Regulation of the DNA damage Response

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DESCRIPTION (provided by applicant): Eukaryotic cells face constant challenges to the integrity of their genome, and sophisticated processes have evolved to respond to DMA damage. Cells arrest cell division, repair damage, and in some cases undergo apoptosis, and failure to carry out these processes can lead to genomic instability and cancer. In fact, defects in these processes form the molecular basis for many cancer-prone disorders. DMA damage tolerance mechanisms are another aspect of the DMA damage response that allow the cell to continue replication in the presence of polymerase-blocking lesions, leaving repair of the damage for a later time. The overall goal of this proposal is to understand the molecular mechanisms that regulate DMA damage tolerance. One form of tolerance involves switching from high-fidelity replicative polymerases to translesion synthesis (TLS) polymerases at the site of damage. This switch is thought to involve monoubiquitination of the replicative sliding clamp PCNA at the stalled replication fork. This form of tolerance is error prone because the TLS polymerases are of lower fidelity than the replicative polymerases. A second, error-free form of DMA damage tolerance also involves the ubiquitination of PCNA. In order to understand the mechanisms regulating DMA damage tolerance and TLS, we will: (1) Determine the mechanism by which single-stranded DMA regulates the ubiquitination of PCNA; (2) Investigate the relationship between PCNA ubiquitination and the switch between replicative and TLS polymerases at the replication fork; and (3) Determine the role of the ATR-mediated checkpoint in TLS and DNA-damage induced mutagenesis. We will explore these questions using a cell free system derived from the eggs of Xenopus laevis that recapitulates many aspects of DNA damage tolerance. These extracts provide a tractable system for analyzing the complex pathways regulating TLS and damage repair. The experiments described in this proposal should help us understand the mechanisms used by cells to regulate DNA damage tolerance process. Because defects in these processes lead to loss of genomic integrity, cancer and cell death, these studies will provide valuable insight into how cancer develops and may ultimately point the way to new approaches for the treatment or detection of cancer.