ANALYSIS OF A DNA DAMAGE INDUCIBLE CHECKPOINT COMPLEX

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Cells continuously experience DNA damage caused by replication errors, metabolically induced oxidative damage, and exogenous mutagens. DNA damage triggers orchestrated cellular responses that include mobilization of repair machinery and activation of cell cycle checkpoints, which arrest cells in G1 and G2/M and slow progression through S phase. Genetic and pharmacologic data demonstrate that in organisms as diverse as yeast and humans, disruption of checkpoint function leads to genetic instability, which correlates with acquisition of oncogenic mutations in mammals. Genetic studies in yeast show that inactivation of checkpoint genes prevents DNA damage-induced cell cycle arrest and sensitizes the yeast to genotoxins. Although many of the yeast checkpoint genes have been identified, our understanding of DNA damage-induced checkpoint activation in mammals has lagged behind. To identify potential regulators of the mammalian DNA- damage response, we cloned hRad1, hRad9, and hHus1, which are human homologs of the S. pombe checkpoint genes, rad1, rad9, and hus1, and we initiated a study to examine the biochemical and cellular functions of these proteins in mammals. Our results demonstrate that these proteins form a complex in human cells. Two members of the complex, hRad9 and hRad1, are phosphorylated and associated with chromatin in response to DNA damage, thus demonstrating that these proteins form a damage-responsive checkpoint complex. Moreover, we provide new data demonstrating that DNA damage provokes a redistribution of hRad9 into nuclear foci. We now propose to extend these findings and define the biochemical functions of hRad9 and identify its roles in cellular checkpoint responses. The Specific Aims of the project are to: 1) determine whether nuclear retention of hRad9 after DNA damage reflects a biochemical sensing mechanism for damaged DNA; 2) analyze the role of hRad9 in checkpoint signaling activation and cell cycle arrest following DNA damage; 3) identify the basal and DNA damage-induced hRad9 phosphorylation sites and determine what role(s) they play in mediating interactions with the hRad1/hHus1 1 heterodimer and in checkpoint activation. Collectively, these studies will reveal novel insights into the pathways that recognize DNA damage and activate intracellular signaling pathways that regulate cell cycle arrest and maintain genome integrity. Additionally, understanding the molecular details of this pathway will identify novel targets to sensitize cells to therapeutic DNA-damaging agents, such as ionizing radiation and anti-tumor chemotherapeutics.