THE BUDDING YEAST RAD9 CHECKPOINT PROTEIN IS SUBJECTED TO MEC1 TEL1-DEPENDENT HYPERPHOSPHORYLATION AND INTERACTS WITH RAD53 AFTER DNA-DAMAGE/

The Saccharomyces cerevisiae RAD9 checkpoint gene is required for tran sient cell-cycle arrests and transcriptional induction of DNA repair g enes in response to DNA damage, Polyclonal antibodies raised against t he Rad9 protein recognized several polypeptides in asynchronous cultur es, and in cells arrested in S or G(2)/M phases while a single form wa s observed in G(1)-arrested cells. Treatment with various DNA damaging agents, i,e, UV, ionizing radiation or methyl methane sulfonate, resu lted in the appearance of hypermodified forms of the protein. All modi fications detected during a normal cell cycle and after DNA damage wer e sensitive to phosphatase treatment, indicating that they resulted fr om phosphorylation, Damage-induced hyperphosphorylation of Rad9 correl ated with check-point functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent. In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATE and ATM genes. In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addi tion to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage sp ecificity of the checkpoint genes in this response to DNA damage. Anal ysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.