Nitrogen oxides such as nitrite, nitrosamines, and nitric oxide are common environmental and endogenous mutagens that deaminate DNA bases and form alkylating agents. E. coli generates them during normal nitrate-based hypoxic respiration and is therefore an ideal model organism for the study of relevant cellular defenses. In E. coli, endonuclease V (Endo V) initiates the excision repair of DNA containing nitrosatively deaminated adenine or guanine (i.e., hypoxanthine or xanthine). Endo V is induced by nitrite, by low pH, or by growth limitation. Proposed studies are in three areas. (1) Mechanisms of nirtosative mutagensis. Endogenous nitrosative mutagenesis will be measured by studying the enhancement, by an nfi (Endo V) mutation, of nitrateinduced A:T to G:C transitions. Answers will be sought to the following questions: (a) Are intracellular nitrosating agents derived mostly from nitric oxide plus oxygen or directly from nitrous acid? (b) Do polyamines prevent or enhance nitrosative mutagenesis? (c) Do nitrite and acid induction of Endo V share a common pathway? (d) What translesion polymerases mediate the mutagenic bypass of xanthine in DNA? (2) Global responses to nitrate/nitrite metabolism. New DNA repair enzymes, defense proteins, and regulatory circuits that defend against nitrosative mutagenesis will be sought (a) by DNA array technology in E. coli and in yeast with Paul Doetsch, and (b) by locating the regulatory genes for nfi and identifying other members of its regulon(s). (3) Enzymes that act on deaminated purines in E. coli and in yeast (with Y.W. Kow and Wolfram Siede). New enzymes that are discovered will be purified and characterized. These will include inducible putative repair enzymes detected during the above microarray studies and an unidentified hypoxanthine DNA glycosylase of E. coli. Corresponding E. coli. mutants will be produced and examined. Mutations for E. coli and yeast dITPase will be studied to determine the enzyme's possible role in resistance to nitrosative mutagenesis, and its possible regulation will be studied with gene fusions and gene arrays.
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