Arrest, Recovery, and Adaptation from DNA Damage

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DESCRIPTION (provided by applicant): The induction and maintenance of the DNA damage response (DDR) plays a central role in protecting genome integrity, allowing cells more time to repair chromosomal double-strand breaks (DSBs) or to eliminate cells that fail to accomplish repair. In budding yeast, creation of a single DSB is sufficient to activate the Mec1 (ATR) and Tel1 (ATM) checkpoint protein kinases that both modify chromatin around the DSB and trigger a cascade of phosphorylations that result in the arrest of cell cycle progression prior to anaphase. This proposal investigates three major aspects of the checkpoint response: the activation and maintenance of the checkpoint, the modification of chromatin by formation of g-H2AX and - as we have discovered - g-H2B, and a synergistic interaction of the DDR with the Spindle Assembly Checkpoint that is responsible for the prolongation of the arrested state. In the first Aim, how Mec1-dependent G2/M cell cycle arrest is maintained will be investigated by studying newly-discovered mutations of phosphorylation sites on Mec1 that are required to turn the checkpoint off; consequently cells fail to adapt and resume mitosis after 12-15 h when there is no DSB repair. How Mec1 monitors the presence of DNA damage will be examined, focusing on the relation between 5' to 3' resection of the DSB ends by exonucleases and maintenance of the checkpoint. Previous results suggest that the ability of cells to resume cell cycle progression after a DSB is repaired depends on how long cells have been arrested, which may correlate with the strength of the checkpoint signal. The strength of checkpoint signaling will be examined by augmenting the response by artificially tethering the Ddc1 and Ddc2 (ATRIP) activators of Mec1 to elicit a response independent of a DSB. A second Aim will focus on the Mec1- and Tel1-dependent phosphorylation of the C-terminus of histone H2B (g-H2B) that resembles the well-studied g- H2AX modification but appears to have separate roles in DNA damage signaling and repair. A third Aim will focus on the synergy between the DNA damage checkpoint and the Spindle Assembly Checkpoint, following up our findings that deleting Mad2 shortens checkpoint arrest in wild type cells and suppresses the permanent arrest of adaptation-defective mutations and that this suppression can be mimicked by deleting the centromere on the chromosome suffering an unrepaired DSB.