FORMALDEHYDE AS A PROBE OF DNA-STRUCTURE .4. MECHANISM OF INITIAL REACTION OF FORMALDEHYDE WITH DNA
FORMALDEHYDE AS A PROBE OF DNA-STRUCTURE .4. MECHANISM OF INITIAL REACTION OF FORMALDEHYDE WITH DNA
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DOI：
10.1021/bi00634a002
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发表时间：
1977-01-01
期刊：
影响因子：
2.9
通讯作者：
VONHIPPEL, PH
中科院分区：
文献类型：
--
作者：
MCGHEE, JD;VONHIPPEL, PH
甲醛被用作天然DNA动态行为的探针。初始变性率可能随温度、甲醛浓度、DNA解链温度和DNA分子量而变化。来自反应初始阶段的DNA的EM证实变性起始于DNA分子内部的AT富集区域。整体变性率随pH值的增加而增加。由于唯一的pH依赖性化学反应是在胸腺嘧啶（和鸟嘌呤）的亚氨基直接位于沃森-克里克螺旋的中间，链间H-键可能会打破反应前。通过研究变性速率作为温度和pH的函数，在通常的反应条件下，变性可能涉及与胸腺嘧啶和腺嘌呤的官能团形成加合物。胸腺嘧啶反应（其是快速可逆的）在高温和高pH条件下主导变性;相反，腺嘌呤反应可以被认为是有效不可逆的，并且在腺嘌呤反应主导的条件下分析反应速率是一个巨大的简化。通过检查与单链多核苷酸的反应速率作为温度的函数，在与甲醛反应之前可能必须解堆;解堆之后，反应速率与单核苷酸的反应速率基本相同。还证明了单羟甲基化腺嘌呤可以与胸腺嘧啶形成一个碱基对，羟甲基与碱基对共面，并突出到双螺旋DNA结构的大沟中。这种取代的碱基对是. apprx。1.5 kcal/mol比未反应的AT对更不稳定。这种稳定性差异可以定量地归因于简单的立体化学，并且可以用于确定由于1个化学反应而变性的相邻碱基对的数目。甲醛与双螺旋DNA中的腺嘌呤部分的初始反应如下进行：DNA碱基对的小序列变性（即，链间H键断裂和碱基解堆积）;暴露于这种自发波动中的腺嘌呤残基的环外氨基在可比的反应条件下以与游离单核苷酸相同的速率反应;反应后的腺嘌呤要么重新形成（较不稳定的）羟甲基化AT碱基对，或保持未堆叠和未氢键键合，这取决于温度和其他环境因素。考虑到这3个步骤，反应速率预测从简单的螺旋线圈理论使用实验确定的环路加权函数的Gralla和Crothers。计算和观察之间的一致性对于变性速率在因子2内，并且在表观活化能为1 kcal/mol。相反，实验数据可以用来获得独立的估计描述的行为在DNA中的小开环的环权重函数，以及计算与甲醛在内部切口和螺旋末端的反应的相对速率。从这些计算得出的中心结论是，在低于Tm的温度下，DNA最可能的瞬时变性状态由仅含有1个开放（非堆叠和非氢键）碱基对的环组成。在这一系列论文中，DNA与甲醛的初始反应机制的定义应该作为基因组调控过程中所涉及的复杂分子途径的部分模型，例如解链蛋白与最初的天然DNA序列或RNA聚合酶与最初封闭的启动子区域的相互作用。
Formaldehyde is used as a probe of the dynamic behavior of native DNA. Initial denaturation rates probably varied with temperature, formaldehyde concentration, DNA melting temperature and DNA MW. EM of DNA from the initial phases of the reaction verifies that denaturation initiates at AT-rich regions in the interior of the DNA molecule. The overall denaturation rate increased with increasing pH. Since the only pH-dependent chemical reaction is at the imino group of thymine (and guanine) located directly in the middle of the Watson-Crick helix, interchain H-bonds probably break prior to reaction. By studying denaturation rates as a function of temperature and pH, under the usual reaction conditions denaturation probably involves adduct formation with the functional groups of thymine and adenine. The thymine reaction (which is rapidly reversible) dominates the denaturation under conditions of high temperature and high pH; conversely, the adenine reaction can be considered to be effectively irreversible and analysis of reaction rates under adenine-reaction-dominated conditions is a vast simplification. By examining reaction rates with single-stranded polynucleotides as a function of temperature, probably must unstack prior to reaction with formaldehyde; following unstacking, the reaction rates are essentially identical to those of mononucleotides. It is also demonstrated that monohydroxymethylated adenine can forn a base pair with thymine, the hydroxymethyl group lying coplanar with the base pair and protruding into the major groove of the double-helical DNA structure. Such a substituted base pair is .apprx. 1.5 kcal/mol less stable than an unreacted AT pair. This stability difference can be quantitatively ascribed to simple stereochemistry and can be used to determine the number of neighboring base pairs which are denatured as a consequence of 1 chemical reaction. The initial reaction of formaldehyde with an adenine moiety in double-helical DNA proceeds as follows: a small sequence of DNA base pairs denatures (i.e., interchain H-bonds break and bases unstack) as a consequence of a local thermal fluctuation; the exocyclic amino group of an adenine residue exposed in this spontaneous fluctuation reacts at the same rate as the free mononucleotide under comparable reaction conditions; and the reacted adenine either re-forms into a (less stable) hydroxymethylated AT base pair, or remains unstacked and unhydrogen bonded, depending on temperature and other environmental factors. Taking these 3 steps into account, reaction rates are predicted from simple helix-coil theory using the experimentally determined loop-weighting functions of Gralla and Crothers. Agreement between calculations and observations is within a factor of 2 for denaturation rates and within .apprx. 1 kcal/mol for apparent activation energies. Conversely, the experimental data can be used to obtain independent estimates of loop-weighting functions describing the behavior of small open loops in DNA, as well as to calculate relative rates of reaction with HCHO at internal nicks and helix ends. The central conclusion from these calculations is that the most probable transiently denatured state of DNA at temperatures below Tm consists of loops containing only 1 open (unstacked and unhydrogen bonded) base pair. The definition, in this series of papers, of the initial reaction mechanism of DNA with formaldehyde should serve as a partial model for the complex molecular pathways involved in processes of genome regulation, such as the interaction of melting proteins with initially native DNA sequences or RNA polymerase with initially closed promoter regions.