ESCHERICHIA-COLI RUVC PROTEIN IS AN ENDONUCLEASE THAT RESOLVES THE HOLLIDAY STRUCTURE
ESCHERICHIA-COLI RUVC PROTEIN IS AN ENDONUCLEASE THAT RESOLVES THE HOLLIDAY STRUCTURE
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DOI：
10.1002/j.1460-2075.1991.tb05016.x
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发表时间：
1991-12-01
期刊：
影响因子：
11.4
通讯作者：
SHINAGAWA, H
中科院分区：
文献类型：
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作者：
IWASAKI, H;TAKAHAGI, M;SHINAGAWA, H
遗传学证据表明，大肠杆菌ruvC基因参与了DNA修复和Ress和recf途径重组的后期过程。为了研究RuvC蛋白的生化性质，我们对RuvC蛋白进行了高产量和高纯度的研究。通过使用模型底物，我们研究了RuvC蛋白是一种内切酶的可能性，它解析Holliday结构，Holliday结构是基因重组的中间产物，在这种结构中，两个双链DNA分子通过单链交叉连接在一起。RuvC蛋白通过在具有相同极性的链中引入Nick来切割十字形连接，十字连接是由超螺旋质粒中的反向重复序列挤出形成的，在结构上类似于Holliday连接。缺口末端由大肠杆菌或T4DNA连接酶连接。对裂解位点的分析表明，决定裂解位点的是DNA拓扑而不是特定的序列。RuvC蛋白还裂解在RecA蛋白作用下形成的有缝隙的环状和线状双链DNA之间的Holliday连接。然而，它不会切割臂之间没有同源性的合成四向连接。凝胶过滤研究表明，RuvC蛋白的活性形式为二聚体。这在机械上适用于参与在交叉连接处交换DNA链的核酸内切酶。根据RuvC蛋白的这些性质和ruvC突变体的表型，我们推测RuvC蛋白是一种在体内解析Holliday结构的内切酶。
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.