Yoshikawa M, Iwasaki H, Kinoshita K, Shinagawa H
Department of Molecular Microbiology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.
Genes Cells. 2000 Oct;5(10):803-13. doi: 10.1046/j.1365-2443.2000.00371.x.
Crystallographic and mutational studies of Escherichia coli RuvC Holliday junction resolvase have revealed that a catalytic site of each subunit is composed of four acidic residues at the bottom of the putative DNA-binding cleft, whose surface contains eight basic residues.
To elucidate the functional roles of the basic residues on the cleft surface, we constructed a series of mutant ruvC genes and characterized their properties in vivo and in vitro. Among them, two RuvC mutants with a single alteration, K107A and K118A, were defective in UV-repair and showed a dominant negative effect. The purified K107A and K118A proteins showed reduced binding activity to the junction DNA in the presence of Mg2+ under high salt conditions. Mn2+ increased both the junction binding and cleaving activities of the mutant proteins. In the absence of a divalent cation, the wild-type, K107A and K118A proteins did not bind to junction DNA under high salt conditions, but the D7N mutant, with an alteration of the catalytic centre, was able to bind to the junction efficiently.
The results presented here, in conjunction with previous crystallographic studies, suggest that the catalytic complex which is formed through interactions of acidic residues, Mg2+ and a cleavable phosphodiester bond, is stabilized by Lys-107 and Lys-118 via electrostatic interactions with the DNA backbone, a process which is critically important for the cleavage reaction to take place. One or two basic residues near the catalytic centre have also been found in other RNase H superfamily proteins, indicating that this is the conserved reaction mechanism in this superfamily.
对大肠杆菌RuvC霍利迪连接体解离酶的晶体学和突变研究表明，每个亚基的催化位点由假定的DNA结合裂隙底部的四个酸性残基组成，其表面含有八个碱性残基。
为了阐明裂隙表面碱性残基的功能作用，我们构建了一系列突变型ruvC基因，并在体内和体外对其特性进行了表征。其中，两个单突变的RuvC突变体K107A和K118A在紫外线修复方面存在缺陷，并表现出显性负效应。纯化的K107A和K118A蛋白在高盐条件下存在Mg2+时，与连接体DNA的结合活性降低。Mn2+增加了突变蛋白的连接体结合和切割活性。在没有二价阳离子的情况下，野生型、K107A和K118A蛋白在高盐条件下不与连接体DNA结合，但催化中心发生改变的D7N突变体能够有效地与连接体结合。
本文给出的结果与先前的晶体学研究相结合，表明通过酸性残基、Mg2+和可切割的磷酸二酯键相互作用形成的催化复合物，通过Lys-107和Lys-118与DNA主链的静电相互作用而稳定，这一过程对于切割反应的发生至关重要。在其他核糖核酸酶H超家族蛋白中也发现了催化中心附近的一个或两个碱性残基，表明这是该超家族中保守的反应机制。
