Analysis of a DNA Damage-Inducible Checkpoint Complex

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DESCRIPTION (provided by applicant): The integrity of our genomes is under continual assault from errors generated during replication, metabolically produced reactive oxygen species, spontaneous depurination, and endogenous and exogenous agents that damage DNA. Replication stress and DNA damage activate checkpoint signaling pathways that help maintain gemonic stability by orchestrating DNA repair, gene transcription, and cell cycle arrest. A critical check point signaling pathway is the Atr-Chkl signaling pathway, which is activated by replication stress and other types of DNA damage and plays an important role in maintaining genomic stability. Key partcipants in the Atr-Chkl signaling pathway are Rad9, Husl, and Radl, which form a Rad9- Husl-Radl (911) complex, and are required for optimal Chkl activation as well as other cellular responses to DNA damage. The 911 complex forms a PCNA-like clamp that is loaded around DNA at sites of damage. Although the clamp is loaded around DNA and is required for optimal Chkl activation, the clamp's actual role in Chkl activation and orchestrating other cellular events remains unknown. In Aim 1 of this proposal, we have identified a novel Rad9-interacting protein that binds the C terminus of Rad9, a region required for Chkl activation, and we propose to examine the role of this interacting protein on Rad9 function and Chkl activation. In Aim 2, we describe Rad9B, a new Rad9 paralog, and demonstrate that Rad9, Rad9B, Husl, HuslB (a Husl paralog), and Radl combinatorially assemble into different clamps. We then propose to examine the roles of each subunit in cellular responses to a panel of genotoxic agents to identify whether each 911 subunit has unique and/or overlapping roles. In Aim 3, we show that the 911 complex interacts with a translesion DNA polymerase and we propose experiments to examine the role of 911 subunits in translesion polymerase function. Collectively, these studies will provide new insights into how the 911 complexes direct downstream events that regulate checkpoint activation, cell survival, and genomic stability.