The objective of this research is to investigate the mechanisms by which ionizing radiaton-induced DNA damage results in mutations. We propose two general approaches. The first is to continue the work begun during the last grant period in which organic synthesis and recombinant DNA techniques were being used to construct viral and plasmid DNA molecules that contain, at specific genome sites, the known products of ionizing radiation damage (initially cis-5,6-dihydroxy-6-hydrothymine, thymine glycol). These structurally altered genomes will be introduced to the intracellular enivronment, where the radiation-induced lesions may cause mutation. Conceivably this will occur through misreplication or faulty repair of the structrually altered DNA. Finally, we shall characterize quantitatively and qualitatively the mutagenic changes induced in daughter DNA molecules, and attempt to establish formal rules that relate the structure of a lesion with its bioligical effects. Our second proposed approach to assessing the gentic effects of ionizing radiation will be to determine the pattern of mutation, or mutational spectrum, induced by radiation in a plasmid-based mutation assay we developed during the previous grant period. The noteworthy features of this plasmid-based assay are its ability to detect forward mutations, a positive selection for the mutant phenotype, and the fact that the target for mutagenesis is small, approximately 400 bp, making determination of mutational spectra by direct DNA sequencing a resonable objective.
