Bergerat A, Guschlbauer W
Département de Biologie, Centre d'Etudes Nucléaires de Saclay, Gif-sur-Yvette, France.
Nucleic Acids Res. 1990 Aug 11;18(15):4369-75. doi: 10.1093/nar/18.15.4369.
The turnover of DNA-adenine-methylase of E. coli strongly decreases when the temperature is lowered. This has allowed us to study the binding of Dam methylase on 14 bp DNA fragments at 0 degrees C by gel retardation in the presence of Ado-Met, but without methylation taking place. The enzyme can bind non-specific DNA with low affinity. Binding to the specific sequence occurs in the absence of S-adenosyl-methionine (Ado-Met), but is activated by the presence of the methyl donor. The two competitive inhibitors of Ado-Met, sinefungin and S-adenosyl-homocysteine, can neither activate this binding to DNA by themselves, nor inhibit this activation by Ado-Met. This suggests that Ado-Met could bind to Dam methylase in two different environments. In one of them, it could play the role of an allosteric effector which would reinforce the affinity of the enzyme for the GATC site. The analogues can not compete for such binding. In the other environment Ado-Met would be in the catalytic site and could be exchanged by its analogues. We have also visualized conformational changes in Dam methylase induced by the simultaneous binding of Ado-Met and the specific target sequence of the enzyme, by an anomaly of migration and partial resistance to proteolytic treatment of the ternary complex Ado-Met/Dam methylase/GATC.
当温度降低时，大肠杆菌DNA-腺嘌呤甲基化酶的周转显著下降。这使我们能够在0摄氏度下，在存在腺苷甲硫氨酸（Ado-Met）但不发生甲基化的情况下，通过凝胶阻滞法研究Dam甲基化酶与14 bp DNA片段的结合。该酶能以低亲和力结合非特异性DNA。在没有S-腺苷甲硫氨酸（Ado-Met）的情况下，酶也能与特定序列结合，但甲基供体的存在会激活这种结合。Ado-Met的两种竞争性抑制剂，杀稻瘟菌素和S-腺苷同型半胱氨酸，既不能自身激活这种与DNA的结合，也不能抑制Ado-Met的这种激活作用。这表明Ado-Met可能在两种不同的环境中与Dam甲基化酶结合。在其中一种环境中，它可能起到变构效应物的作用，增强酶对GATC位点的亲和力。类似物无法竞争这种结合。在另一种环境中，Ado-Met会处于催化位点，并且可以被其类似物取代。我们还通过Ado-Met/Dam甲基化酶/GATC三元复合物迁移异常和对蛋白水解处理的部分抗性，观察到了由Ado-Met和该酶的特定靶序列同时结合所诱导的Dam甲基化酶构象变化。
