The primary goals of this proposal are to understand the activity and the regulation of human DNA polymerase eta (pol eta) in the context of its response to anticancer therapeutic agents. Pol eta is the key enzyme that replicates through UV irradiation-induced DNA damage. Genetic defects in pol eta result in Xeroderma Pigmentosum Variant (XP-V). XP-V patients are highly prone to cancer development, and cells derived from XP-V patients are hypermutable by UV irradiation. Biochemical studies have shown that pol eta can also manage different DNA lesions introduced by AraC, gemcitabine (dFdCyd), and cisplatin. Given that pol eta is expressed in many different tissues and that its role in tissues other than skin is not understood, it is necessary to further our understanding of the role of pol eta when cells are being challenged with these therapeutic agents. Although pol eta is important for cell survival, it is a low fidelity mutagenic enzyme. Therefore, the regulation of polymerase switching between pol eta and high fidelity replicative polymerases is critical for a proper balance between cell survival and preventing mutagenesis. We observe that pol eta deficient cells are more sensitive to nucleoside analogs AraC, dFdC, and cisplatin. Difference in cellular sensitivity may arise from the ability of pol eta to extend or bypass AraC or dFdC. In addition to their value as therapeutics, AraC and dFdC possess subtle structural differences that make them powerful tools for studying the mechanistic properties of pol eta, such as of its preference for different nucleotides during elongation. Therefore, we will characterize the interactions between nucleoside analogs and pol eta and identify key active site residues that confer upon it low-fidelity status at both biochemical and cellular levels. We also observed that the level of phosphorylated pol eta increased after UV irradiation, which implies that the in vivo activity of pol eta is regulated by phosphorylation. Therefore, we are proposing to explore the impact of phosphorylation on cellular pol eta activity in response to UV irradiation and therapeutic agents.
The specific aims are: 1. Characterize the biochemical properties of pol eta on nucleoside analogs. 2. Examine the relationship between pol eta and nucleoside analogs in cells. 3. Study the molecular mechanism that regulates the activity of pol eta in response to gemcitabine and cisplatin combination treatment. The project promises to broaden our understanding of pol eta, from its basic mechanism of action to its regulation. The information obtained will provide new insights into the cellular responses to UV irradiation and therapeutic DNA damaging agents at both molecular and cellular levels. ? ? ?
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