RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.

Studies in the budding yeast, Saccharomyces cerevisiae, have demonstrated that a substantial fraction of double-strand break repair following acute radiation exposure involves homologous recombination between repetitive genomic elements. We have previously described an assay in S. cerevisiae that allows us to model how repair of multiple breaks leads to the formation of chromosomal translocations by single-strand annealing (SSA) and found that Rad59, a paralog of the single-stranded DNA annealing protein Rad52, is critically important in this process. We have constructed several rad59 missense alleles to study its function more closely. Characterization of these mutants revealed proportional defects in both translocation formation and spontaneous direct-repeat recombination, which is also thought to occur by SSA. Combining the rad59 missense alleles with a null allele of RAD1, which encodes a subunit of a nuclease required for the removal of non-homologous tails from annealed intermediates, substantially suppressed the low frequency of translocations observed in rad1-null single mutants. These data suggest that at least one role of Rad59 in translocation formation by SSA is supporting the machinery required for cleavage of non-homologous tails.