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.
The Escherichia coli ruvA and ruvB genes are involved in DNA repair and in the late step of homologous genetic recombination. We have demonstrated previously that the RuvA-RuvB protein complex in the presence of ATP promotes reabsorption of cruciform structures extruded from a supercoiled plasmid with an inverted repeat sequence. Because the cruciform structure is topologically analogous to the Holliday structure, we have proposed that the role of the RuvA and RuvB proteins in recombination is to promote a strand exchange reaction at the Holliday junction. Here, we studied the specific interaction of the RuvA-RuvB complex with the Holliday structure using synthetic analogs prepared by annealing four oligonucleotides. The affinities of the RuvA protein for synthetic Holliday junctions are much higher (>20-fold) than for duplex DNA, and the affinities of the RuvA protein for the junctions are further enhanced (>4-fold) by the interaction with the RuvB protein. The RuvA-RuvB protein complex in the presence of ATP promotes dissociation of the synthetic Holliday junction with homology in the central core into two halves by catalyzing branch migration to the DNA ends, but it does not affect the structure of the synthetic Holliday junction without the homology. The separation of the synthetic Holliday junction is a result of the activity of the RuvA-RuvB complex that promotes strand exchange and DNA unwinding. Furthermore, RuvA and RuvB promote the strand exchange reaction at the Holliday junctions made by RecA. These results provide further evidence that the RuvA-RuvB complex recognizes the Holliday junction and promotes branch migration in homologous recombination.
The Escherichia coli ruvC gene is involved in DNA repair and recombination and encodes an endonuclease that resolves Holliday structure in vitro. The 2.8-kb chromosomal DNA fragment that encompasses the ruvC gene and its flanking regions was cloned and sequenced. Four open reading frames were identified in the order orfl7-orJ26-ruvC-orfJ3 immediately upstream of the ruvAB operon, and their orientations are the same as the ruvAB operon, except for orf23. Proteins encoded by orfl7, or426, and ruvC (orfl9) were identified by the maxicell method, and their sizes agreed with those predicted from the DNA sequences. Among the open reading frames in this region, only ruvC is involved in the repair of UV-damaged DNA. ruvC appeared to be regulated by at least two promoters, but, in contrast to the ruvAB operon, ruvC is not regulated by the SOS system as demonstrated by operon fusions.Escherichia coli strains carrying mutations in the ruv locus were isolated initially as mutants which were sensitive to mitomycin C (19) or which showed reduced ability of recombination between duplicated gal genes (32). These mutants are also sensitive to various DNA-damaging agents such as UV, ionizing radiation, and some chemical mutagens, and they form multinucleated cells after treatment with low doses of such agents (18)(19)(20)32). Although ruv mutations have little effect on conjugal recombination in otherwise wild-type strains, they reduce the ability of recombination in recBC sbcA and recBC sbcBC strains (11)(12)(13). This evidence suggests that the ruv locus may be involved in recombinational repair by the RecE and the RecF pathways.The ruv locus contains two genes, ruvA and ruvB, that constitute an operon regulated by the SOS system (3,27,29). Recently these genes were also shown to be involved in mutagenesis induced by UV and X irradiation and by some chemicals (9, 24). The purified RuvB protein possesses weak ATPase activity (7), which is stimulated by the RuvA protein in the presence of DNA (26). The RuvA-RuvB complex in the presence of ATP renatures cruciform structure in supercoiled DNA with a palindromic sequence, indicating that it may promote strand exchange reactions in homologous recombination (26).While we were cloning the chromosomal DNA fragment that complemented the repair defect in the ruv mutants, we noticed that the phenotype of one of the ruv mutants we analyzed, CS85 (ruv-53), was not complemented by the plasmid carrying the ruvAB operon, but was complemented by the chromosomal DNA region upstream of the ruvAB operon. Sharples et al. (25) have identified the approximate location of this mutation on the physical map of this region and designated it ruvC. The ruvC mutants, as with the other ruv mutants, are defective in DNA repair and recombination of the RecE and the RecF pathways (1,12,19,25,30). In this study we analyzed the nucleotide sequence of the ruvC gene and its flanking regions and identified the proteins encoded by the ruvC region.RuvC protein has been highly purified recently and has an * Correspondin...
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