Although great strides have been made in definition of the genes involved in nucleotide excision repair, relatively little is known regarding the genes that govern sensitivity to ionizing radiation. The long term goal of our work is to identify and characterize genes that govern radiation sensitivity in mammalian cells. Recent work from our laboratory has demonstrated that the radiation sensitive phenotype of the hamster mutant irs-2 is suppressed in strains transfected with the human poly(ADP- ribose) polymerase (PARP) cDNA or selected for resistance to 3- aminobenzamide (3-AB, an inhibitor of PARP). These observations suggest that PARP may play an important role in the response of cells to ionizing radiation and suggest PARP as the candidate gene that is altered in irs- 2.
The specific aims of the work proposed here are: i) to determine whether mutations of PARP are responsible for the radiation sensitive phenotype of irs-2 and the mechanism by which It confers this sensitivity. This will be pursued by both DNA sequence analysis of the PARP coding sequences in irs-2 and 3-AB resistant strains and biochemical analysis of PARP activity by Western and activity blots. To further confirm that mutations of PARP cause suppression of the radiation sensitive phenotype, mutants of irs-2 resistant to other more specific inhibitors of PARP will be selected. We will also attempt to suppress the radiation sensitive phenotype of irs-2 by transfecting wild type and mutant forms of PARP. To investigate the mechanism by which these mutations confer sensitivity, the interaction of PARP with other proteins will be examined. ii) to determine whether the suppression of radiation sensitivity is specific for irs-2. Radiation-sensitive strains deficient in double stand break repair, base excision repair and strains proficient in these pathways will be transfected with the human PARP cDNA to determine whether they show a similar suppression of radiation sensitivity. iii) to determine whether the human PARP cDNA complements the radiation sensitive phenotype of cells from Ataxia-telangiectasia (A- T) patients. Considering that irs-2 is the most A-T like of the hamster radiation sensitive mutantS, we will attempt to complement the radiation sensitive phenotype of A-T cells by transfecting them with the human PARP cDNA. iv) to identify and clone the human gene complementing or suppressing the radiation sensitive phenotype of irs-2. This will be accomplished by successive rounds of transfection of either purified human DNA or metaphase chromosomes to-complement the irs-2 phenotyPe followed by cloning of human specific sequences. Alternatively we will transfect expressing cDNA libraries followed by rapid recovery of the complementing cDNA. The significance of this work lies in the identification of genes that govern sensitivity to ionizing radiation. This may ultimately enable us to predict and alter the response of tumor cells to radiation therapy.
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