the ability of Rad52 to interact with Rad51 and Rfa1 but do not affect the interaction with Rfa2. We suggest a model in which DNA binding plays a role in facilitating the protein interactions that define the putative recombinosome.