Requirement for End-Joining and Checkpoint Functions, but Not RAD52 -Mediated Recombination, after Eco RI Endonuclease Cleavage of Saccharomyces cerevisiae DNA

Abstract

ABSTRACT RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae . Analysis of Eco RI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G 1 -phase Rad + cells. Continuous Eco RI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G 2 phase. Surprisingly, rad52 mutants were not more sensitive to Eco RI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when Eco RI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52 -mediated recombination and by recombination-independent complementary end joining in yeast cells.