Thursday, November 21, 2024
11/21/2024
Project Summary Replication protein A (RPA) is required for nearly every DNA repair and replication process. RPA binds single- stranded DNA and interacts with dozens of proteins at sites of DNA maintenance. We are interested in the protein complexes that form between RPA and other proteins when performing specific DNA repair tasks. The current project examines RPA’s role as a central scaffold in uracil base excision repair. Our approach is to manipulate the binding of RPA to Uracil DNA Glycosylase (UNG2) to examine the relevance of their interaction. We have developed a strategy to covalently tether together RPA and UNG2 to form mini DNA repair complexes that resemble the architecture of the two proteins when they interact in cells. Our Preliminary Data demonstrates our success at forming RPA-UNG2 protein complexes in both cellular and recombinant systems. This allows us to definitively determine how RPA functions as part of a protein complex with UNG2, and complementary studies examine the effects of weakening their association. Specifically, Aim 1 uses purified RPA and UNG2 constructs to examine the activity of the proteins alone or as part of a complex. Binding and enzymatic experiments using synthetic DNA substrates will determine the structural nature of their substrates in vivo. We focus specifically on the action of RPA and UNG2 at ssDNA-dsDNA junctions, their known substrates, by preparing uracilated fork-like DNA structures that resemble replication forks found in the nucleus. Aim 2 examines the RPA-UNG2 protein complex in human colorectal cells under conditions of uracil stress that are induced by commonly used chemotherapeutic agents. The strength of RPA’s interaction with UNG2 will be controlled and correlated with UNG2’s uracil excision efficiency at the replication fork. Finally, additional cellular proteins that associate with RPA and UNG2 will be identified. The function of these proteins will be examined in the context of multi-protein complexes that form during uracil base excision repair. The targeted scope of this project examining RPA in base excision repair will facilitate our methodological development, which will be widely adaptable for examining RPA-containing protein complexes in other DNA repair processes.
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