Powell L M, Dryden D T, Willcock D F, Pain R H, Murray N E
Institute of Cell and Molecular Biology, University of Edinburgh, U.K.
J Mol Biol. 1993 Nov 5;234(1):60-71. doi: 10.1006/jmbi.1993.1563.
The methyltransferase of the EcoK type I restriction/modification system is trimeric, M2S1, where the S subunit determines the sequence specificity of the enzyme. The methyltransferase has a strong preference for hemimethylated substrate DNA and, therefore, we have investigated the effect of the methylation state of DNA on binding by the enzyme, together with the effects on binding of the cofactor S-adenosyl-L-methionine. Our results indicate that the methyltransferase has two non-interacting S-adenosyl-L-methionine binding sites, each with a dissociation constant of 3.60 (+/- 0.42) microM determined by equilibrium dialysis, or 2.21 (+/- 0.29) microM determined by the displacement of a fluorescent probe. Ultraviolet light-induced crosslinking showed that S-adenosyl-L-methionine binds strongly only to the modification (M) subunits. Changes in the sedimentation velocity of the methyltransferase imply a protein conformational change due to S-adenosyl-L-methionine binding. Gel retardation results show that the binding of S-adenosyl-L-methionine to the methyltransferase enhances binding to both specific and non-specific DNAs, but the enhancement is greater for the specific DNA. Differences in binding affinities contribute to the recognition of the specific nucleotide sequence AAC(N)6GTGC by the methyltransferase in preference to a non-specific sequence. In contrast, although the complexes of unmodified and hemimethylated DNAs with the methyltransferase have different mobilities in non-denaturing gels, there appears to be no contribution of binding affinity to the distinction between these two substrates. Therefore, the preference for a hemimethylated substrate must be due to a difference in catalysis.
EcoK I 型限制/修饰系统的甲基转移酶是三聚体，即M2S1，其中S亚基决定了该酶的序列特异性。该甲基转移酶对半甲基化的底物DNA有强烈偏好，因此，我们研究了DNA甲基化状态对该酶结合的影响，以及辅因子S-腺苷-L-甲硫氨酸对结合的影响。我们的结果表明，甲基转移酶有两个不相互作用的S-腺苷-L-甲硫氨酸结合位点，通过平衡透析测定，每个位点的解离常数为3.60（±0.42）μM，通过荧光探针的置换测定为2.21（±0.29）μM。紫外线诱导的交联表明，S-腺苷-L-甲硫氨酸仅与修饰（M）亚基强烈结合。甲基转移酶沉降速度的变化意味着由于S-腺苷-L-甲硫氨酸的结合导致蛋白质构象发生变化。凝胶阻滞结果表明，S-腺苷-L-甲硫氨酸与甲基转移酶的结合增强了对特异性和非特异性DNA的结合，但对特异性DNA的增强作用更大。结合亲和力的差异有助于甲基转移酶优先识别特定核苷酸序列AAC(N)6GTGC而非非特异性序列。相比之下，尽管未修饰和半甲基化DNA与甲基转移酶的复合物在非变性凝胶中有不同的迁移率，但结合亲和力似乎对区分这两种底物没有贡献。因此，对半甲基化底物的偏好一定是由于催化作用的差异。
