Saito A, Iwasaki H, Ariyoshi M, Morikawa K, Shinagawa H
Department of Molecular Microbiology, Osaka University, Japan.
Proc Natl Acad Sci U S A. 1995 Aug 1;92(16):7470-4. doi: 10.1073/pnas.92.16.7470.
Escherichia coli RuvC protein is a specific endonuclease that resolves Holliday junctions during homologous recombination. Since the endonucleolytic activity of RuvC requires a divalent cation and since 3 or 4 acidic residues constitute the catalytic centers of several nucleases that require a divalent cation for the catalytic activity, we examined whether any of the acidic residues of RuvC were required for the nucleolytic activity. By site-directed mutagenesis, we constructed a series of ruvC mutant genes with similar amino acid replacements in 1 of the 13 acidic residues. Among them, the mutant genes with an alteration at Asp-7, Glu-66, Asp-138, or Asp-141 could not complement UV sensitivity of a ruvC deletion strain, and the multicopy mutant genes showed a dominant negative phenotype when introduced into a wild-type strain. The products of these mutant genes were purified and their biochemical properties were studied. All of them retained the ability to form a dimer and to bind specifically to a synthetic Holliday junction. However, they showed no, or extremely reduced, endonuclease activity specific for the junction. These 4 acidic residues, which are dispersed in the primary sequence, are located in close proximity at the bottom of the putative DNA binding cleft in the three-dimensional structure. From these results, we propose that these 4 acidic residues constitute the catalytic center for the Holliday junction resolvase and that some of them play a role in coordinating a divalent metal ion in the active center.
大肠杆菌RuvC蛋白是一种特异性核酸内切酶，在同源重组过程中负责拆分霍利迪连接体。由于RuvC的核酸内切酶活性需要二价阳离子，并且由于3个或4个酸性残基构成了几种需要二价阳离子来发挥催化活性的核酸酶的催化中心，因此我们研究了RuvC的任何酸性残基是否是核酸酶活性所必需的。通过定点诱变，我们构建了一系列ruvC突变基因，在13个酸性残基中的1个残基处进行了类似的氨基酸替换。其中，在Asp-7、Glu-66、Asp-138或Asp-141处发生改变的突变基因不能互补ruvC缺失菌株的紫外线敏感性，并且多拷贝突变基因导入野生型菌株时表现出显性负性表型。对这些突变基因的产物进行了纯化并研究了它们的生化特性。它们都保留了形成二聚体以及特异性结合合成霍利迪连接体的能力。然而，它们对连接体没有核酸酶活性，或者核酸酶活性极低。这4个分散在一级序列中的酸性残基，在三维结构中位于假定的DNA结合裂隙底部附近。根据这些结果，我们提出这4个酸性残基构成了霍利迪连接体拆分酶的催化中心，并且其中一些残基在活性中心中协调二价金属离子方面发挥作用。
