Molecular analysis of the Escherichia coli ruvC gene, which encodes a Holliday junction-specific endonuclease

Abstract

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...

Escherichia coli orf17 Codes for a Nucleoside Triphosphate Pyrophosphohydrolase Member of the MutT Family of Proteins

“…Its sequence agrees with Takahagi et al (4) except for the original GTG start site, which we modified to ATG as a matter of convenience. Expression of the gene from pETorf17 results in the appearance of a major band on SDS gels corresponding to a 17-kDa polypeptide ( Fig.…”

“…The solution structure of the MutT protein reveals this signature sequence to be located in the loop 1-helix 1 motif of an ␣ ϩ ␤ sandwich (30). Recently, crystals of Orf17 dATPase have been obtained suitable for x-ray analysis, 4 and it will be of interest to see how the structural features of the signature sequence common to both enzymes compare in these otherwise very diverse proteins. At present, no specific physiological function can be ascribed to the Orf 17 dATPase.…”

“…5B three additional proteins with the highly homologous MutT signature sequence, yet having no nucleoside triphosphatase activity and no apparent role in preventing mutations. Orf257 and Orf1.9 are newly discovered enzymes, which hydrolyze NADH (10) and GDP-mannose (11), respectively, and the Orf257 is an NADH pyrophosphatase, Orf1.9 is a GDP-mannose hydrolase, and A(p) 4 Aase is a P 1 -P 4 -di(adenosine-5Ј)-tetraphosphatase. Numerical superscripts refer to the position of each polypeptide within the complete protein.…”

“…This conserved amino acid signature sequence was found, by computer search, to be present in a number of open reading frames broadly distributed throughout nature, from viruses to humans (1,2), and one of these, orf17 (GenBank D10165, 1992), is the subject of this paper. The orf17 gene is located at 41 min on the E. coli chromosome (3) just upstream of the ruvC gene (4,5), which codes for a Holliday junction-specific endonuclease (4).…”

Evidence that Phenylalanine 69 in Escherichia coliRuvC Resolvase Forms a Stacking Interaction during Binding and Destabilization of a Holliday Junction DNA Substrate

“…Genetic analyses indicate that RuvC resolvase plays a defined role in processing recombination and repair intermediates (7)(8)(9)(10)(11). In vitro studies confirm that RuvC specifically interacts with and resolves Holliday junctions by endonucleolytic cleavage (12,13).…”