Inhibition of DNA synthesis prevents mitotic entry through the action of the S-phase checkpoint. We have isolated S-phase arrest-defective {sad) mutants that show lethality in the presence of the DNA synthesis inhibitor hydroxyurea (HU). Several of these mutants show phenotypes consistent with inappropriate mitotic entry in the presence of unreplicated DNA, indicating a defect in the S-phase checkpoint, sadl mutants are additionally defective for the G^ and Gj DNA damage checkpoints, and for

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... damage-induced transcription of RNR2 and RNR3. The transcriptional response to DNA damage requires activation of the Dunl protein kinase. Activation of Dunl in response to replication blocks or DNA damage is blocked in sadl mutants. The HU sensitivity of sadl mutants is suppressed by mutations in CKSl, a subunit of the p34^"*^^* kinase, further establishing a link between cell cycle progression and lethality, sadl mutants are allelic to rad53, a radiation-sensitive mutant. SADl encodes an essential protein kinase. The observation that SADl controls three distinct checkpoints suggests a common mechanism for cell cycle arrest at these points. Together, these observations implicate protein phosphorylation in the cellular response to DNA damage and replication blocks.