Xeroderma pigmentosum (XP) patients have an inherited predisposition to sunlight-induced skin cancer, and the cells from the majority of these patients have been shown to be deficient in nucleotide excision repair. Lack of repair of UV-induced DNA damage can explain why these cells are extremely sensitive to mutations induced by UV. However, one class of XP patients, designated variant, are not deficient in excision repair, and yet their cells exhibit an even greater sensitivity to UV-induced mutations than those of classic excision repair-deficient XP patients. Data from UV-induced mutational spectra suggest that the cancer predisposition of XP variant patients results from their cells having a defective DNA replication complex, so that when they replicate DNA containing photoproducts they make frequent errors, and these errors are of a very distinctive kind. Recent data, derived from an in vitro DNA replication assay comparing XP variant and HeLa cell extracts, confirm that this is true. This replication-competent extract from XP variant cells represents the first and, as yet, the only example of a human DNA replication complex that demonstrates error-prone bypass of DNA damage. These cells represent a unique opportunity to investigate the mechanisms by which normal cells achieve fidelity when they replicate damaged DNA, and how errors in replication lead to mutations. Since mutations are causally involved in the process of neoplastic transformation, this study has direct relevance to the study of tumorigenesis. It is proposed to study the mechanism of bypass of a number of lesions by extracts from normal cells and from XP variant cells. It is also proposed to investigate a variety of replication proteins to determine if they may contribute to, or be responsible for, the replication defect in XP variant cells. Specifically, it is proposed: 1) to test whether cell extracts from normal cells give the same mutation spectrum as HeLa cell extracts when replication UV damaged templates in vitro; 2) to expand a current database of UV-induced mutations mediated by replication with XP variant cell extracts; 3) to switch the origin of replication of the replication substrate relative to the target gene, to confirm that XP variant replication complex processes photoproducts asymmetrically, in a strand-specific manner; 4) to test whether the addition of RP-A, RF-C, PCNA, or pol alpha correct the XP variant defect in vitro, and to sequence the coding region of the pol delta gene from 2 XP variant cell lines; 5) to prepare extracts from a variety of XP variant cell lines, to test for complementation; and 6) to examine bypass parameters by normal and XP variant extracts of the following lesions placed site-specifically in the same sequence context: T-T dimers, T-T 6-4s, N2-dG-(+)-trans-anti-benzo[a]pyrene, C8-dG-1-aminopyrine, or C8-dG-2-aminofluorene.
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