Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage
Oxidative demethylation by Escherichia coli AlkB directly reverts DNA base damage
复制标题
DOI：
10.1038/nature00908
复制
发表时间：
2002-09-12
期刊：
影响因子：
64.8
通讯作者：
Sedgwick, B
中科院分区：
文献类型：
--
作者：
Trewick, SC;Henshaw, TF;Sedgwick, B
甲基化剂产生细胞毒性和诱变性DNA损伤。细胞使用3-甲基腺嘌呤-DNA糖基酶从DNA中去除一些甲基化碱基，并使用自杀式的o -6-甲基鸟嘌呤-DNA甲基转移酶将其他病变的烷基转移到半胱氨酸残基上（1,2）。在这里，我们报道了高度保守的AlkB蛋白通过一种前所未有的机制修复DNA烷基化损伤。AlkB没有检测到核酸酶、DNA糖基化酶或甲基转移酶活性；然而，大肠杆菌alkB突变体在处理单链DNA产生的甲基化损伤方面存在缺陷（3-5）。理论上的蛋白质折叠识别表明，AlkB类似于Fe(II)-和α -酮戊二酸依赖的双加氧酶(6)，它们使用铁氧中间体氧化化学惰性化合物（7,8）。我们在这里证明纯化的AlkB在依赖于氧、α -酮戊二酸盐和铁（II）的反应中修复单链和双链DNA中的1-甲基腺嘌呤和3-甲基胞嘧啶的细胞毒性损伤。AlkB酶将α -酮戊二酸的氧化脱羧与DNA中这些甲基化碱基的羟基化偶联，导致直接还原到未修饰的碱基并释放甲醛。
Methylating agents generate cytotoxic and mutagenic DNA damage. Cells use 3-methyladenine-DNA glycosylases to excise some methylated bases from DNA, and suicidal O-6-methyl-guanine- DNA methyltransferases to transfer alkyl groups from other lesions onto a cysteine residue(1,2). Here we report that the highly conserved AlkB protein repairs DNA alkylation damage by means of an unprecedented mechanism. AlkB has no detectable nuclease, DNA glycosylase or methyltransferase activity; however, Escherichia coli alkB mutants are defective in processing methylation damage generated in single-stranded DNA(3-5). Theoretical protein fold recognition had suggested that AlkB resembles the Fe(II)- and alpha-ketoglutarate-dependent dioxygenases (6), which use iron-oxo intermediates to oxidize chemically inert compounds(7,8). We show here that purified AlkB repairs the cytotoxic lesions 1-methyladenine and 3-methylcytosine in single- and double-stranded DNA in a reaction that is dependent on oxygen, alpha-ketoglutarate and Fe( II). The AlkB enzyme couples oxidative decarboxylation of alpha-ketoglutarate to the hydroxylation of these methylated bases in DNA, resulting in direct reversion to the unmodified base and the release of formaldehyde.