Cells throughout the phylogenetic tree respond to DNA damaging agents by modulating gene expression, and in some cases this is due to alterations at the level of transcription initiation. The precise mechanisms of regulation of transcription are central to our ability, in the future, to manipulate cells with pharmacological and other agents to enhance cell resistance to DNA damaging materials. The core promoter for the small mammalian DNA repair enzyme DNA beta-polymerase (beta-pol) presents an ideal model system in which to study transcriptional aspects of the DNA damage response mechanism, and also an ideal system for study of basic mechanisms of transcription initiation for a mammalian DNA repair gene. The transfected human beta-pol """"""""core promoter"""""""", defined as 114 nucleotides 5' of the major transcription initiation site, shows a strong transcriptional response to treatment of cells with the DNA methylating agent MNNG. This response is mediated through the single ATF/CRE-binding site in the core promoter, and the response requires a functional protein kinase A signal transduction pathway in the cells. We believe the most productive approach at this stage for further study of a transcription factor mediated event, such as beta-pol promoter response to DNA damage by MNNG, is to use in vitro transcription systems. Thus, we will use in vitro transcription assays to discover the mechanism by which proteins binding at the ATF/CRE-binding site, and the protein kinase A signal transduction pathway act in concert to confer DNA damage regulation to the beta-pol gene.
Specific Aims of this Proposal involve creating an efficient in vitro model system for study of the mechanism of beta-pol promoter activation secondary to DNA alkylation. Kinetic assays of transcription initiation in vitro will be combined with biochemical studies of proteins binding at the ATF/CRE-binding site and of other proteins involved in the transcription machinery. Our preliminary research has indicated the feasibility of creating such in vitro systems, both with a HeLa cell nuclear extract and with purified proteins from bovine tissue (testes). From this approach, we expect to learn how a specific DNA damage-induced modification in the ATF/CREB protein (or other preinitiation complex protein) accelerates transcript initiation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
1R01ES006492-01
Application #
3254732
Study Section
Chemical Pathology Study Section (CPA)
Project Start
1993-09-01
Project End
1997-08-31
Budget Start
1993-09-01
Budget End
1994-08-31
Support Year
1
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Type
Organized Research Units
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
He, Feng; Yang, Xiao-Ping; Srivastava, Deepak K et al. (2003) DNA polymerase beta gene expression: the promoter activator CREB-1 is upregulated in Chinese hamster ovary cells by DNA alkylating agent-induced stress. Biol Chem 384:19-23
Chen, K H; Srivastava, D K; Wilson, S H (2001) Relationship between base excision repair capacity and DNA alkylating agent sensitivity in mouse monocytes. Mutat Res 487:121-6
Latham, G J; Forgacs, E; Beard, W A et al. (2000) Vertical-scanning mutagenesis of a critical tryptophan in the ""minor groove binding track"" of HIV-1 reverse transcriptase. Major groove DNA adducts identify specific protein interactions in the minor groove. J Biol Chem 275:15025-33
Narayan, S; Wilson, S H (2000) Kinetic analysis of Sp1-mediated transcriptional activation of a TATA-containing promoter. Biochemistry 39:818-23
Narayan, S; Wilson, S H (2000) Kinetic analysis of Sp1-mediated transcriptional activation of the human DNA polymerase beta promoter. Oncogene 19:4729-35
Chen, K H; Srivastava, D K; Singhal, R K et al. (2000) Modulation of base excision repair by low density lipoprotein, oxidized low density lipoprotein and antioxidants in mouse monocytes. Carcinogenesis 21:1017-22
Chen, K H; Reece, L M; Leary, J F (1999) Mitochondrial glutathione modulates TNF-alpha-induced endothelial cell dysfunction. Free Radic Biol Med 27:100-9
Chen, K H; Yakes, F M; Srivastava, D K et al. (1998) Up-regulation of base excision repair correlates with enhanced protection against a DNA damaging agent in mouse cell lines. Nucleic Acids Res 26:2001-7
Forgacs, E; Latham, G; Beard, W A et al. (1997) Probing structure/function relationships of HIV-1 reverse transcriptase with styrene oxide N2-guanine adducts. J Biol Chem 272:8525-30
Strauss, P R; Beard, W A; Patterson, T A et al. (1997) Substrate binding by human apurinic/apyrimidinic endonuclease indicates a Briggs-Haldane mechanism. J Biol Chem 272:1302-7

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