IONIZING RADIATION INDUCED DNA DAMAGE AND REPAIR

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The goal of this project is to increase our understanding of how ionizing radiation causes cancer, mutation and cell death by elucidating the enzymology and genetics of mammalian X-ray- induced DNA repair. A direct approach for the development of human genetics for the enzymes of repair of X-ray-induced DNA repair is described. This project will define the primary structure and genetics of human and bovine enzymes required for repair of ionizing radiation-induced DNA damage. Bovine tissue (calf thymus) will be used as a source of preparation of homogeneous enzymes for amino acid sequencing and antibody production. The cDNA sequences coding for repair enzymes will be selected from lambda gt11 libraries by immunoscreening with rabbit anti-enzyme antibodies or from lambda gt10 libraries by screening with mixed oligonucleotides. Once the cDNA for the bovine enzyme is obtained the human cDNA will be selected from lambda gt10 or lambda gt11 libraries through its nucleic acid homology or immunologic cross reactivity, respectively. Apurinic endonuclease has been chosen as the initial target for cloning in this program for the following reasons; 1) Apurinic sites in DNA are known to mutagenic (and possibly carcinogenic) 2) apurinic sites are common to many types of DNA damage and repair including X-rays 3) the issue of whether a single gene or multiple mammalian genes code for AP endonuclease is unresolved 4) the requirements for this approach have been fulfilled a) homogeneous enzyme b) amino-acid sequence data c) antibody probes and d) cDNA libraries for the tissue of origin are all available. Knowledge of the primary structure of the enzymes of X-ray induced DNA repair will be useful in understanding how such enzymes recognize damaged DNA, the sequence of events during repair and how failures of repair might lead to the biological effects of X-rays. The availability of molecular probes for these genes will allow studies of their role in the inherited defects of DNA repair such as ataxia telangiectasia and in the susceptibility of individuals and tumors to X-rays effects.