Repair of endonuclease-induced double-strand breaks, in Saccharomyces cervisiae: Essential role for genes associated with nonhomologous end-joining

Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous e nd-joining (NHEJ) is not well characterized in the yeast Saccharomyces cere visiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Ga lactose-induced expression of EcoRI endonuclease in rad50 nrel1, or xrs2 mu tants, which are deficient in plasmid DSB end-joining and some forms of rec ombination, resulted in G2 arrest and rapid cell killing. Endonuclease synt hesis also produced moderate cell killing in sir4 strains. In contrast, Eco RI caused prolonged cell-cycle arrest of recombination-defective rad51, rad 52, rad54, md55, and rad57 mutants, bur cells remained viable. Cell-cycle p rogression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phen otypic responses of additional mutants, including exo1, srs2, rad5, and rdh 54 strains, suggest roles in recombinational repair, but not in NHEJ. Inter estingly, the rapid cell killing in haploid rad50 and mrel1 strains was lar gely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cyc le in the diploid mutants. These results demonstrate essential but separabl e roles for NHEJ pathway genes in the repair of chromosomal DSBs that are s tructurally similar to those occurring during cellular development.