Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation.
Kinetic studies of Escherichia coli AlkB using a new fluorescence-based assay for DNA demethylation.
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DOI：
10.1093/nar/gkm1031
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发表时间：
2007
影响因子：
14.9
通讯作者：
Bhagwat, A S
中科院分区：
文献类型：
--
作者：
Roy, Todd W;Bhagwat, A S
大肠杆菌AlkB蛋白催化直接逆转DNA烷基化损伤；主要是由内源性或环境烷化剂造成的 1-甲基腺嘌呤 (1mA) 和 3-甲基胞嘧啶 (3mC) 损伤。 AlkB 是非血红素铁 (II) α-酮戊二酸依赖性双加氧酶超家族的成员，它通过氧化消除甲基作为甲醛来去除烷基。我们开发了一种基于荧光的检测方法来检测该酶家族的脱烷基化活性。它使用甲醛脱氢酶将甲醛转化为甲酸，并使用荧光监测 NADH 类似物的产生。该检测方法比现有的 DNA 去甲基化检测方法有很大改进，因为它连续、快速且不需要放射性标记材料。它还可用于研究其他去甲基化反应，包括组蛋白去甲基化。我们用它来确定 AlkB 的动力学常数，发现它们与之前报道的值有些不同。结果表明，AlkB 以相当的效率对 1mA 和 3mC 进行去甲基化，并且相对于双链 DNA 底物，对单链 DNA 底物的偏好程度较低。
The Escherichia coli AlkB protein catalyzes the direct reversal of alkylation damage to DNA; primarily 1-methyladenine (1mA) and 3-methylcytosine (3mC) lesions created by endogenous or environmental alkylating agents. AlkB is a member of the non-heme iron (II) α-ketoglutarate-dependent dioxygenase superfamily, which removes the alkyl group through oxidation eliminating a methyl group as formaldehyde. We have developed a fluorescence-based assay for the dealkylation activity of this family of enzymes. It uses formaldehyde dehydrogenase to convert formaldehyde to formic acid and monitors the creation of an NADH analog using fluorescence. This assay is a great improvement over the existing assays for DNA demethylation in that it is continuous, rapid and does not require radioactively labeled material. It may also be used to study other demethylation reactions including demethylation of histones. We used it to determine the kinetic constants for AlkB and found them to be somewhat different than previously reported values. The results show that AlkB demethylates 1mA and 3mC with comparable efficiencies and has only a modest preference for a single-stranded DNA substrate over its double-stranded DNA counterpart.