PubMed Health⌕ Search

Biomedical subjects

G J Sharples

Publications and source records attributed to G J Sharples.

33 records · Page 2Linked to original sources
PubMed1994

A mutation in helicase motif III of E. coli RecG protein abolishes branch migration of Holliday junctions.

The RecG protein of Escherichia coli catalyses branch migration of Holliday junctions made by RecA and dissociates synthetic X junctions into duplex products in reactions that require hydrolysis of ATP. To investigate the mode of action of this enzyme a chromosomal mutation that inactivates recG (recG162) was cloned and sequenced. The recG162 mutation is a G:C to A:T transition, which produces an Ala428 to Val substitution in the protein. This change affects a motif (motif III) in the protein that is highly conserved in DNA and RNA helicases. RecG162 protein was purified and shown to retain the ability to bind synthetic X and Y junctions. However, it does not dissociate these junctions and fails to catalyse branch migration of Holliday junction intermediates purified from a RecA strand exchange reaction. RecG162 retains a DNA-dependent ATPase activity, but this is much reduced relative to the wild-type protein, especially with single-stranded DNA as a co-factor. These results suggest that branch migration by RecG is related to a junction-targeted DNA helicase activity.

Adenosine Triphosphatases↗
PubMed1993

An E. coli RuvC mutant defective in cleavage of synthetic Holliday junctions.

Escherichia coli RuvC protein is a specific endonuclease that resolves recombination intermediates into viable products. The structural features needed for RuvC activity were investigated by sequencing three ruvC mutations and relating the base pair changes identified to the activity of the mutant proteins. Each of the three mutations is a single base-pair substitution. ruvC51 converts glycine-15 to an aspartic acid residue. The product of ruvC51 was purified and shown to retain the ability to bind junctions, albeit with a slightly reduced affinity. However, it has lost the ability to resolve these structures by symmetrical cleavage. A multicopy ruvC51 plasmid confers sensitivity to UV light in a ruvC+ strain. The ruvC53 allele causes a glycine-17 to serine substitution while ruvC55 produces a stop codon. Neither of these genes produces a stable product. The results suggest that the N-terminal domain of RuvC may be concerned with cleavage of junctions.

Amino Acid Sequence↗
PubMed1993

Processing of recombination intermediates by the RecG and RuvAB proteins of Escherichia coli.

The RuvAB, RuvC and RecG proteins of Escherichia coli process intermediates in recombination and DNA repair into mature products. RuvAB and RecG catalyse branch migration of Holliday junctions, while RuvC resolves these structures by nuclease cleavage around the point of strand exchange. The overlap between RuvAB and RecG was investigated using synthetic X- and Y-junctions. RuvAB is a complex of RuvA and RuvB, with RuvA providing the DNA binding subunit and RuvB the ATPase activity that drives branch migration. Both RuvA and RecG form defined complexes with each of the junctions. The gel mobilities of these complexes suggests that the X-junction attracts two tetramers of RuvA, but mainly monomers of RecG. Dissociation of the junction in the presence of ATP requires high levels of RuvAB. RecG is shown to have a much higher specific activity to the extent that very little of this protein would be required to match RuvAB in vivo. Both proteins also dissociate a Y-junction, which is consistent with helicase activity. However, RecG shows no ability to unwind more conventional substrates and the suggestion is made that its helicase activity is directed towards specific DNA structures such as junctions.

Amino Acid Sequence↗
PubMed1993

Dissociation of synthetic Holliday junctions by E. coli RecG protein.

The RecG protein of Escherichia coli is needed for normal levels of recombination and for repair of DNA damaged by ultraviolet light, mitomycin C and ionizing radiation. The true extent of its involvement in these processes is masked to a large degree by what appears to be a functional overlap with the products of the three ruv genes. RuvA and RuvB act together to promote branch migration of Holliday junctions, while RuvC catalyses the resolution of these recombination intermediates into viable products by endonuclease cleavage. In this paper, we describe the overproduction and purification of RecG and demonstrate that the overlap extends to the biochemistry. We show that the 76 kDa RecG protein is a DNA-dependent ATPase, like RuvB. Using gel retardation assays we demonstrate that it binds specifically to a synthetic Holliday junction, like RuvA and RuvC. Finally, we show that in the presence of ATP and Mg2+, RecG dissociates these junctions to duplex products, like RuvAB. We suggest that RecG and RuvAB provide alternative activities than can promote branch migration of Holliday junctions in recombination and DNA repair.

Adenosine Triphosphatases↗
PubMed1993

Resolution of Holliday intermediates in recombination and DNA repair: indirect suppression of ruvA, ruvB, and ruvC mutations.

The ruvA, ruvB, and ruvC genes of Escherichia coli provide activities that catalyze branch migration and resolution of Holliday junction intermediates in recombination. Mutation of any one of these genes interferes with recombination and reduces the ability of the cell to repair damage to DNA. A suppressor of ruv mutations was identified on the basis of its ability to restore resistance to mitomycin and UV light and to allow normal levels of recombination in a recBC sbcBC strain carrying a Tn10 insertion in ruvA. The mutation responsible was located at 12.5 min on the genetic map and defines a new locus which has been designated rus. The rus suppressor works just as well in recBC sbcA and rec+ sbc+ backgrounds and is not allele specific. Mutations in ruvB and ruvC are suppressed to an intermediate level, except when ruvA is also inactive, in which case suppression is complete. In all cases, suppression depends on RecG protein, a DNA-dependent ATPase that catalyzes branch migration of Holliday junctions. The rus mutation activates an additional factor that probably works with RecG to process Holliday junction intermediates independently of the RuvAB and RuvC proteins. The possibility that this additional factor is a junction-specific resolvase is discussed.

Bacterial Proteins↗
PubMed1992

Genetic analysis of recombination in prokaryotes.

Bacteria provide a simple system for the genetic analysis of homologous recombination. More than twenty genes have been identified in Escherichia coli. The enzymatic activities associated with the products of many of these genes have been revealed by studies with model DNA substrates. It is now possible to pair homologous molecules in vitro and process these through defined intermediates into mature recombinants of the types predicted by genetic crosses.

Bacteria↗
PubMed1991

Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product.

In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts. We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose. The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants. Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro. This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity. The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.

Bacterial Proteins↗
PubMed1991

Location of a mutation in the aspartyl-tRNA synthetase gene of Escherichia coli K12.

A mutation (tls-1) that confers a temperature-sensitive growth phenotype in Escherichia coli was shown by DNA cloning and sequencing to be an allele of aspS, the gene for aspartyl-tRNA synthetase. The mutation, which lies near minute 41 on the genetic map, was located some 2.3 kb from the 5' end of the ruvAB operon. A DNA fragment encoding the carboxy-terminus of AspRS was found to be sufficient to allow growth of a tls-1 strain at the non-permissive temperature.

Alleles↗
PubMed1991

Molecular organization and nucleotide sequence of the recG locus of Escherichia coli K-12.

The nucleotide sequence of the Escherichia coli K-12 recG gene was determined. recG was identified as an open reading frame located between the spoT operon and the convergent gltS gene. It encodes a polypeptide of 693 amino acids which was identified as a 76-kDa protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after it was labeled with [35S]methionine in maxicells. The sequence determined revealed no obvious promoter. Synthesis of RecG by plasmids carrying the intact gene varied with the orientation of the insert relative to the vector promoter and with the extent of upstream spoT operon sequence included in the construction. It is concluded that recG is the fourth and last gene in the spoT operon, although a possible promoter for independent transcription of spoU and recG was identified near the end of the spoT gene. The primary sequence of RecG revealed that it is related to proteins that act as helicases and has a well-conserved motif identified with ATP binding.

Amino Acid Sequence↗
PubMed1991

Resolution of Holliday junctions in Escherichia coli: identification of the ruvC gene product as a 19-kilodalton protein.

The ruvC gene of Escherichia coli specifies a nuclease that resolves Holliday junction intermediates in genetic recombination (B. Connolly, C.A. Parsons, F.E. Benson, H.J. Dunderdale, G.J. Sharples, R.G. Lloyd, and S.C. West, Proc. Natl. Acad, Sci. USA 88:6063-6067, 1991). The gene was located between aspS and the ruvAB operon by DNA sequencing and deletion analysis of ruvC plasmids and was shown to encode a protein of 18,747 Da. Analysis of the DNA flanking ruvC indicated that the gene is transcribed independently of the LexA-regulated ruvAB operon and is not under direct SOS control. ruvC lies downstream of an open reading frame, orf-33, for a protein which migrates during sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a 33-kDa polypeptide. These two genes probably form an operon. However, expression of ruvC was found to be very poor relative to that of orf-33. A double ribosomal frameshift between these genes is proposed as a possible reason for the low level of RuvC. Two further open reading frames of unknown function were identified, one on either side of the orf-33-ruvC operon.

Amino Acid Sequence↗
PubMed1990

A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes.

We report the discovery of a novel group of highly conserved DNA sequences located within the intergenic regions of the chromosomes of Escherichia coli, Salmonella typhimurium and other bacteria. These intergenic repeat units (IRUs) are 124-127 nucleotides long and have the potential to form stable stem-loop structures. The location of these sequences within the intergenic regions is variable with respect to known or putative signals for transcription and translation of the flanking genes. Some of the IRU sequences are transcribed, others are probably not. The structure and possible functions of these sequences are discussed in relation to palindromic units and other repeated DNA sequences in bacteria.

Base Sequence↗
PubMed1990

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination.

Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.

Blotting, Southern↗
PubMed1988

Nucleotide sequencing of the ruv region of Escherichia coli K-12 reveals a LexA regulated operon encoding two genes.

The nucleotide sequence of a 2505 bp region of the Escherichia coli chromosome containing the LexA regulated ruv gene has been determined. A sequence of 1631 bp encoding two non-overlapping open reading frames that constitute a single operon and which specify polypeptides with predicted molecular weights of 22172 daltons and 37177 daltons respectively, was identified as the most probable sequence for ruv. Each of the two open reading frames, designated ruvA and ruvB, is preceded by a reasonable Shine-Dalgarno sequence. Two 16 bp sequences (SOS boxes) that match the consensus sequence for binding LexA protein are located 5' to ruvA in a region that provides a possible single promoter for expression of both ruvA and ruvB, with the second SOS box overlapping the putative -35 region. A possible transcriptional terminator is located 137 bp downstream of ruvB. The amino acid sequence predicted for RuvB contains a region that matches a highly conserved sequence found in several DNA repair and recombination proteins that bind ATP.

Amino Acid Sequence↗
PubMedDate not supplied

Formation and resolution of recombination intermediates by E. coli RecA and RuvC proteins.

The recombination of DNA molecules has been reconstituted in vitro using two purified enzymes from Escherichia coli. RecA protein catalyses homologous pairing and strand exchange reactions to form intermediate DNA structures that are acted upon by RuvC. The newly identified RuvC protein resolves the intermediates by specific endonucleolytic cleavage to produce recombinant DNA molecules.

Bacterial Proteins↗