The goal of this project is to explain how ionizing radiation causes cancer, mutation and cell death by elucidating the chemistry and enzymology of X-ray-induced DNA damage and repair in human cells. New approaches to the study of X-ray-induced single-strand breaks and base-modifications are described. We will use a repeated sequence (alpha sequence) in human DNA to 1) determine the location (sequence) of strand breaks after gamma-irradiation in vitro and in vivo; 2) determine the effect of oxygenation, pH, temperature, and free radical scavengers on the location and extent of strand breaks; 3) determine the location (sequence) of alkali-labile sites in gamma-irradiated DNA; 4) determine the level of production of a newly described X-ray lesion, 3' phosphoglycolate termini, in DNA irradiated in vitro and in intact cells. The approach to study of X-ray induced base modifications uses a sensitive """"""""post-modification labeling"""""""" method. This method will be used to determine 1) levels of formation of particular base-modifications after gamma-irradiation and 2) the rates and extent of repair of such base-modifications after gamma irradiation. The approach chosen to study human repair enzymes uses DNA substrates which contain single, defined and radiolabeled lesions of the types induced by X-rays. We will 1) determine the presence of human enzymes specific for DNA containing thymine glycols, HMU and 3' phosphoglycolate termini; 2) utilize such substrates for isolation and characterization of such enzymes; 3) determine whether a deficiency of such an enzyme can explain the susceptibility of ataxia telangiectasia cells to X-rays and radiomimetic drugs. Knowledge of the processes of X-ray DNA damage and repair will be useful in developing more rational approaches to cancer prevention and therapy and in predicting the effects of low level radiation exposure.
