Excision repair is generally regarded as the major process by which mammalian cells reduce the cytotoxic, mutagenic, and carcinogenic effects of DNA damage produced by ultraviolet radiation and chemical carcinogens. The long-term objective of this project is to investigate in detail the molecular mechanisms of excision repair of carcinogen damage and the ways in which the repair process is modulated by the chromatin structure of mammalian cells. Understanding the details of the excision repair process is of particular interest in light of recent studies indicating that different regions of mammalian genomes are repaired at different rates. The first goal of the present application is to explore the role of ATP in the early steps of excision repair in cells damaged with several different carcinogens. The possibility that the ATP requirement is related to involvement of a DNA topoisomerase will be specifically examined. This application also proposes studies of damage and repair-associated modifications of chromatin proteins. Such modifications will be identified using appropriate radioactively labeled precursors. In addition, the temporal relation of the chromatin modifications to the pre- and post-incision phases of the repair process will be explored and the physical relation of modified chromatin proteins to sites of excision repair will be examined. These studies will be conducted using a permeable human fibroblasts in which excision repair proceeds in a fashion essentially identical to that seen in intact cells, but is accessible at all stages to biochemical manipulation. The project also involves development of a technique using biotin-modified dUTP and affinity chromatography which for the first time will permit isolation and direct analysis of chromatin undergoing excision repair. The proposed studies should generate biochemical data which will be directly applicable to understanding the process of excision repair in vivo.
