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首页> 美国卫生研究院文献>Molecules >The Role of Active-Site Residues Phe98 His239 and Arg243 in DNA Binding and in the Catalysis of Human Uracil–DNA Glycosylase SMUG1
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The Role of Active-Site Residues Phe98 His239 and Arg243 in DNA Binding and in the Catalysis of Human Uracil–DNA Glycosylase SMUG1

机译：活性位点残基Phe98His239和Arg243在DNA结合和人尿嘧啶-DNA糖基化酶SMUG1催化中的作用

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摘要

Human SMUG1 (hSMUG1) hydrolyzes the N-glycosidic bond of uracil and some uracil lesions formed in the course of epigenetic regulation. Despite the functional importance of hSMUG1 in the DNA repair pathway, the damage recognition mechanism has been elusive to date. In the present study, our objective was to build a model structure of the enzyme–DNA complex of wild-type hSMUG1 and several hSMUG1 mutants containing substitution F98W, H239A, or R243A. Enzymatic activity of these mutant enzymes was examined by polyacrylamide gel electrophoresis analysis of the reaction product formation and pre-steady-state analysis of DNA conformational changes during enzyme–DNA complex formation. It was shown that substitutions F98W and H239A disrupt specific contacts generated by the respective wild-type residues, namely stacking with a flipped out Ura base in the damaged base-binding pocket or electrostatic interactions with DNA in cases of Phe98 and His239, respectively. A loss of the Arg side chain in the case of R243A reduced the rate of DNA bending and increased the enzyme turnover rate, indicating facilitation of the product release step.
机译：人类SMUG1（hSMUG1）水解尿嘧啶的N-糖苷键和表观遗传调控过程中形成的一些尿嘧啶损伤。尽管hSMUG1在DNA修复途径中具有功能重要性，但迄今为止，损伤识别机制尚不清楚。在本研究中，我们的目标是建立野生型hSMUG1和几个包含取代F98W，H239A或R243A的hSMUG1突变体的酶-DNA复合物的模型结构。通过对反应产物形成的聚丙烯酰胺凝胶电泳分析和酶-DNA复合物形成过程中DNA构象变化的稳态分析，检查了这些突变酶的酶活性。结果表明，取代F98W和H239A破坏了由相应野生型残基产生的特异性接触，即在Phe98和His239情况下，在受损的碱基结合口袋中与倒置的Ura碱基堆叠或与DNA的静电相互作用。在R243A的情况下，Arg侧链的损失降低了DNA弯曲的速率并增加了酶的转化速率，表明促进了产物释放步骤。

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