The Mec1 and Rad53 protein kinases are essential for budding yeast cell viability and are also required to activate the S-phase checkpoint, which supports DNA replication under stress conditions. Whether these two functions are related to each other remains to be determined, and the nature of the replication stress-dependent lethality of mec1 and rad53 mutants is still unclear. We show here that a decrease in cyclin-dependent kinase 1 (Cdk1) activity alleviates the lethal effects of mec1 and rad53 mutations both in the absence and in the presence of replication stress, indicating that the execution of a certain Cdk1-mediated event(s) is detrimental in the absence of Mec1 and Rad53. This lethality involves Cdk1 functions in both G 1 and mitosis. In fact, delaying either the G 1 /S transition or spindle elongation in mec1 and rad53 mutants allows their survival both after exposure to hydroxyurea and under unperturbed conditions. Altogether, our studies indicate that inappropriate entry into S phase and segregation of incompletely replicated chromosomes contribute to cell death when the S-phase checkpoint is not functional. Moreover, these findings suggest that the essential function of Mec1 and Rad53 is not necessarily separated from the function of these kinases in supporting DNA synthesis under stress conditions. T he integrity of the genome is constantly challenged by DNA damage caused by environmental and intracellular factors. Aberrant DNA replication is a major source of mutations and chromosome rearrangements that can lead to cancer and other diseases in metazoans (reviewed in reference 23). Replication fork progression can be hampered by exogenous or endogenous DNA damage. Furthermore, faithful replication depends on a balanced supply of deoxyribonucleotides (deoxyribonucleoside triphosphates [dNTPs]), whose levels are maintained during S-phase through the action of the ribonucleotide reductase (RNR) activity that converts the ribonucleotides to dNTPs (reviewed in reference 37). Indeed, replication fork pausing can be experimentally induced by genotoxic drugs, such as hydroxyurea (HU), which reduces dNTP pools by inhibiting RNR activity, and the DNA alkylating agent methyl methanesulfonate (MMS) that causes intra-S damage.Eukaryotic cells respond to replication interference through a complex signal-transduction pathway, known as the S-phase checkpoint, whose key players in the budding yeast Saccharomyces cerevisiae are the Mec1 and Rad53 kinases (reviewed in references 5 and 63). Mec1, together with its interacting protein Ddc2, is recruited to stalled forks, where it activates the effector kinase Rad53. Both kinases act in various ways to respond to replication interference. They are needed to complete DNA replication after exposure to HU or MMS (16, 55) by maintaining the integrity and/or activity of the replication forks (11,15,26,34). Furthermore, they stimulate dNTP production (1, 25, 64, 65) and the transcription of several MCB binding factor (MBF)-regulated genes that are involved in DNA replication...