Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB
Transient kinetic analysis of oxidative dealkylation by the direct reversal DNA repair enzyme AlkB
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DOI：
10.1074/jbc.ra120.013517
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发表时间：
2020-05-22
影响因子：
4.8
通讯作者：
O'Brien, Patrick J.
中科院分区：
文献类型：
--
作者：
Baldwin, Michael R.;Admiraal, Suzanne J.;O'Brien, Patrick J.
AlkB是细菌Fe（II）？和2-酮戊二酸盐作为对外源核酸适应性反应的一部分，修复DNA和RNA中广泛的烷基化核碱基的依赖性双加氧酶？烷基化剂虽然有大肠杆菌AlkB及其同系物的结构和特异性的长期利益，在测定其修复活动的困难，限制了我们的理解，他们的底物特异性和动力学机制。在这里，我们使用定量动力学方法来确定AlkB识别和修复烷基化DNA的瞬时动力学。这些实验表明，AlkB是一种比以前报道的快得多的烷基化修复酶，并且它明显快于识别和切除一些相同碱基损伤的DNA修复糖基化酶。我们观察到，而1，N-6-乙烯基腺嘌呤可以修复AlkB在单链和双链DNA中具有相似的效率，1-甲基腺嘌呤优先修复单链DNA。我们的研究结果为今后AlkB及其人类同系物ALKBH 2和ALKBH 3的研究奠定了基础。
AlkB is a bacterial Fe(II)? and 2-oxoglutarate?dependent dioxygenase that repairs a wide range of alkylated nucleobases in DNA and RNA as part of the adaptive response to exogenous nucleic acid?alkylating agents. Although there has been longstanding interest in the structure and specificity of Escherichia coli AlkB and its homologs, difficulties in assaying their repair activities have limited our understanding of their substrate specificities and kinetic mechanisms. Here, we used quantitative kinetic approaches to determine the transient kinetics of recognition and repair of alkylated DNA by AlkB. These experiments revealed that AlkB is a much faster alkylation repair enzyme than previously reported and that it is significantly faster than DNA repair glycosylases that recognize and excise some of the same base lesions. We observed that whereas 1,N-6-ethenoadenine can be repaired by AlkB with similar efficiencies in both single- and double-stranded DNA, 1-methyladenine is preferentially repaired in single-stranded DNA. Our results lay the groundwork for future studies of AlkB and its human homologs ALKBH2 and ALKBH3.