Taylor C, Ford K, Connolly B A, Hornby D P
Krebs Institute for Biomolecular Research, Department of Molecular Biology, Sheffield University, U.K.
Biochem J. 1993 Apr 15;291 ( Pt 2)(Pt 2):493-504. doi: 10.1042/bj2910493.
The cloning and overexpression of the MspI DNA methyltransferase as a functional fusion with glutathione S-transferase is described. The fusion enzyme retains full biological activity and has been used to investigate the interaction of substrates and inhibitors with MspI DNA methyltransferase. The fusion enzyme has been purified to homogeneity in a single step on GSH-agarose and is free from contaminating exonuclease activity. The enzyme can be photolabelled with S-adenosyl-L-methionine and the level of incorporation of label is enhanced by the presence of a nonspecific DNA duplex. In the presence of a cognate oligodeoxynucleotide, no photolabelling was observed since methyl transfer occurs instead. The inclusion of a mechanism-based inhibitor of C-5 deoxycytidine DNA methylation (an oligodeoxynucleotide containing the base 2-pyrimidinone-1-beta-D-2'-deoxyribofuranoside in the position of the deoxycytidine to which methyl addition occurs), which is thought to form a covalent interaction with the reactive cysteine of such enzymes, led to an enhancement of S-adenosyl-L-methionine photolabelling which suggests that, in contrast with results obtained with EcoRII DNA methyltransferase [Som and Friedman (1991) J. Biol. Chem. 266, 2937-2945], methylcysteine is not the photolabelled product. The implications of the results obtained with this mechanism-based inhibitor are discussed with respect to other C-5-specific DNA methyltransferases. Gel-retardation assays in the presence of cognate oligodeoxynucleotides that contain the reactive pyrimidinone base in place of the deoxycytidine target base are described. These demonstrate that most probably a stable covalent bond is formed between the methyltransferase and this oligodeoxynucleotide. However, the alternative of extremely tight non-covalent binding cannot be rigorously excluded. Furthermore, the results from these experiments indicate that the reaction mechanism proceeds in a manner similar to that of HhaI DNA methyltransferase with sequence-specific DNA binding being followed by addition of S-adenosyl-L-methionine and concomitant isomerization of the ternary complex leading to methyl transfer. S-Adenosyl-L-homocysteine appears to inhibit the reaction pathway as a result of either competition with the methyl donor and potentiation of a high-affinity interaction between the enzyme and DNA in an abortive ternary complex or through an allosteric interaction.
本文描述了MspI DNA甲基转移酶与谷胱甘肽S-转移酶功能性融合后的克隆及过表达。融合酶保留了完整的生物活性，并已用于研究底物和抑制剂与MspI DNA甲基转移酶的相互作用。该融合酶可通过一步在谷胱甘肽琼脂糖上纯化至均一，且无外切核酸酶活性污染。该酶可用S-腺苷-L-甲硫氨酸进行光标记，非特异性DNA双链体的存在可提高标记掺入水平。在同源寡脱氧核苷酸存在的情况下，未观察到光标记，因为发生了甲基转移。加入一种基于机制的C-5脱氧胞苷DNA甲基化抑制剂（一种在发生甲基化的脱氧胞苷位置含有碱基2-嘧啶酮-1-β-D-2'-脱氧呋喃核糖苷的寡脱氧核苷酸），据认为该抑制剂会与此类酶的活性半胱氨酸形成共价相互作用，这导致S-腺苷-L-甲硫氨酸光标记增强，这表明与用EcoRII DNA甲基转移酶获得的结果[Som和Friedman（1991）《生物化学杂志》266, 2937 - 2945]相反，甲基半胱氨酸不是光标记产物。讨论了用这种基于机制的抑制剂获得的结果对其他C-5特异性DNA甲基转移酶的意义。描述了在同源寡脱氧核苷酸存在下进行的凝胶阻滞分析，这些寡脱氧核苷酸含有反应性嘧啶酮碱基以取代脱氧胞苷靶碱基。这些结果表明，甲基转移酶与该寡脱氧核苷酸之间很可能形成了稳定的共价键。然而，不能完全排除极其紧密的非共价结合的可能性。此外，这些实验结果表明，反应机制的进行方式与HhaI DNA甲基转移酶类似，即先进行序列特异性DNA结合，随后加入S-腺苷-L-甲硫氨酸并伴随三元复合物的异构化导致甲基转移。S-腺苷-L-高半胱氨酸似乎通过与甲基供体竞争并增强酶与DNA在无效三元复合物中的高亲和力相互作用，或通过变构相互作用来抑制反应途径。
