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Genotoxicity and Repair of Tobacco-Specific Nitrosamine DNA Adducts

烟草特异性亚硝胺 DNA 加合物的基因毒性和修复

基本信息

批准号：
10626468
负责人：
Thomas E Spratt
金额：
$ 1.28万
依托单位：
PENNSYLVANIA STATE UNIV HERSHEY MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-20 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10626468
关键词：
Active Sites Binding Sites Biological Assay Biology Bypass Carcinogens Cell Cycle Cell Survival Cells Cellular biology Chemicals Chemistry DNA DNA Adducts DNA Crosslinking Agent DNA Damage DNA Repair DNA Sequence DNA biosynthesis DNA replication fork DNA-Directed DNA Polymerase Databases Deoxyguanosine Eukaryotic Cell Fiber Flow Cytometry Fluorescence Microscopy Genome Genomics Goals Guanosine Triphosphate In Vitro Individual Kinetics Knowledge Location Malignant Neoplasms Measures Methodology Methods Nitrosamines Nuclear Nucleotide Excision Repair Nucleotides Organ Play Polymerase Positioning Attribute Predisposition Process Proteins Regulation Research Personnel Risk Role S phase Scientist Specificity Techniques Technology Testing Time Tobacco Tobacco-Related Carcinoma adduct analog chemotherapeutic agent crosslink deoxyguanosine triphosphate design experimental study fluorophore genotoxicity innovation mutant next generation sequencing prevent protein protein interaction public health relevance repaired response tool

项目摘要

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.

项目总结