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 (UV) and chemical carcinogens. The object of this proposal is to explore the involvement of ATP and of changes in chromatin structure in excision repair. Previous studies have revealed that, in normal human cells, ATP is required for excision repair of DNA damaged by ultraviolet radiation (UV) at or before the incision step. ATP is also required for repair of UV damage in xeroderma pigmentosum (XP) cells complemented with T4 UV endonuclease. One objective of this proposal is to characaterize specifically the ATP requirement for excision repair in UV endonuclease-complemented XP cells and to compare that requirement enzymologically with the ATP requirement in normal cells. Other ATP-related questions to be studies are: i) whether these ATP requirements are related to involvement of a DNA topisomerase in excision repair; ii) whether any of the incompletely repair deficient XP cell lines show altered ATP requirements; and iii) whether ATP is required for excision repair induced by other carcinogenic DNA damaging agents, e.g., N-methyl-N-nitrosourea (MNU), an alkylating agent, and x-ray, a strand breaking agent. Chromatin changes accompanying repair of UV, MNU and x-ray damage will also be studied: i) determining whether excision repair is accompanied by acetylation, phosphorylation, and/or methylation of specific nuclear proteins; ii) determining at which step in the excision repair pathway poly ADP ribose added to nuclear proteins in response to DNA damage is turned over or degraded; iii) determing whether specific chromatin proteins are gained or lost during the process of excision repair; and iv) determining whether chromatin modifications associated with DNA damage and repair are specifically localized in the regions of chromatin underdoing repair. All of the studies proposed will utilize a permeable cell system in which excision repair proceeds in a fashion essentially identical to that in intact cells, but is accessible at all stages to experimental manipulation. In addition, a novel approach involving biotin-modified dUTP and avidin-agarose chromatography will be used to isolate and directly analyze chromatin undergoing repair. These experiments should generate biochemical data which will be directly applicable to understanding excision repair in vivo, contributing to a more complete molecular description of this important biological process.
