The experiments proposed here address fundamental issues concerning molecular mechanisms of DNA repair and mutagenesis by taking advantage of i) my own lab's extensive experience with DNA repair and mutagenesis in E. coli and our familiarity with R. meliloti genetics and ii) some of the many possibilities for innovative interactions that have developed within our group over the past several years. The proposed lines of research take full advantage of the high resolution experimentation that is possible with E. coli and R. meliloti and offer possibilities for detailed insights concerning mechanisms. To obtain an evolutionarily diverged analog of the E. coli O8-methylguanine-DNA methyltransferases, we will clone and sequence the gene for the R. meliloti O8-methlguanine-DNA methyltransferases, that we characterized during the previous funding period. We will then analyze this gene in a variety of genetic studies that will include constructing deletions to define the minimal functional unit of the protein, creating chimeric proteins between the R. meliloti O8-methylguanine-DNA methyltransferase and the corresponding E. coli protein(s), and carrying out selected site-directed mutagenesis studies to probe the function of certain key conserved amino acids. These studies will take advantage of mutants constructed by Bruce Demple's and Leona Samson's lab and of the """"""""HiFi"""""""" PCR technology developed in Bill Thilly's lab. We will take advantage of developments from my lab and those of Leona Samson and Bruce Demple to explore a novel phenomenon we have observed which indicates that mismatch repair can influence the amount of mutagenesis caused by alkylating agents. We will follow up on our observations from the previous funding period by isolating mismatch repair deficient mutants of R. meliloti, an organisms that lacks the dam-dependent methylation so important in E. coli mismatch repair. Finally, in collaboration with John Essigman, we will explore the basic mechanism of umuD+umuC+ -dependent mutagenesis by alkylating and oxidizing agents. We will do this by exploring the effect of UmuC and activated UmuD on the behavior of DNA polymerases on monoadducted templates carrying lesions introduced by alkylating or oxidizing agents.
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