Ahmad I, Krishnamurthy V, Rao D N
Department of Biochemistry, Indian Institute of Science, Bangalore.
Gene. 1995 May 19;157(1-2):143-7. doi: 10.1016/0378-1119(95)00671-r.
The DNA-binding properties of the EcoP15I DNA methyltransferase (M.EcoP15I; MTase) were studied using electrophoretic mobility shift assays. We show by molecular size-exclusion chromatography and dimethyl suberimidate cross-linking that M.EcoP15I is a dimer in solution. While M.EcoP15I binds approx. threefold more tightly to its recognition sequence, 5'-CAGCAG-3', than to non-specific sequences in the presence of AdoMet or its analogs, the discrimination between specific and non-specific sequences significantly increases in presence of ATP. These results suggest for the first time a role for ATP in DNA recognition by type-III restriction-modification enzymes. Furthermore, we show that although c2 EcoPI mutant MTases are defective in AdoMet binding, they are still able to bind DNA in a sequence-specific manner.
使用电泳迁移率变动分析研究了EcoP15I DNA甲基转移酶（M.EcoP15I；MTase）的DNA结合特性。我们通过分子排阻色谱法和亚氨二甲酸二琥珀酰亚胺酯交联表明，M.EcoP15I在溶液中是二聚体。虽然在存在腺苷甲硫氨酸（AdoMet）或其类似物的情况下，M.EcoP15I与其识别序列5'-CAGCAG-3'的结合比与非特异性序列的结合紧密约三倍，但在存在ATP的情况下，特异性和非特异性序列之间的区分显著增加。这些结果首次表明ATP在III型限制修饰酶识别DNA中发挥作用。此外，我们表明，虽然c2 EcoPI突变型MTases在AdoMet结合方面存在缺陷，但它们仍然能够以序列特异性方式结合DNA。
