A single double-strand break (DSB) in a chromosome of the model organism, Saccharomyces cerevisiae, is sufficient to trigger the DNA damage checkpoint, causing cells to arrest prior to mitosis and allowing them more time to repair the lesion. The response to a DSB is controlled by two protein kinases, Mecl and Tell, which are the budding yeast homologues of the key checkpoint protein kinases ATR and ATM in mammalian cells. A cascade of protein kinases results in G2/M arrest. The response to a DSB includes extensive chromatin modification around the break by phosphorylation of histone H2AX and concomitant recruitment of cohesin proteins around the DSB site. These responses can be studied by synchronously inducing a single, unrepaired DSB in all cells in the population by the HO endonuclease. The goals of this grant are first to understand the sequence of events, soon after the DSB is created, that lead to the establishment of the DNA damage checkpoint and, second, to understand how the checkpoint is maintained. The role of the Cdkl cell cycle kinase, Cdc28, in controlling 5' to 3' resection of DSB ends is of particular interest, as is the role of the single-strand DNA binding complex, RPA. A further goal is to understand both how the phsophorylation of H2AX (y-H2AX) spreads over a 50-kb region and how Y-H2AX is both maintained and how it is removed when damage is repaired. Finally, this proposal will continue to address the two ways in which cells can resume cell cycle progression after prolonged arrest. In adaptation, cells resume mitosis in spite of the continued presence of a broken chromosome. The role of the spindle checkpoint proteins Mad2-Bubl in maintaining the arrest of several adaptation-defective mutations will be pursued along with further analysis of adaptation-defective mutations. In contrast, cells that repair the DSB after activating the checkpoint undergo recovery, which can be prevented by at least two recovery-defective mutations. The identification of additional recovery-defective mutations will be undertaken.
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