The proposed research is aimed at understanding the DNA excision repair process and its significance in fibroblasts from xeroderma pigmentosum complementation group C (XP-C) patients, and the relation to excision repair in normal human fibroblasts. These are steps to understanding the repair defect in XP-C cells and its relation to the high incidence of cancer in XP patients. Nondividing XP-C cells exposed to ultraviolet light (UV, 254 nm) efficiently repair large unique regions of DNA that represent 5 to 10% of the genome, but do not repair damage elsewhere. Associated with this preferential repair is a greater resistance to lethal UV effects. The repair process will be characterized with respect to the genetic content, physical structure, and rate of repair of the unique domains, and its relation to normal repair mechanisms. These goals will be achieved through an identification of genes associated with the repaired DNA, a definition of genetic and physical maps of repaired domains and a determination of repair rates of associated and unassociated genes in normal and XP cells. The biological significance of this repair will be assessed by studying the effect of UV and subsequent excision repair on transcription of domain-associated genes in normal, XP-C and other XP-A strains that are UV sensitive and exhibit repair at random locations. A genomic fraction enriched in preferentially repaired DNA has been isolated. Genes associated with it, such as the beta- actin gene, are detected by filter hybridization methods. Genes chosen for investigation include transcriptionally-active and inactive ones, to test the hypothesis that repair domains are transcriptionally active domains. A cosmid library will be constructed as the means to defining genetic, structural and transcriptional maps of the beta-actin and other repair domains. This library will provide cloned DNA representing a biologically significant human genomic subfraction and should be useful to many other human molecular genetic studies.
