Modification of DNA Polymerase Delta by a Novel Mechanism During Replication Stre

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DESCRIPTION (provided by applicant): DNA polymerase delta (Pol d) is a key enzyme that is essential for eukaryotic chromosomal DNA replication. Mammalian Pol d consists of four subunits, all of which are required for its full function in vitro. This proposal is based on the novel discovery that levels of the human Pol d p12 subunit are dramatically depleted after DNA damage by UV and other genotoxic agents that activate the ATR/Chk1 mediated S-phase checkpoint. We have shown that this results in the conversion of Pol d from a tetramer into a trimer, Pol d3. The biochemical properties of pol d3 will be compared to those of the parent enzyme. We will examine its kinetic properties, its abilities to bypass template lesions, and its abilities to act as a proof reading enzyme. Our working hypothesis is that the conversion to Pol d3 prevents Pol d from bypassing template lesions, thereby allowing repair processes to take place. The spatiotemporal aspects of the localization of Pol d3 to DNA damage foci will be studied by immunofluorescence microscopy and laser scanning cytometry after UV damage and compared to the recruitment of other DNA damage proteins. The role of ubiquitination in the depletion of pol d will be studied. The identity of the ubiquitination system will be determined using several different approaches, including siRNA knockdown of candidate ubiquitination proteins, identification of p12 binding proteins, and isolation of E3 ligases that act to ubiquitinate p12 using in vitro assays.