THE SAD1 RAD53 PROTEIN-KINASE CONTROLS MULTIPLE CHECKPOINTS AND DNA DAMAGE-INDUCED TRANSCRIPTION IN YEAST/

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 c onsistent with inappropriate mitotic entry in the presence of unreplic ated DNA, indicating a defect in the S-phase checkpoint. sad1 mutants are additionally defective for the G(1) and G(2) DNA damage checkpoint s, and for DNA damage-induced transcription of RNR2 and RNR3. The tran scriptional response to DNA damage requires activation of the Dun1 pro tein kinase. Activation of Dun1 in response to replication blocks or D NA damage is blocked in sad1 mutants. The HU sensitivity of sad1 mutan ts is suppressed by mutations in CKS1, a subunit of the p34(CDC28) kin ase, further establishing a link between cell cycle progression and le thality. sad1 mutants are allelic to rad53, a radiation-sensitive muta nt. SAD1 encodes an essential protein kinase. The observation that SAD 1 controls three distinct checkpoints suggests a common mechanism for cell cycle arrest at these points, Together, these observations implic ate protein phosphorylation in the cellular response to DNA damage and replication blocks.