DNA recognition by the methyl-CpG binding domain of MeCP2
DNA recognition by the methyl-CpG binding domain of MeCP2
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DOI：
10.1074/jbc.m007224200
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发表时间：
2001-02-02
影响因子：
4.8
通讯作者：
Bird, AP
中科院分区：
文献类型：
--
作者：
Free, A;Wakefield, RID;Bird, AP
转录抑制因子MeCP 2的甲基-CpG结合结构域（MBD）已被提出通过带正电荷的DNA结合表面上的疏水补丁识别单个对称甲基化的CpG碱基对。我们已经测试了这种结合模型的突变衍生物的MeCP 2 MBD的电泳迁移率变动分析补充核磁共振结构分析。发现结合面上暴露的精氨酸侧链，特别是Arg-III，对于结合是关键的。Arg-111被发现与保守的天冬氨酸侧链Asp-121相互作用，其被提议定向精氨酸侧链以允许与DNA的特异性接触。无序的B-C环区域的构象灵活性，形成结合面的一部分，也被证明是重要的。相比之下，暴露的疏水侧链的突变对DNA结合的影响不太严重。这表明Arg-III侧链可能有助于对CpG位点的序列特异性识别，而不是简单地与磷酸骨架进行非特异性接触。在人类遗传性疾病Rett综合征中发现的MBD内的大多数错义突变显示或预测影响结构域的折叠，而不是直接影响DNA识别事件。
The methyl-CpG binding domain (MBD) of the transcriptional repressor MeCP2 has been proposed to recognize a single symmetrically methylated CpG base pair via hydrophobic patches on an otherwise positively charged DNA binding surface. We have tested this binding model by analysis of mutant derivatives of the MeCP2 MBD in electrophoretic mobility shift assays complemented by NMR structural analysis. Exposed arginine side chains on the binding face, in particular Arg-lll, were found to be critical for binding. Arg-111 was found to interact with the conserved aspartate side chain Asp-121, which is proposed to orientate the arginine side chain to allow specific contacts with the DNA. The conformational flexibility of the disordered B-C loop region, which forms part of the binding face, was also shown to be important. In contrast, mutation off the exposed hydrophobic side chains had a less severe effect on DNA binding. This suggests that the Arg-lll side chain may contribute to sequence-specific recognition of the CpG site rather than simply making nonspecific contacts with the phosphate backbone. The majority of missense mutations within the MBD found in the human genetic disorder Rett syndrome were shown or predicted to affect folding of the domain rather than the DNA recognition event directly.