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 compensating 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. These mutations (designated mec1-srf for synthetic lethality with rad-fifty-two) 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 that Mec1p is required for the normal progression of DNA synthesis. sml1 suppressor mutations suppress both the accumulation of DNA synthesis intermediates and the requirement for Rad52p in mec1-srf mutants, but they do not suppress 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-stranded DNA breaks that are not rapidly repaired in rad52 mutants. Taken together, these data suggest that the RAD52 recombinational repair pathway is required to prevent or repair double-stranded DNA breaks caused by defective DNA replication in mec1-srf mutants.
Merrill
B J
BJ
Department of Pharmacology, Division of Cellular and Molecular Medicine, Center for Molecular Genetics, University of California, San Diego, California 92093-0651, USA.
Holm
C
C
D004260:DNA Repair / Q000235:genetics*; D004261:DNA Replication / Q000235:genetics*; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D004277:DNA, Single-Stranded / Q000096:biosynthesis / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D005838:Genotype; D006918:Hydroxyurea / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D016296:Mutagenesis; D017346:Protein Serine-Threonine Kinases; D051721:Rad52 DNA Repair and Recombination Protein; D011995:Recombination, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D047908:Intracellular Signaling Peptides and Proteins; C497197:RAD52 protein, S cerevisiae; D051721:Rad52 DNA Repair and Recombination Protein; D029701:Saccharomyces cerevisiae Proteins; C088937:MEC1 protein, S cerevisiae; D017346:Protein Serine-Threonine Kinases; D006918:Hydroxyurea