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.

National Institute of Health (NIH)
National Institute of Environmental Health Sciences (NIEHS)
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Massachusetts Institute of Technology
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Memisoglu, A; Samson, L (2000) Contribution of base excision repair, nucleotide excision repair, and DNA recombination to alkylation resistance of the fission yeast Schizosaccharomyces pombe. J Bacteriol 182:2104-12
Wyatt, M D; Samson, L D (2000) Influence of DNA structure on hypoxanthine and 1,N(6)-ethenoadenine removal by murine 3-methyladenine DNA glycosylase. Carcinogenesis 21:901-8
Ekstrom, P O; Borresen-Dale, A L; Qvist, H et al. (1999) Detection of low-frequency mutations in exon 8 of the TP53 gene by constant denaturant capillary electrophoresis (CDCE). Biotechniques 27:128-34
Opperman, T; Murli, S; Smith, B T et al. (1999) A model for a umuDC-dependent prokaryotic DNA damage checkpoint. Proc Natl Acad Sci U S A 96:9218-23
Hickman, M J; Samson, L D (1999) Role of DNA mismatch repair and p53 in signaling induction of apoptosis by alkylating agents. Proc Natl Acad Sci U S A 96:10764-9
Li-Sucholeiki, X C; Khrapko, K; Andre, P C et al. (1999) Applications of constant denaturant capillary electrophoresis/high-fidelity polymerase chain reaction to human genetic analysis. Electrophoresis 20:1224-32
Bennett, R A (1999) The Saccharomyces cerevisiae ETH1 gene, an inducible homolog of exonuclease III that provides resistance to DNA-damaging agents and limits spontaneous mutagenesis. Mol Cell Biol 19:1800-9
Glassner, B J; Posnick, L M; Samson, L D (1998) The influence of DNA glycosylases on spontaneous mutation. Mutat Res 400:33-44
Glassner, B J; Rasmussen, L J; Najarian, M T et al. (1998) Generation of a strong mutator phenotype in yeast by imbalanced base excision repair. Proc Natl Acad Sci U S A 95:9997-10002
Masuda, Y; Bennett, R A; Demple, B (1998) Dynamics of the interaction of human apurinic endonuclease (Ape1) with its substrate and product. J Biol Chem 273:30352-9

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