ENVIRONMENTAL MUTAGENESIS AND DNA REPAIR

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The long term objective of our research is an analysis of alkylation damage and mutagenesis in Drosophila as a model multicellular animal system in which genetics, molecular genetics and biochemistry can be systematically exploited for studies of DNA repair and mutation induction. By isolating mutagen-sensitive (mus) mutants, we aim to identify DNA repair genes and their products and determine the roles of these mus genes in constitutive and inducible DNA repair pathways and mutagenesis. Our methods involve both in vivo and in vitro experiments. In vitro experiments are used to isolate mus mutants and examine the effects of these mutants on mutation induction, using either the sex-linked recessive lethal test or the rosy single gene system. The sex-linked recessive lethal test provides an objective and rapid technique for examining DNA repair-deficient mutants for alterations in the frequency of mutation induction. The rosy single gene system, in combination with powerful recombinant DNA techniques provides a method for detailed molecular analysis of mutants induced in the presence of absence of known DNA repair deviciencies. Preliminary studies with this latter system have demonstrated the feasibility of this system for the molecular analysis of mutagenesis in this higher eukaryote. In vitro experiments utilizing cell cultures prepared from the mus mutants can identify alkylation repair defects. Measurements of unscheduled DNA synthesis provide a method for the general survey of mus strins for repair deficiencies, and current studies have identified a number of loci deficient in UDS induced by several alkylating agents. These and other indirect studies have indicated the need for more sensitive methods for the analysis of alkylation-induced NDA damage. Preliminary studies have demonstrated the feasibility of direct chromatographic analysis for alkylation-induced base lesions in Drosophila DNA. Because high performance liquid chromatography provides the most sensitive direct method for detailed analysis of the nature of alkylation repair defects, we propose further experiments whichrely on this technology. The immediate importance of continued study of this system as an animal model with implications for human health has been amply demonstrated: defects in DNA repair ability have been directly associated with a number of major human diseases.