Skin cancer is the most prevalent form of cancer and has been steadily rising over the past 20 years. Recently, there has been great concern that the incidence of skin cancer may increase even more rapidly with the discovery that the ozone layer which shields the earth from the highly mutagenic ultraviolet radiation in sunlight,is slowly becoming depleted. Compelling evidence that uv light is the primary carcinogen comes from the strong correlation between mutations in the ras protooncogenes and p53 tumor suppressor genes of skin cancers with dipyrimidine sites, the principal sites for uv-induced DNA photoproduct formation. Though the major photoproducts of dipyrimidine sites are known to be the cis-syn, (6-4) and Dewar products, which if any of these are responsible for the observed mutations and the molecular details of the steps leading to the mutations are not. We propose to elucidate the precise structure-activity relationships of the major photoproducts of dipyrimidine sites by a combined synthetic, physical, enzymatic and biological approach: We are particularly interested in understanding the molecular basis of DNA damage recognition by repair enzymes, and that of the mutations that result from the replicative bypass of DNA photoproducts by DNA polymerases. Our primary focus will be on unraveling the origin of the C->T mutation at TpdC sites, the major mutation induced by uv light in both bacteria and mammals and mutations at T-tracts, one of the major sites of frameshift mutations. The ability of many of the dC-containing products, particularly the cis-syn dimers, to spontaneously deaminate and tautomerize has led to a number of distinct proposals for the origin of their mutagenicity, and will be a target of study. Likewise, T-tracts have many photoproduct sites, and can result in the formation of products between non-adjacent dimers, making it difficult to ascribe the observed frameshifts with a given photoproduct or site. To determine the precise structure-activity relationships, we propose to prepare pure, well characterized, oligonucleotides containing the photoproducts of these sites, some of which are unstable or new and will require us to develop new methodology for this purpose. Once in hand, the site-specific photoproduct-containing oligonucleotides will be incorporated into (a) short duplexes for melting temperature studies and high resolution structural studies by NMR and x-ray crystallography: (b) polymers for gel electrophoretic assays of bending and unwinding; (c) long duplex DNA fragments for in vitro repair enzyme studies; (d) long templates for in vitro replication enzyme studies; and (e) bacteriophage and viral DNA for in vivo mutagenesis studies. These photoproduct containing DNAs will also be used to optimize and develop new chemical, enzymatic, and antibody-based assays needed to assay for these photoproducts both in vitro and in vivo. The proposed studies are expected to result in new information and fundamental new insights into the mechanisms of mutagenesis that could ultimately enhance prevention of skin cancer and other cancers.
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