Iwasaki H, Takahagi M, Shiba T, Nakata A, Shinagawa H
Department of Experimental Chemotherapy, Osaka University, Japan.
EMBO J. 1991 Dec;10(13):4381-9. doi: 10.1002/j.1460-2075.1991.tb05016.x.
Genetic evidence suggests that the Escherichia coli ruvC gene is involved in DNA repair and in the late step of RecE and RecF pathway recombination. To study the biochemical properties of RuvC protein, we overproduced and highly purified the protein. By employing model substrates, we examined the possibility that RuvC protein is an endonuclease that resolves the Holliday structure, an intermediate in genetic recombination in which two double-stranded DNA molecules are linked by single-stranded crossover. RuvC protein cleaves cruciform junctions, which are formed by the extrusion of inverted repeat sequences from a supercoiled plasmid and which are structurally analogous to Holliday junctions, by introducing nicks into strands with the same polarity. The nicked ends are ligated by E.coli or T4 DNA ligases. Analysis of the cleavage sites suggests that DNA topology rather than a particular sequence determines the cleavage site. RuvC protein also cleaves Holliday junctions which are formed between gapped circular and linear duplex DNA by the function of RecA protein. However, it does not cleave a synthetic four-way junction that does not possess homology between arms. The active form of RuvC protein, as studied by gel filtration, is a dimer. This is mechanistically suited for an endonuclease involved in swapping DNA strands at the crossover junctions. From these properties of RuvC protein and the phenotypes of the ruvC mutants, we infer that RuvC protein is an endonuclease that resolves Holliday structures in vivo.
遗传学证据表明，大肠杆菌ruvC基因参与DNA修复以及RecE和RecF途径重组的后期步骤。为了研究RuvC蛋白的生化特性，我们过量表达并高度纯化了该蛋白。通过使用模型底物，我们研究了RuvC蛋白是否是一种核酸内切酶，能够解析霍利迪结构，即遗传重组过程中的一种中间体，其中两个双链DNA分子通过单链交叉连接。RuvC蛋白通过在具有相同极性的链上引入切口来切割十字形连接，十字形连接是由超螺旋质粒中反向重复序列的挤出形成的，在结构上类似于霍利迪连接。切口末端由大肠杆菌或T4 DNA连接酶连接。对切割位点的分析表明，DNA拓扑结构而非特定序列决定了切割位点。RuvC蛋白还能切割由RecA蛋白功能在缺口环状和线性双链DNA之间形成的霍利迪连接。然而，它不能切割臂之间不具有同源性的合成四向连接。通过凝胶过滤研究发现，RuvC蛋白的活性形式是二聚体。从机制上讲，这适合于一种参与在交叉连接处交换DNA链的核酸内切酶。根据RuvC蛋白的这些特性以及ruvC突变体的表型，我们推断RuvC蛋白是一种在体内解析霍利迪结构的核酸内切酶。
