Wyszynski M W, Gabbara S, Bhagwat A S
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Nucleic Acids Res. 1992 Jan 25;20(2):319-26. doi: 10.1093/nar/20.2.319.
The proposed mechanism for DNA (cytosine-5)-methyltransferases envisions a key role for a cysteine residue. It is expected to form a covalent link with carbon 6 of the target cytosine, activating the normally inactive carbon 5 for methyl transfer. There is a single conserved cysteine among all DNA (cytosine-5)-methyltransferases making it the candidate nucleophile. We have changed this cysteine to other amino acids for the EcoRII methylase; which methylates the second cytosine in the sequence 5'-CCWGG-3'. Mutants were tested for their methyl transferring ability and for their ability to form covalent complexes with DNA. The latter property was tested indirectly with the use of a genetic assay involving sensitivity of cells to 5-azacytidine. Replacement of the conserved cysteine with glycine, valine, tryptophan or serine led to an apparent loss of methyl transferring ability. Interestingly, cells carrying the mutant with serine did show sensitivity to 5-azacytidine, suggesting the ability to link to DNA. Unexpectedly, substitution of the cysteine with glycine results in the inhibition of cell growth and the mutant allele can be maintained in the cells only when it is poorly expressed. These results suggest that the conserved cysteine in the EcoRII methylase is essential for methylase action and it may play more than one role in it.
针对DNA（胞嘧啶-5）-甲基转移酶提出的作用机制设想了一个半胱氨酸残基的关键作用。预计它会与目标胞嘧啶的C6形成共价连接，激活通常无活性的C5以进行甲基转移。在所有DNA（胞嘧啶-5）-甲基转移酶中存在一个单一的保守半胱氨酸，使其成为候选亲核试剂。我们已将EcoRII甲基转移酶中的这个半胱氨酸替换为其他氨基酸；EcoRII甲基转移酶可使序列5'-CCWGG-3'中的第二个胞嘧啶甲基化。对突变体进行了甲基转移能力以及与DNA形成共价复合物能力的测试。后者的特性通过涉及细胞对5-氮杂胞苷敏感性的遗传测定进行间接测试。将保守半胱氨酸替换为甘氨酸、缬氨酸、色氨酸或丝氨酸导致甲基转移能力明显丧失。有趣的是，携带丝氨酸突变体的细胞确实对5-氮杂胞苷敏感，表明其具有与DNA连接的能力。出乎意料的是，用甘氨酸替代半胱氨酸会导致细胞生长受到抑制，并且只有在突变等位基因表达不佳时才能在细胞中维持。这些结果表明，EcoRII甲基转移酶中的保守半胱氨酸对于甲基转移酶的作用至关重要，并且它可能在其中发挥不止一种作用。
