We propose to use a sensitive genetic reversion assay developed in our laboratory to study rates of DNA mutation induced by nitric oxide (NO) and its metabolites under experimentally controlled and physiologically relevant conditions. Recently, we have found that the chemical messenger, nitric oxide (NO), is able to induce C--T mutations in DNA at rates that far exceed other known deaminating compounds like bisulfite. In the present proposal, we propose that No may be important source of C--T and other mutations in DNA, and we propose to use our sensitive genetic assay to sort out the complex chemistry and kinetics of NO reactivity with DNA. First, we will use our genetic reversion assay to determine the rates of DNA deamination (of C, G and A bases) induced by nitric oxide (NO) and its postulated active species, peroxynitrite, under a variety of conditions. In contrast to previous studies, our experiments will be carried out at close to physiological concentrations of nitric oxide. By utilizing both reversion and forward assays, we will be able to determine dose response curves and mutational spectra produced by NO in DNA, in addition to rate constants. Second, we will sort out the role of oxygen in activating NO mutagenesis by carrying out reactions under anaerobic and controlled oxygen tensions and at differing NO concentrations. With our system we can carefully control the concentrations of O2 generating species and radicals, such as H202, OH and O2 and measure directly the mutational response. If oxidative damage is implicated, DNA will be treated before and after NO treatment with appropriate repair enzymes like Endo III, Fapy-DNA glycosylase, etc., to probe for oxidative damage. Third, we will examine the role of catalysts, like nitrites and thiocyanate, and other cellular nucleophiles (amines and thiol compounds) in moderating mutation by nitric oxide. We will also examine the synergistic effect of metal ions and inhibitory effect of antioxidants on rates of mutations. Fourth, we will study the role of histone (nucleosomes) and rec A proteins in protecting DNA from nitric oxide mutagenesis. Such studies should assist in determining the metabolic pathways of nitric oxide in vivo and the relative importance in mutagenesis. In summary, we expect to determine the type of mutations induced by nitric oxide and its metabolites under a variety of experimentally controlled and physiologically relevant conditions, and derive rate constants for these mutational processes. In this way, we hope to be able to make predictions about NO mutagenicity and toxicity that can later be tested in animal systems.
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