DNA recognition by the methyl-CpG binding domain of MeCP2

被引:79
作者
Free, A
Wakefield, RID
Smith, BO
Dryden, DTF
Barlow, PN
Bird, AP
机构
[1] Univ Edinburgh, Inst Cell & Mol Biol, Edinburgh EH9 3JR, Midlothian, Scotland
[2] Univ Edinburgh, Edinburgh Ctr Prot Technol, Edinburgh EH9 3JJ, Midlothian, Scotland
关键词
D O I
10.1074/jbc.M007224200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:3353 / 3360
页数:8
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