Exposure to environmental DNA-damaging agents can lead to mutations in the genome, potentially providing the initiating step in the process of carcinogenesis. Environmentally-induced skin cancer, in particular squamous cell carcinoma and melanoma, represents an increasingly important health problem, both as a result of increased recreational exposure to sunlight, and of depletion of the protective ozone layer of the atmosphere. At least 500,000 new cases of skin cancer are reported in the U.S. each year, resulting in 8,000 deaths. Much of the recent progress in understanding the molecular basis of skin cancer has come from the study of patients with the disease xeroderma pigmentosum (XP). XP is a genetic disease, which occurs at a frequency of about 1 in 250,000 in the U.S. The disease is characterized clinically by sun sensitivity, and an accelerated onset of a variety of skin cancers in sun-exposed areas of the body. There are seven XP complementation groups, termed A-G, and a variant (V) form. While the molecular defect in groups A-G has been identified as a defect in one of the proteins required for the repair of UV-induced DNA damage, the molecular defect in the XP variant remains unknown. Since the molecular defect in XPV cells is not known (but appears to be at the level of replication of damaged DNA rather than in DNA repair) this proposal aims to use an in vitro approach to determine whether the DNA replication complex from these cells has an alteration in the process of replication and mutagenesis during DNA synthesis on UV-damaged DNA templates. Cell-free extracts of XPV cells will be used to replicate plasmid containing either a single UV-induced pyrimidine dimer or (6-4) photoproduct. If there is a difference between XPV and normal extracts in the ability to replicate these lesions, biochemical complementation of the altered reaction will be carried out, with the ultimate aim to identify the protein which is defective in XPV cells. The proposed study has the potential to elucidate the molecular basis of the XPV phenotype, and also to provide insight into how mutation fixation occurs during replication of damaged DNA in normal cells, with implications for the health of individuals exposed to environmental DNA-damaging agents.
