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 y east, 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 tim e for DNA repair to take place. Mec1 and Rad53 also slow S phase progressio n in response to DNA alkylation(4), although the mechanism for this and its relative importance in protecting cells from DNA damage have not been dete rmined. Here we show that the DNA-alkylating agent methyl methanesulphonate (MMS) profoundly reduces the rate of DNA replication fork progression; how ever, this moderation does not require Rad53 or Mec1. The accelerated S pha se in checkpoint mutants(4), therefore, is primarily a consequence of inapp ropriate initiation events(5-7). Wild-type cells ultimately complete DNA re plication in the presence of MMS. In contrast, replication forks in checkpo int 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 replica tion fork catastrophe seems to be a primary mechanism by which checkpoints preserve viability in the face of DNA alkylation.