The damage of DNA by ultraviolet (UV) light from the sun is assumed to be responsible for the induction of mutations and the development of skin cancer in humans. To understand UV carcinogenesis, a more detailed knowledge of the molecular mechanisms of UV damage, repair and mutagenesis is necessary. In this competing continuation application, we will address the following issues: In Specific Aim I, we will characterize the DNA damaging and mutagenic properties of UVA irradiation, a component of the solar spectrum that has been linked to melanoma. In Specific Aim II, we will try to demonstrate a molecular link between sunlight exposure and melanoma. We will attempt to identify a UVB signature in the human p16 gene of melanoma patients and a UVA signature in the BRAF gene. In Specific Aim III, we will test the hypothesis that specific lesion-tolerant DNA polymerases and additional factors are involved in UV mutagenesis. We will determine the in vivo roles of DNA polymerases eta, iota, kappa, beta, RAD3/RAD7 and REV1, as well as other proteins in UV mutagenesis. These studies will employ siRNA technology to knock down specific proteins involved in UV mutagenesis. The experiments will use supF plasmids containing CPDs or (6-4) photoproducts, which are replicated in cells treated with siRNA against the same polymerases. These studies will be complemented by ones using mouse embryonic fibroblasts carrying mutation reporter transgenes and derivatives of these cells expressing photoproduct-specific photolyases and specific siRNAs. In Specific Aim IV we will test the hypothesis that 5-methylcytosine plays a crucial role in UV mutagenesis in mammalian cells. We will study the effects of 5-methylcytosine and 5-methylcytosinebinding proteins (mCpG-binding proteins, MeCP2, MBD2, MBD1) on UV damage induction and repair in vitro, on the deamination of 5-methylcytosine within cyclobutane pyrimidine dimers, and on mutagenesis in a CpG-methylated mutation reporter gene. Altogether, these studies should increase our understanding of the mechanisms of UV-induced DNA damage, its repair and the mechanisms of mutagenesis that lead to alterations in genes that are implicated in nonmelanoma and melanoma skin cancers.