An allele of RFA1 suppresses RAD52-dependent double-strand break repair in Saccharomyces cerevisae

Abstract

An allele of RFA1, the largest subunit of the single-stranded DNA- binding complex RP-A, was identified as a suppressor of decreased direct- repeat recombination in rad1 rad52 double mutants. In this study, we used two LEU2 direct-repeat assays to investigate the mechanism by which the rfa1- D228Y allele increases recombination. We found that both intrachromatid and sister chromatid recombination are stimulated in rfa1-D228Y strains. In a rad1 rad52 background, however, the majority of the increased recombination is caused by stimulation of deletion events by an intrachromatid recombination mechanism that is likely to be single-strand annealing. Studies in which an HO endonuclease cut was introduced between the two leu2 copies indicate that the rfa1-D228Y mutation partially suppresses the rad52 defect in recovering recombination products. Furthermore, molecular analysis of processing and product formation kinetics reveals that, in a rad52 background, the rfa1-D228Y mutation results in increased levels of recombinant products and the disappearance of large single-stranded intermediates characteristic of rad52 strains. On the basis of these results, we propose that in the absence of wild-type Rad52, the interaction of RP-A with single-stranded DNA inhibits strand annealing, and that this inhibition is overcome by the rfa1-D228Y mutation.