This renewal revision proposal focuses on a key problem in radiation biology, elucidating molecular determinants of checkpoint and repair responses to G1 DNA damage. Our model system is the budding yeast S. cerevisiae. We have studied the cell cycle delay induced by ionizing radiation in G1 and shown that a bona fide checkpoint response arrests irradiated yeast cells before DNA replication until repair is initiated. In dissecting the signal transduction pathway in G1, we have discovered novel roles for chromatin modification in signaling and DNA damage tolerance. Importantly, the chromatin modifiers, checkpoint regulators, cell cycle machinery and DNA repair mechanisms are all broadly conserved among eukaryotes. Thus, understanding yeast DNA damage response pathways promises broad insight into homologous mechanisms in human cells. Preliminary studies. We have pursued genetic determinants of the G1 checkpoint, revealing novel effectors that link DNA damage to checkpoint signaling and cell cycle arrest. Our recent studies have linked activation of Rad9 and Rad53 to Rad9 assembly onto chromatin at sites of DNA damage, mediated by recognition of two specific chromatin marks, histone H3 dimethyl-K79 and histone H2A phospho-S129. We have also discovered links between G1 checkpoint response and histone H4 acetylation and histone H2AZ deposition. These data suggest a key role for chromatin in checkpoint activation, persistence, recovery and adaptation. Recent data suggest that assembly of a Rad9 signaling domain rather than formation of ssDNA is necessary and sufficient for G1 checkpoint signaling.
Specific Aims. During this project period, we intend to 1) dissect the determinants of Rad9 recruitment and function at G1 DSBs, 2) characterize roles for NuA4-dependent histone acetylation in persistence of the Rad9-dependent G1 checkpoint arrest and 3) determine the role of single strand DNA formation in G1 checkpoint response. Significance. Proper checkpoint and/or DNA repair responses are often lacking in cancer cells, contributing to genomic instability. The powerful tools available in yeast will facilitate dissection of protein-protein interactions between conserved checkpoint factors and chromatin that determine G1 checkpoint response. These studies may define highly conserved molecular targets that will allow modulation of ionizing radiation effects on cancer and normal tissue.
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