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Your Environment. Your Health.

GENOTOXICITY AND REPAIR OF TOBACCO-SPECIFIC NITROSAMINE DNA ADDUCTS

Export to Word (http://www.niehs.nih.gov//portfolio/index.cfm/portfolio/grantdetail/grant_number/R01ES021762/format/word)
Principal Investigator: Spratt, Thomas E.
Institute Receiving Award Pennsylvania State Univ Hershey Med Ctr
Location Hershey, PA
Grant Number R01ES021762
Funding Organization National Institute of Environmental Health Sciences
Award Funding Period 20 Jul 2012 to 31 May 2023
DESCRIPTION (provided by applicant): 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.     
Science Code(s)/Area of Science(s) Primary: 03 - Carcinogenesis/Cell Transformation
Publications See publications associated with this Grant.
Program Officer Frederick Tyson
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