NITRIC OXIDE MUTAGENESIS AND DEAMINATION OF DNA

我们建议使用我们开发的一种敏感的基因逆转试验 实验室研究一氧化氮(NO)诱导的DNA突变率 及其代谢产物在实验控制和生理状态下 相关条件。最近，我们发现这种化学物质 信使，一氧化氮(NO)，能够诱导DNA的C-T突变 速度远远超过其他已知的脱氨基化合物，如亚硫酸氢盐。 在本提案中，我们提出NO可能是 C-T和其他DNA突变，我们建议使用我们的敏感 用遗传分析方法研究NO的复杂化学和动力学 与DNA的反应性。 首先，我们将使用我们的基因逆转试验来确定 一氧化氮(NO)诱导的DNA脱氨基(C、G和A碱基)和 其假定的活性物种过氧亚硝酸盐，在各种不同的 条件。与之前的研究相比，我们的实验将是 在接近生理浓度的一氧化氮下进行。 通过利用反向和正向分析，我们将能够 测定一氧化氮的剂量效应曲线和致突变谱 DNA，除了速率常数。第二，我们将梳理角色 氧在激活NO诱变中的作用 厌氧和控制的氧分压和在不同的NO 浓度。有了我们的系统，我们可以小心地控制 产生氧气的物种和自由基的浓度，如H202，OH 和O2，并直接测量突变反应。如果氧化损伤 被牵连，DNA将在没有治疗之前和之后接受治疗 适当的修复酶，如Endo III，Fapy-DNA糖基酶等，以 探查氧化损伤。第三，我们将研究 催化剂，如亚硝酸盐和硫氰酸盐，以及其他细胞亲核剂 (胺和硫醇化合物)调节一氧化氮的突变。我们 还将考察金属离子的协同作用和抑制作用 抗氧化剂对突变率的影响。第四，我们将研究 组蛋白(核小体)和rec A蛋白在保护DNA中的作用 一氧化氮诱变。这类研究应有助于确定 一氧化氮在体内的代谢途径及其相对重要性 在突变过程中。总而言之，我们希望确定突变的类型 一氧化氮及其代谢物在多种条件下诱导的 实验控制的和生理相关的条件，以及 推导出这些突变过程的速率常数。通过这种方式，我们 希望能够做出无致突变性和毒性的预测 稍后可以在动物系统中进行测试。

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.