INDUCED DEAMINATION OF CYTOSINE AND 5-METHY1C IN DNA

我们的实验室一直在研究导致DNA脱氨基的因素。 我们已经使用模型核酸碱基和聚合物系统研究了两个 相互关联的假说：(L)电离和质子化碱基对可能 被诱导在DNA中形成，以及(2)它们可以提供一种途径， 会导致DNA中的诱导性脱氨。我们假设质子化 碱基对可能来自几种不同类型的DNA改变，以及 质子化胞嘧啶的脱氨基可能包括先前的 未确定的和重要的基因突变来源。为了 研究了诱导脱氨，我们开发了一种灵敏的逆转试验 可用来评估单个地点的脱氨率。 各种条件。在目前的拨款中，我们建议延长我们的 从三个方面进行研究。 首先，我们将测量5-甲基胞嘧啶(5mC)的脱氨率。 在37℃的特定位置，单链(Ss)和双链 (DS)DNA，并比较脱氨率和未甲基化的速率 胞嘧啶在相同的位置和相似的条件下。5mC脱氨基 直接转化为胸腺嘧啶。该反应的动力学参数从来没有 在生理条件下测量的。我们的网站有三个 目标DNA可以在哪里甲基化，我们可以在哪里测量 脱氨基5mC。 第二，我们观察到CC--&gt；TT串联双突变发生在 长时间孵育的双链DNA异常高频率 一段时间。结果是不寻常的，因为CC--&gt；TT频率 甚至高于单个C--&gt；T突变体和病变 推测可能涉及尿嘧啶中间体，因为串联双突变 在ung+菌株中几乎被淘汰。基于时间的诱导方法 双重突变体可能代表某种类型的诱导突变现象， 以前没有被描述过。我们要寻找原因(S) 串联双突变体，确定它们被不同种类的 代理人，并确定病变(S)的责任。 第三，我们将完成对食品添加剂效果的研究， 亚硫酸氢根对胞嘧啶脱氨速率及机理的影响 双链DNA。这将在生理温度和 PH，在厌氧条件下，以及在存在和不存在游离态- 激进的抑制剂。

Our laboratory has been studying factors that induce deamination in DNA. We have used model nucleic acid base and polymer systems to examine two inter-related hypotheses: (l) that ionized and protonated base pairs may be induced to form in DNA, and (2) that they may provide one pathway that can lead to induced deamination in DNA. We hypothesize that protonated base pairs may arise from several different kinds of DNA alterations, and that the deamination of protonated cytosines may comprise a previously undetermined and significant source of genetic mutations. In order to study induced deamination, we have developed a sensitive reversion assay by which the rates of deamination at a single site can be assessed under a variety of conditions. In the present grant, we propose to extend our studies in three ways. First, we will measure the rates of deamination of 5-methylcytosine (5mC) at 37 degrees C at specific locations in single (ss) and double-stranded (ds) DNA and compare the rates of deamination with those of unmethylated cytosine at the same site and under similar conditions. 5mC deaminates directly to thymine. Kinetic parameters for this reaction have never been measured under physiological conditions. There are three sites in our target where DNA can be methylated, and where we can measure the deamination of 5mC. Second, we have observed that CC-->TT tandem double mutations occur with unusually high frequency in double-stranded DNA incubated for long periods of time. The results are unusual in that the CC-->TT frequency is higher than even that of single C-->T mutants and the lesion presumably involves a uracil intermediate because tandem double mutations are nearly eliminated in an ung+ strain. The time dependent induction of double mutants may represent some type of induced mutation phenomena that has not previously been characterized. We will search for the cause(s) of tandem double mutants, determine their rates of induction by various agents, and identify the lesion(s) responsible. Third, we will complete our studies on the effect of the food additive, bisulfite ion, on the rate and mechanism of deamination of cytosine in double stranded DNA. This will be done at physiological temperatures and pH, under anaerobic conditions, and in the presence and absence of free- radical inhibitors.