Ford K, Taylor C, Connolly B, Hornby D P
Krebs Institute, Department of Molecular Biology, University of Sheffield, U.K.
J Mol Biol. 1993 Apr 5;230(3):779-86. doi: 10.1006/jmbi.1993.1200.
MspI methyltransferase (M.MspI) catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C-5 position of the outer deoxycytidine base in the DNA sequence 5'-CCGG-3'. Recombinant M.MspI when expressed and purified as a translational fusion with glutathione-S-transferase, shows all of the properties of the wild-type enzyme. We report the kinetic analysis of M.MspI binding to DNA, which suggests a two-stage methylation process, whose initial DNA binding rate is governed by the presence of a positively charged sulphonium centre on the cofactor. Results are also presented that indicate that M.MspI binds preferentially to hemi-methylated DNA and that full methylation of either deoxycytidine on both strands significantly impairs sequence-specific protein-DNA interactions. Furthermore, the importance of the 4-amino group of the inner deoxycytidine for sequence-specific protein-DNA interactions is demonstrated by substituting deoxycytidine with 2-pyrimidinone-1-beta-D-2-deoxyriboside. In addition, we detail the intrinsic structural elements of a cofactor, required to enhance the binding of M.MspI to its recognition sequence, by using S-adenosyl-L-methionine and a range of derivatives.
MspI甲基转移酶（M.MspI）催化甲基从S-腺苷-L-甲硫氨酸转移至DNA序列5'-CCGG-3'中外部脱氧胞苷碱基的C-5位。重组M.MspI与谷胱甘肽-S-转移酶作为翻译融合体表达并纯化后，表现出野生型酶的所有特性。我们报告了M.MspI与DNA结合的动力学分析，结果表明存在一个两阶段甲基化过程，其初始DNA结合速率受辅因子上带正电荷的锍中心的影响。研究结果还表明，M.MspI优先结合半甲基化DNA，两条链上任何一个脱氧胞苷的完全甲基化都会显著损害序列特异性蛋白质-DNA相互作用。此外，通过用2-嘧啶酮-1-β-D-2-脱氧核糖核苷取代脱氧胞苷，证明了内部脱氧胞苷的4-氨基对于序列特异性蛋白质-DNA相互作用的重要性。此外，我们通过使用S-腺苷-L-甲硫氨酸及其一系列衍生物，详细阐述了辅因子的内在结构元件，这些元件可增强M.MspI与其识别序列的结合。
