The goal of this research is to understand radiation-induced DNA strand breaks in terms of their biochemistry and not simply as interruptions in the DNA double helix. New biochemical and molecular biological techniques, such as Fast Protein Liquid Chromatography, end-group analysis by DNA electrophoresis, and retroviral expression vectors, will be employed. Experimentation will determine the biochemical basis for the production, processing, and ultimate repair of these lesions in irradiated human cells. This information will add to our basic knowledge of cellular radiation effects. It should also contribute to our understanding of radiation carcinogenesis, and may allow better use of radiotherapy in cancer treatment. The hypothesis, to be tested, is that DNA strand breaks are of fundamental importance to repair mechanisms for radiation-damaged DNA. Not only are they DNA lesions themselves, but they also represent intermediate states in the repair of other damage, and may serve a regulatory function for DNA repair in the human system. The study of strand breaks, therefore, should provide basic insight into how the cell maintains its genetic stability. This research will expand upon knowledge about strand breaks gained through in vitro and bacterial studies, by directly studying these lesions in human cells. The most notable difference in the human system is the added involvement of chromatin, which can affect both the induction and repair of DNA lesions. For this reason, the relationship between DNA strand breaks and chromatin will be studied, particularly with respect to the chromatin-associated enzyme poly(ADP-ribose) polymerase. The research plan has three parts. First, the different DNA strand breaks produced in human cells will be biochemically identified and quantitated, and repair rates will be determined. Second, the human enzyme(s) responsible for repairing these different lesions will be purified and characterized. Third, the ability of different DNA strand breaks to stimulate poly(ADP-ribosylation) will be studied and evaluated to determined the possible mechanism of poly(ADP-ribose) polymerase activation: and the effect of poly(ADP-ribose) polymerase activity on strand-break repair will be assessed using expression vectors to vary intracellular levels of the enzyme. This work should help us to better understand the molecular mechanisms of radiation action in the human system.
