Kinetic mechanisms and interaction of rat liver DNA methyltransferase with defined DNA substrates.
Kinetic mechanisms and interaction of rat liver DNA methyltransferase with defined DNA substrates.
复制标题
大鼠肝脏 DNA 甲基转移酶与特定 DNA 底物的动力学机制和相互作用。
DOI：
10.1007/bf00219397
复制
发表时间：
1987
影响因子：
4.3
通讯作者：
Lapeyre,JN
中科院分区：
文献类型：
--
作者：
Ruchirawat,M;Noshari,J;Lapeyre,JN
DNA 底物类似物由聚 (dC-dG)、M13 和 XP12 DNA 构建，不包含混合类型的甲基化位点。这些用于使用高度纯化的大鼠肝脏 DNA (胞嘧啶-5)-甲基转移酶 (DMase−) 制剂来区分维持和从头甲基化的不同动力学机制。发现单链 (ss) 和双链 (ds) DNA 上的从头甲基化遵循 Michaelis-Menten 动力学，而半甲基化位点的甲基化根据半甲基化区域的大小而显示出差异。在类似于新复制的 DNA 的维持甲基化的长延伸上，无法实现饱和，并且动力学显示出非理想的正协同动力学，而短延伸则显示出非 Michaelis-Menten 动力学和快速饱和。两种类型的 DMase-DNA 复合物可以通过 DNA-琼脂糖基质上的亲和层析和预孵育测定来区分。后来的复合物参与甲基转换，表现出增强的稳定性。人们发现，不同结构的 DNA 的竞争性与复合物形成的稳定性相似，例如，分别是单链 DNA、半 DNA 和双链 DNA。在利用 5-溴脱氧尿苷的研究中，胸腺嘧啶类似物保持基本反应机制不变，但增加了 km 和 S0.5，同时降低了这些反应的速度。
DNA substrate analogs were constructed from poly(dC-dG), M13, and XP12 DNA which do not contain a mixture of types of methylation sites. These were used to distinguish different kinetic mechanisms for maintenance andde novomethylation using a highly purified rat liver DNA (cytosine-5)-methyltransferase (DMase−) preparation.De novomethylation on single (ss) and double-stranded (ds) DNA was found to obey Michaelis-Menten kinetics while methylation of hemimethylated sites showed differences depending on size of the hemimethylated region. On long stretches analogous to maintenance methylation of newly replicated DNA, saturation could not be achieved and the kinetics showed non-ideal positive cooperative kinetics, while short stretches showed non-Michaelis-Menten kinetics and rapid saturation. Two types of DMase-DNA complexes could be distinguished by means of affinity chromatography on DNA-agarose matrices and in preincubation assays. The later complex, which is engaged in methyl group turnover, exhibited enhanced stability. The competitiveness of variously configured DNAs was found to parallel the stability of complex formation, e.g., ss, hemi- and ds DNA, respectively. In studies utilizing 5-bromodeoxyuridine, the thymine analog left the basic reaction mechanisms unchanged but increased the kmand S0.5while reducing the velocity of these reactions.