Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint

The checkpoint kinase proteins Mec1 and Rad53 are required in the budding yeast, Saccharomyces cerevisiae, to maintain cell viability in the presence of drugs causing damage to DNA or arrest of DNA replication forks(1-3). It is thought that they act by inhibiting cell cycle progression, allowing time for DNA repair to take place. Mec1 and Rad53 also slow S phase progression in response to DNA alkylation(4), although the mechanism for this and its relative importance in protecting cells from DNA damage have not been determined. Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; however, this moderation does not require Rad53 or Mec1. The accelerated S phase in checkpoint mutants(4), therefore, is primarily a consequence of inappropriate initiation events(5-7). Wild-type cells ultimately complete DNA replication in the presence of MMS. In contrast, replication forks in checkpoint mutants collapse irreversibly at high rates. Moreover, the cytotoxicity of MMS in checkpoint mutants occurs specifically when cells are allowed to enter S phase with DNA damage. Thus, preventing damage-induced DNA replication fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation.