Genotoxicity and Repair of Tobacco-Specific Nitrosamine DNA Adducts

Project Summary Translesion DNA synthesis (TLS) polymerases are critical to cell survival by replacing high fidelity polymerases during roadblocks that occur during DNA repair and replication. One difficulty in elucidating the multiple roles of these polymerases is that it is impossible to identify which polymerase is active in a specific situation. Here we propose a chemical biology approach in which we can measure the activity of DNA polymerase kappa. We have designed and synthesized N2-­benzyl-­2′-­deoxyguanosine and analogs that are highly select toward pol kappa. We have previously shown that in vitro, N2-­benzyl-­GTP reacts with pol κ 105-­fold more efficiently than pol eta, iota, beta, nu, and delta, and in cells, the incorporation of N2-­4-­ethynylbenzyl-­dG into the DNA is dependent on pol kappa. With this tool we will examine the multiple roles of pol kappa in two following specific aims: (1) Determine the role of pol kappa in NER, and (2) determine the role pol kappa plays during S-­phase. In aim 1, we will examine the NER activity of pol kappa with respect to DNA damage, protein-­protein interactions, and the location of the activity in the genome. In aim 2 we will examine pol kappa activity in S-­ phase with respect to bypassing DNA damage and replication of non-­B DNA sequences. In particular we will examine the polymerase switch mechanisms at the replication fork, the role of protein-­protein interactions in activation of pol kappa activity, and the location of pol kappa activity in the genome. Similar techniques will be employed in the two aims. (i) Activity assays will be performed utilizing N2-­4-­ethynylbenzyl-­dG and Click Chemistry to attach a fluorophore. The activity will be analyzed by fluorescence microscopy to examine nuclear/cytoplasmic localization of 4-­ethynylbenzyl-­dG, while flow cytometry will be used to examine cell-­cycle activity. (ii) These two techniques will be combined with mutant-­inactive-­proteins to determine the critical proteins and interactions involved in the activity. (iii) iPOND-­like experiments will be performed to identify proteins associated with the activity in an unbiased manner. (iv) DNA strand fiber assays will be employed to distinguish between the polymerase switch mechanism and post-­gap repair during S-­phase. (v) Next generation sequencing will be utilized to probe the genomic identity of the activity. This proposal is very innovative in creating a new method by which scientists will be able to examine the activity of a single DNA polymerase in a cell. PUBLIC HEALTH RELEVANCE. Differences in activity of DNA polymerase have a major impact on the ability of an individual to respond to DNA damaging agents. This methodology may be used in identifying the susceptibility of individual or organs to carcinogens and the efficacy of DNA damaging chemotherapeutic agents.