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.
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