IONIZED BASE PAIRS AND CROSS STRAND DEAMINATION

我们实验室一直在研究引起DNA脱氨的因素。我们 已启动了一系列关于模型核酸碱基和聚合物的研究 系统检查两个相互关联的假设：（1）电离的碱基对 可能存在于DNA中，（2）它们可能提供一种途径，可以导致 诱导DNA脱氨。我们假设质子化碱基对可能 由几种不同类型的DNA改变引起， 这些碱基中质子化胞嘧啶的脱氨基作用可包括 以前未确定的基因突变来源。为了研究 诱导脱氨，我们已经开发了一种逆转试验， 可以在各种条件下评估脱氨基的程度。在 在目前的拨款下，我们建议从几个方面扩展我们的研究。我们将 测试的假设，一个异常的基地，如O（6）-甲基鸟嘌呤在一个 DNA链将诱导相反方向的胞嘧啶脱氨基 三螺旋中质子化胞嘧啶残基可 与正常的B型DNA相比，以加速的速率脱氨。第一、 我们将继续我们的合成和定点诱变研究， 确定O（6）-中胞嘧啶脱氨基的速率和条件。 烷基鸟嘌呤：DNA中的胞嘧啶碱基对。通过改变 O（6）-烷基取代基，我们可以考察给电子和 吸电子基团对诱导脱氨基作用的影响。这样，我们希望 区分质子诱导脱氨的两种可能机制： 也就是说，局部变性与捕获质子。第二，我们将研究 胞嘧啶在隆起或定位时脱氨基的倾向 与某些修饰过的碱基如2-氨基嘌呤相对。三是 研究DNA二级结构对脱氨基作用的影响，包括 A-型或Z-型构象或发夹或十字形的胞嘧啶 结构，我们将询问脱氨作用是否在动力学上增强， 胞嘧啶可以被质子化的DNA构象，如 三股螺旋第四，我们将继续核磁共振和量热 研究模型DNA核苷的目的是获得准确的 氢键相互作用的势能和自由能。的 一系列O（6）-烷基取代基对这些的电子效应 互动将被评估。

Our laboratory has been studying factors that induce deamination in DNA. We have initiated a series of studies on model nucleic acid base and polymer systems to examine two interrelated hypotheses: (1) that ionized base pairs may be found in DNA, and (2) 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 in these bases may comprise a previously undetermined source of genetic mutations. In order to study induced deamination, we have developed a reversion assay by which the rates of deamination can be assessed under a variety of conditions. In the present grant, we propose to extend our studies in several ways. We will test the hypotheses that an aberrant base like O(6)-methylguanine in one strand of DNA will induce deamination of the cytosine in the opposite strand, and that protonated cytosine residues in triple helices may deaminate with accelerated rates as compared to normal B-form DNA. First, we will continue our synthetic and site-directed mutagenesis studies to determine the rate and conditions under which cytosine deaminates in O(6)- alkylguanine:cytosine base pairs in DNA. By varying the nature of the O(6)-alkyl substituent, we can examine the effect of electron-donating and electron withdrawing groups on induced deamination. In this way, we hope to discriminate between two possible mechanisms of proton-induced deamination: i.e., local denaturation vs. trapped proton. Second, we will examine the propensity of cytosine to deaminate when it is bulged or is positioned opposite certain modified bases like 2-aminopurine. Third, we will investigate the effect of DNA secondary structure on deamination, including cytosine in an A-form or Z-form conformation or in a hairpin or cruciform structure,. We will ask whether the deamination is kinetically enhanced in DNA conformations where the cytosine can be protonated, as in triple-stranded helices. Fourth, we will continue NMR and calorimetric studies on model DNA nucleosides with the intent of obtaining accurate enthalpies and free energies of hydrogen bonding interactions. The electronic effects of a series of O(6)-alkyl substituents on these interactions will be evaluated.