OXYGEN AND SPONTANEOUS MUTAGENESIS IN ESCHERICHIA COLI

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DESCRIPTION: (adapted from the investigators abstract) Active species of oxygen are normally generated by respiring cells and are known to be toxic, damaging proteins, membrane lipids and DNA. These toxic byproducts of aerobic respiration, which include superoxide radical, hydrogen peroxide and hydroxyl radical, are thought to be possible causative agents in mutagenesis, carcinogenesis and aging. Although detailed pathways by which oxygen species cause the above conditions have yet to be clearly defined, interaction with DNA is an obvious component. Among the multiple forms of DNA injury, the production of mutational events is amenable to genetic analysis. Using the genetically well-characterized facultative aerobic bacterium, Escherichia coli, measurement of forward (LacI-, rifampin resistance and valine resistance) and back (TrpA+) mutation frequencies reveal the proportion of spontaneous mutagenesis that can be attributed to oxygen. Aerobic and anaerobic (anerobic chamber with reduced media) conditions will reveal the proportion of spontaneous mutagenesis that can be attributed to oxygen. Aerobic and anaerobic mutational spectra will be determined using the trpA reversion system and the DNA sequencing of lacI(-) mutations; such spectra provide insights into mutational mechanisms. Aerobic and anaerobic frequencies (and possibly mutational spectra) will be measured in mutant E. coli strains defective in enzymatic activity that normally protects against oxidative damage (catalase {katG, kat} and superoxide dismutase {sodA, sodB}) and DNA repair (recA, polA, xthA and mutL). Anaerobic mutation frequencies and spectra will be determined using the most stable of the active oxygen species, hydrogen peroxide, as a mutagen. The goals of this project are to: (1) compare aerobic and anaerobic mutation frequencies in E. coli to establish the contribution of oxygen species toward the load of spontaneous mutations, (2) define mutational spectra in wild type and mutant E. coli strains, to provide models of oxygen-induced mutational mechanisms, and (3) elucidate the mechanisms(s) of hydrogen peroxide-induced mutagensis.