We propose a coordinate study of the process of mutagenesis induced by alkylating and oxidizing agents in biological systems. Project 5 proposes development of sensitive means to map and measure DNA adducts and proposes experiments to discover total input (adduct spectra) and output (point mutational spectra) for MNNG and H2)2 (W.G. Thilly). Project 3 proposes applying the technology of site specific DNA adduction to create DNA vectors which are to be used in experiments in bacteria and human cells to discover the precise genetic outcome of a particular alkyl-or aryl-adduct in a particular DNA sequence (J.M. Essigman). Project 1, 4, and 6 represent applications of the tools of molecular genetics to isolate and study the effects and regulation of particular genes and gene products in the mutational process. In Project 6 the specifics of O6-alkyl-transferase activity will be examined in terms of enzyme structure, the peculiar phenotype of Rhizobium will be used to probe the importance of mismatch repair, and the role of umuCumuD dependent pathways in alkylation and oxidizing agent mutagenesis will be extended (G. Walker). In project 1 oxidizing agent mutational pathways will be dissected using the newly cloned yeast AP endonuclease combined genetically with superoxide dismutase phenotypes for studies in yeasts, bacteria and human cells (B. Demple). Project 4 examines the role of the alk B gene product in modulating the mutational outcome of alkylation damage in bacterial and human cells and also extends knowledge of the importance of demelkylation pathways in human cells (Samson). Project 7 uses the new technology of mutational spectrometry to trace the early DNA events and subsequent mutational results of exposure of animals to alkylating agents with particular emphasis on oncogene sequences. Together the seven projects span the events of initial DNA reaction, processing by the DNA repair systems, and eventual appearance of specific genetic change in bacteria, yeasts, human cells and rodent tissue. As a programmatic unit we link the efforts of molecular geneticists interested in mechanistic steps in mutagenesis to the systems commonly used by toxicologists to evaluate potential hazards for human health.
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