A requirement for recombinational repair in Saccharomyces cerevisiae is caused by DNA replication defects of mec1 mutants

To examine the role of the RAD52 recombinational repair pathway in compensa ting for DNA replication defects in Saccharomyces cerevisiae, we performed a genetic screen to identify mutants that require Rad52p for viability. We isolated 10 mec1 mutations that display synthetic lethality with rad52. The se mutations (designated mec1-srf for synthetic lethality with rad-fifty-tw o) simultaneously cause two types of phenotypes: defects in the checkpoint function of Mec1p and defects in the essential function of Mec1p. Velocity sedimentation in alkaline sucrose gradients revealed that mec1-srf mutants accumulate small single-stranded DNA synthesis intermediates, suggesting th at Mec1p is required for the normal progression of DNA synthesis. sml1 supp ressor mutations suppress both the accumulation of DNA synthesis intermedia tes and the requirement for Rad52p in mec1-srf mutants, but they do not sup press the checkpoint defect in mec1-srf mutants. Thus, it appears to be the DNA replication defects in mec1-srf mutants that cause the requirement for Rad52p. By using hydroxyurea to introduce similar DNA replication defects, we found that single-stranded DNA breaks frequently lead to double-strande d DNA breaks that are not rapidly repaired in rad52 mutants. Taken together , these data suggest that the RAD52 recombinational repair pathway is requi red to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.