Skin cancer is the most prevalent form of cancer. The key event in uv- induced skin cancer induction is the formation of a mutation that results from DNA synthesis past a DNA photo product by a DNA pommies. The overall aim of this proposal is to understand how and why some polymers are able to synthesize past DNA photo products while others are not, and the mechanisms by which polymerase bypass DNA photo products while others and select the nucleotide that are inserted opposite them. We plan to approach this problem by a combined chemical, physical, enzymatic, and biological approach, which relies our ability to synthesize photo product- containing DNA substrates for detailed study. Specifically, we propose to develop new routes for the synthesis of photo product and analogs which can test hypotheses regarding nucleotide insertion selectivity opposite DNA photo products. The structure, H-bonding, proton exchange rates, dynamics, and thermodynamics of photoduct- containing duplexes will be studied to further understand the molecular basis for nucleotide insertion preferences opposite DNA photo products by polymerase. To better understand the difference between replicative and DNA damage bypass polymerase, we will carry out a detailed kinetic and physical analysis of the individual steps in the bypass of DNA photo products. We will investigate the role of sequence context on bypass rates and nucleotide insertion selectivity opposite DNA photo products by DNA polymerase, and the extent to which polymerase can switch a primer from one temple late to another at replication fork. We will investigate the molecular basis for the A-rule in bypass of DNA photo products, and the effect of sequence context on bypass rate and nucleotide insertion selectivity. A number of chemical and physical agents will be investigating as probes for how DNA polymerase bind to native and photo damaged template primers at various steps in the elongation process. Replication of SV40 vectors containing photo products and analogs will also be studied in normal and XPV human cell extracts as a function of leading and lagging strand synthesis, and sequence context.
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