The incidence of skin cancer, the most common type of human cancer, is increasing in the United States and worldwide. The damage of DNA bases by ultraviolet (UV) radiation causes mutations and UV light is strongly implicated in the development of human basal and squamous cell carcinoma as well as the more lethal melanoma. To understand UV carcinogenesis, a more detailed knowledge of the molecular mechanisms of UV damage formation, DNA repair processes, and mutation induction is necessary. We will apply several sensitive techniques to map UV-induced lesions [primarily cyclobutane pyrimidine dimers (CPDs) and (6-4) photoproducts (6-4)PPs)] and their repair rates at the DNA sequence level. We will continue to investigate the molecular mechanisms of selective UV damage formation in vivo, and to analyze DNA sequence-specific repair rates in several human genes. As part of these aims, we will attempt to develop new methodology to study repair of (6-4) photoproducts at the DNA sequence level. We will measure the major types of DNA lesions and their sequence distribution in solar light irradiated cells. The specific mechanisms of UV mutagenesis will be determined by using a supF shuttle vector and a lacl transgene to study mutagenesis after irradiation with different UV light sources (UVC, UVB, solar light). These studies will address questions as to how the methylation of CpG sequences changes the mutational spectra, which photoproduct contributes most to mutagenesis, what the contribution of CPD deamination to UV mutagenicity is, and whether the frequency of UV-specific mutations correlates with photoproduct frequencies. The sequence context dependence of lesion bypass will be studied with site-specific CPDs and (6-4) photoproducts in a HeLa cell-free extract system. The interference of UV damage with transcription will be analyzed by determining the effects of (6-4) photoproducts on transcription factor binding and by studying the effects of UV lesions (both CPDs and 6-4PPs) in promoters on transcription.
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