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G C Walker

Publications and source records attributed to G C Walker.

At least 37 records · Page 2Linked to original sources
PubMed2000

The SOS response: recent insights into umuDC-dependent mutagenesis and DNA damage tolerance.

Be they prokaryotic or eukaryotic, organisms are exposed to a multitude of deoxyribonucleic acid (DNA) damaging agents ranging from ultraviolet (UV) light to fungal metabolites, like Aflatoxin B1. Furthermore, DNA damaging agents, such as reactive oxygen species, can be produced by cells themselves as metabolic byproducts and intermediates. Together, these agents pose a constant threat to an organism's genome. As a result, organisms have evolved a number of vitally important mechanisms to repair DNA damage in a high fidelity manner. They have also evolved systems (cell cycle checkpoints) that delay the resumption of the cell cycle after DNA damage to allow more time for these accurate processes to occur. If a cell cannot repair DNA damage accurately, a mutagenic event may occur. Most bacteria, including Escherichia coli, have evolved a coordinated response to these challenges to the integrity of their genomes. In E. coli, this inducible system is termed the SOS response, and it controls both accurate and potentially mutagenic DNA repair functions [reviewed comprehensively in () and also in ()]. Recent advances have focused attention on the umuD(+)C(+)-dependent, translesion DNA synthesis (TLS) process that is responsible for SOS mutagenesis (). Here we discuss the SOS response of E. coli and concentrate in particular on the roles of the umuD(+)C(+) gene products in promoting cell survival after DNA damage via TLS and a primitive DNA damage checkpoint.

Adaptation, Physiological↗
PubMed2000

Intravascular ultrasound-guided interventions in coronary artery disease: a systematic literature review, with decision-analytic modelling, of outcomes and cost-effectiveness.

BACKGROUND: Intravascular ultrasound (IVUS) is the generic name for any ultrasound technology used in vivo within the blood vessels. More specifically, intracoronary ultrasound enables imaging of the coronary arteries from within the lumen. This review concentrates on the role of intracoronary ultrasound as an adjunct to interventional cardiology. OBJECTIVES: (1) To identify the literature on IVUS for guiding coronary interventions, and to synthesise evidence about outcomes compared with outcomes when IVUS guidance has not been used. (2) To use this evidence, together with other information about costs and outcomes, to model the cost effectiveness of IVUS guidance. (3) To synthesise the evidence on the reproducibility of measurements of cross-sectional area made using IVUS. METHODS DATA SOURCES: (1) Electronic searches of MEDLINE, EMBASE, Science Citation Index, Index to Scientific and Technical Proceedings, Engineering Compendex, Engineering Page One, Cochrane Library, Inside (British Library), 1990-98. (2) Contacting experts and centres of expertise, 1990-99. (3) Internet search, 1990-99. METHODS STUDY SELECTION: Studies of IVUS-guided coronary interventions performed on humans were included in the review. Non-English language studies were also included when they covered IVUS-guided stenting or angioplasty. Control evidence regarding outcomes without IVUS guidance was sought only from randomised controlled trials (RCTs). Studies investigating the reproducibility of measurements of cross-sectional area were included only if the results were expressed in terms of the mean and standard deviation of paired differences. METHODS DATA EXTRACTION: Checklists that covered study details, patient characteristics and results were completed independently by three reviewers. Consensus was reached on any disagreements. Local data were gathered on the costs of IVUS-guided stenting. METHODS DATA SYNTHESIS: Overall event rates were calculated by pooling patient results from the included studies. A decision-analytic model was used to combine information from the literature with cost estimates, in order to predict cost-effectiveness in terms of cost per restenosis event avoided by the use of IVUS guidance. The analysis was performed from the perspective of the healthcare provider. Sensitivity analysis was undertaken. A simple extrapolation was made to long-term outcome so that cost-utility (using quality-adjusted life years (QALYs)) could be estimated. The minimum detectable change in cross-sectional area was estimated from the reproducibility results. RESULTS: Only one study on IVUS-guided angioplasty satisfied the inclusion criteria, and there were no studies on IVUS-guided atherectomy or other IVUS-guided interventions that satisfied the inclusion criteria. Of the 15 articles on IVUS-guided stenting that satisfied the inclusion criteria, seven presented data on outcomes at 6 months post-intervention. The angiographic restenosis rate was 16 +/- 1%. This compared with 24 +/- 2% derived from five articles on stenting without IVUS guidance. Data for follow-up periods longer than 6 months were presented in only two studies. Data from a total of five studies were included in the decision-analytic model. The cost per restenosis event avoided was 1545 pound sterling. After extrapolation to long-term outcome, the calculated cost per QALY was 6438 pound sterling. The baseline QALY gain was only 0.03 years. Sensitivity analysis resulted in large differences between the best- and worst-case scenarios, for example, from a saving of 5000 pound sterling to a cost of 24,000 pound sterling restenosis event avoided. The smallest changes in cross-sectional area that could be measured were 1.6 mm2 by a single observer and 1.9 mm2 by different observers. CONCLUSIONS: Implications for healthcare: The evidence available is too weak for there to be any reliable implications for clinical practice. (ABSTRACT TRUNCATED)

Algorithms↗
PubMed1999

Inferences concerning the ATPase properties of DnaK and other HSP70s are affected by the ADP kinase activity of copurifying nucleoside-diphosphate kinase.

Preparations of Escherichia coli DnaK from our lab as well as preparations of DnaK and other HSP70 proteins from several major labs in the field produce a stoichiometric initial burst of [alpha-(32)P]ADP when incubated with [alpha-(32)P]ATP and contain an ADP kinase activity. We determined that the initial burst activity results from the transfer of gamma-phosphate from the radiolabeled substrate [alpha-(32)P]ATP to unlabeled ADP bound by the DnaK and is the same activity that results in ADP phosphorylation. The purification of DnaK from E. coli cells that carry a disrupted ndk gene, ndk::km, results in preparations with greatly reduced ADP kinase activities compared with preparations of DnaK purified from ndk(+) cells. The reduction in the amount of ADP kinase activity in preparations of DnaK purified from ndk::km cells shows that nucleoside-diphosphate kinase (NDP kinase) is responsible for most of the ADP kinase activity present in DnaK preparations isolated from ndk(+) cells. The remaining ADP kinase activity in preparations from ndk::km cells, which varies between preparations, is also a property of NDP kinase, which is most likely expressed because of a low frequency reversion of the disrupted ndk gene. A weak, but measurable physical interaction exists between DnaK and NDP kinase and may be at least partially responsible for the co-purification of NDP kinase with DnaK. The presence of contaminating NDP kinase can explain the range of k(cat) values reported for the ATPase activity of DnaK as well as recent reports of initial burst kinetics by DnaK (Banecki, B., and Zylicz, M. (1996) J. Biol. Chem. 271, 6137-6143) and an ADP-ATP exchange activity of DnaK (Hiromura, M., Yano, M., Mori, H., Inoue, M., and Kido, H. (1998) J. Biol. Chem. 273, 5435-5438).

Adenosine Triphosphatases↗
PubMed1999

The Escherichia coli SOS mutagenesis proteins UmuD and UmuD' interact physically with the replicative DNA polymerase.

The Escherichia coli umuDC operon is induced in response to replication-blocking DNA lesions as part of the SOS response. UmuD protein then undergoes an RecA-facilitated self-cleavage reaction that removes its N-terminal 24 residues to yield UmuD'. UmuD', UmuC, RecA, and some form of the E. coli replicative DNA polymerase, DNA polymerase III holoenzyme, function in translesion synthesis, the potentially mutagenic process of replication over otherwise blocking lesions. Furthermore, it has been proposed that, before cleavage, UmuD together with UmuC acts as a DNA damage checkpoint system that regulates the rate of DNA synthesis in response to DNA damage, thereby allowing time for accurate repair to take place. Here we provide direct evidence that both uncleaved UmuD and UmuD' interact physically with the catalytic, proofreading, and processivity subunits of the E. coli replicative polymerase. Consistent with our model proposing that uncleaved UmuD and UmuD' promote different events, UmuD and UmuD' interact differently with DNA polymerase III: whereas uncleaved UmuD interacts more strongly with beta than it does with alpha, UmuD' interacts more strongly with alpha than it does with beta. We propose that the protein-protein interactions we have characterized are part of a higher-order regulatory system of replication fork management that controls when the umuDC gene products can gain access to the replication fork.

Bacterial Proteins↗
PubMed1999

A model for a umuDC-dependent prokaryotic DNA damage checkpoint.

The products of the Escherichia coli umuDC operon are required for translesion synthesis, the mechanistic basis of most mutagenesis caused by UV radiation and many chemicals. The UmuD protein shares homology with LexA, the repressor of SOS-regulated loci, and similarly undergoes a facilitated autodigestion on interaction with the RecA/single-stranded DNA nucleoprotein filaments formed after a cell experiences DNA damage. This cleavage, in which Ser-60 of UmuD acts as the nucleophile, produces UmuD', the form active in translesion synthesis. Expression of the noncleavable UmuD(S60A) protein and UmuC was found to increase survival after UV irradiation, despite the inability of the UmuD(S60A) protein to participate in translesion synthesis; this survival increase is uvr(+) dependent. Additional observations that expression of the UmuD(S60A) protein and UmuC delayed the resumption of DNA replication and cell growth after UV irradiation lead us to propose that the uncleaved UmuD protein and UmuC delay the resumption of DNA replication, thereby allowing nucleotide excision repair additional time to repair the damage accurately before replication is attempted. After a UV dose of 20 J/m(2), uncleaved UmuD is the predominant form for approximately 20 min, after which UmuD' becomes the predominant form, suggesting that the umuDC gene products play two distinct and temporally separated roles in DNA damage tolerance, the first in cell-cycle control and the second in translesion synthesis over unrepaired or irreparable lesions. The relationship of these observations to the eukaryotic DNA damage checkpoint is discussed.

Bacterial Proteins↗
PubMed1999

Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis.

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuD mutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190-7194, 1990). To identify elements of the UmuD' protein necessary for its role in translesion synthesis, we began with umuD', a modified form of the umuD gene that directly encodes the UmuD' protein, and obtained missense umuD' mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD'2 homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded beta-sheet and appears to interfere with UmuD'2 homodimer formation indirectly by affecting the structure of the UmuD' monomer. When expressed from a multicopy plasmid, the L40R umuD' mutant gene exhibited a dominant negative effect on a chromosomal umuD+ gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD' function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal beta-strand and results in a mutant UmuD' protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD+ strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD'2 homodimer, which are mobile in solution.

Bacterial Proteins↗
PubMed1999

A novel Sinorhizobium meliloti operon encodes an alpha-glucosidase and a periplasmic-binding-protein-dependent transport system for alpha-glucosides.

The most abundant carbon source transported into legume root nodules is photosynthetically produced sucrose, yet the importance of its metabolism by rhizobia in planta is not yet known. To identify genes involved in sucrose uptake and hydrolysis, we screened a Sinorhizobium meliloti genomic library and discovered a segment of S. meliloti DNA which allows Ralstonia eutropha to grow on the alpha-glucosides sucrose, maltose, and trehalose. Tn5 mutagenesis localized the required genes to a 6.8-kb region containing five open reading frames which were named agl, for alpha-glucoside utilization. Four of these (aglE, aglF, aglG, and aglK) appear to encode a periplasmic-binding-protein-dependent sugar transport system, and one (aglA) appears to encode an alpha-glucosidase with homology to family 13 of glycosyl hydrolases. Cosmid-borne agl genes permit uptake of radiolabeled sucrose into R. eutropha cells. Analysis of the properties of agl mutants suggests that S. meliloti possesses at least one additional alpha-glucosidase as well as a lower-affinity transport system for alpha-glucosides. It is possible that the Fix+ phenotype of agl mutants on alfalfa is due to these additional functions. Loci found by DNA sequencing to be adjacent to aglEFGAK include a probable regulatory gene (aglR), zwf and edd, which encode the first two enzymes of the Entner-Doudoroff pathway, pgl, which shows homology to a gene encoding a putative phosphogluconolactonase, and a novel Rhizobium-specific repeat element.

Bacterial Proteins↗
PubMed1999

Structural characterization of the symbiotically important low-molecular-weight succinoglycan of Sinorhizobium meliloti.

The production of succinoglycan by Sinorhizobium meliloti Rm1021 is required for successful nodule invasion by the bacterium of its host plant, alfalfa. Rm1021 produces succinoglycan, an acidic exopolysaccharide composed of an octasaccharide repeating unit modified with acetyl, succinyl, and pyruvyl moieties, in both low- and high-molecular-weight forms. Low-molecular-weight (LMW) succinoglycan, previously thought to consist of monomers, trimers, and tetramers of the repeating unit, has been reported as being capable of promoting the formation of nitrogen-fixing nodules by succinoglycan-deficient derivatives of strain Rm1021. We have determined that the three size classes of LMW succinoglycan species are in fact monomers, dimers, and trimers of the repeating unit and that the trimer is the species active in promoting nodule invasion. A detailed structural analysis of the components of LMW succinoglycan by using various chromatographic techniques, along with nuclear magnetic resonance analyses, has revealed that there is considerable heterogeneity within the LMW succinoglycan oligomers in terms of noncarbohydrate substitutions, and we have determined the structural basis of this heterogeneity.

Chromatography↗
PubMed1999

Citrate synthase mutants of Sinorhizobium meliloti are ineffective and have altered cell surface polysaccharides.

The gltA gene, encoding Sinorhizobium meliloti 104A14 citrate synthase, was isolated by complementing an Escherichia coli gltA mutant. The S. meliloti gltA gene was mutated by inserting a kanamycin resistance gene and then using homologous recombination to replace the wild-type gltA with the gltA::kan allele. The resulting strain, CSDX1, was a glutamate auxotroph, and enzyme assays confirmed the absence of a requirement for glutamate. CSDX1 did not grow on succinate, malate, aspartate, pyruvate, or glucose. CSDX1 produced an unusual blue fluorescence on medium containing Calcofluor, which is different from the green fluorescence found with 104A14. High concentrations of arabinose (0.4%) or succinate (0. 2%) restored the green fluorescence to CSDX1. High-performance liquid chromatography analyses showed that CSDX1 produced partially succinylated succinoglycan. CSDX1 was able to form nodules on alfalfa, but these nodules were not able to fix nitrogen. The symbiotic defect of a citrate synthase mutant could thus be due to disruption of the infection process or to the lack of energy generated by the tricarboxylic acid cycle.

Chromatography, High Pressure Liquid↗
PubMed1999

Genetic analysis of the mobilization and leading regions of the IncN plasmids pKM101 and pCU1.

The conjugative IncN plasmids pKM101 and pCU1 have previously been shown to contain identical oriT sequences as well as conserved restriction endonuclease cleavage patterns within their tra regions. Complementation analysis and sequence data presented here indicate that these two plasmids encode essentially identical conjugal DNA-processing proteins. This region contains three genes, traI, traJ, and traK, transcribed in the same orientation from a promoter that probably lies within or near the conjugal transfer origin (oriT). Three corresponding proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and complementation analysis confirmed that this region contains three tra complementation groups. All three proteins resemble proteins of the IncW plasmid R388 and other plasmids thought to have roles in processing of plasmid DNA during conjugation. The hydropathy profile of TraJ suggests a transmembrane topology similar to that of several homologous proteins. Both traK and traI were required for efficient interplasmid site-specific recombination at oriT, while traJ was not required. The leading region of pKM101 contains three genes (stbA, stbB, and stbC), null mutations in which cause elevated levels of plasmid instability. Plasmid instability was observed only in hosts that are proficient in interplasmid recombination, suggesting that this recombination can potentially lead to plasmid loss and that Stb proteins somehow overcome this, possibly via site-specific multimer resolution.

Amino Acid Sequence↗
PubMed1998

Biosynthetic control of molecular weight in the polymerization of the octasaccharide subunits of succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti.

Succinoglycan, a symbiotically important exopolysaccharide of Rhizobium meliloti, is composed of polymerized octasaccharide subunits, each of which consists of one galactose and seven glucoses with succinyl, acetyl, and pyruvyl modifications. Production of specific low molecular weight forms of R. meliloti exported and surface polysaccharides, including succinoglycan, appears to be important for nodule invasion. In a previous study of the roles of the various exo gene products in succinoglycan biosynthesis, exoP, exoQ, and exoT mutants were found to synthesize undecaprenol-linked fully modified succinoglycan octasaccharide subunits, suggesting possible roles for their gene products in polymerization or transport. Using improved techniques for analyzing succinoglycan biosynthesis by these mutants, we have obtained evidence indicating that R. meliloti has genetically separable systems for the synthesis of high molecular weight succinoglycan and the synthesis of a specific class of low molecular weight oligosaccharides consisting of dimers and trimers of the octasaccharide subunit. Models to account for our unexpected findings are discussed. Possible roles for the ExoP, ExoQ, and ExoT proteins are compared and contrasted with roles that have been suggested on the basis of homologies to key proteins involved in the biosynthesis of O-antigens and of certain exported or capsular cell surface polysaccharides.

Dimerization↗
PubMed1998

Interactions of peptides with DnaK and C-terminal DnaK fragments studied using fluorescent and radioactive peptides.

Monocysteine derivatives of Peptide C (KLIGVLSSLFRPK) were modified with N-((2-(acetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole (ANBD) to introduce a fluorescent probe. Five Peptide C derivatives-PepC-V5C-ANBD, PepC-L6C-ANBD, PepC-S7C-ANBD, PepC-S8C-ANBD, and PepC-L9C-ANBD-were then used to investigate the peptide-binding properties of DnaK. Introduction of the ANBD moiety at positions 8 and 9 of Peptide C yields peptides that bind to DnaK with a high affinity similar to unmodified peptide C. In contrast, the derivative carrying ANBD at position 6, PepC-L6C-ANBD, bound to DnaK with a binding affinity 470 times lower than that of PepC-L9C-ANBD. Peptide C derivatives carrying ANBD at positions 5 or 7 have intermediate DnaK binding affinities. By assaying the binding affinities of PepC-L9C-ANBD and PepNR-S6C-[1-14C]acetamide to DnaK and three C-terminal fragments of DnaK, DnaK 384-638 (residues 384 to 638), DnaK 389-607 (residues 389 to 607) and DnaK 386-561 (residues 386 to 561), we found that the last 31 residues of DnaK (residues 607-638) do not have a significant effect on the peptide binding to DnaK. However, residues 561 to 607, which form the C, D, and E alpha-helices directly adjacent to the peptide binding pocket of DnaK [X. Zhu, X. Zhou, W. F. Burkholder, A. Gragerov, C. M. Ogata, M. E. Gottesman, and W. A. Hendrickson, Science 272, 1606-1614, 1996], play important roles in stabilizing the DnaK/peptide complex. The kinetics of PepC-L9C-ANBD binding to DnaK, DnaK 384-638, DnaK 389-607, and DnaK 386-561 also support this observation.

Bacterial Proteins↗
PubMed1998

Identification of the Rhizobium meliloti alcohol dehydrogenase gene (adhA) and heterologous expression in Alcaligenes eutrophus.

A screen for Rhizobium meliloti genes which improve the growth of Alcaligenes eutrophus on sucrose identified the first alcohol dehydrogenase gene (adhA) isolated from the Rhizobiaceae. R. meliloti adhA is constitutively expressed in A. eutrophus and has alcohol dehydrogenase activity. R. meliloti adhA mutants retain some alcohol dehydrogenase activity.

Alcaligenes↗
PubMed1998

Degradation of carboxy-terminal-tagged cytoplasmic proteins by the Escherichia coli protease HflB (FtsH).

Proteins with short nonpolar carboxyl termini are unstable in Escherichia coli. This proteolytic pathway is used to dispose of polypeptides synthesized from truncated mRNA molecules. Such proteins are tagged with an 11-amino-acid nonpolar destabilizing tail via a mechanism involving the 10Sa (SsrA) stable RNA and then degraded. We show here that the ATP-dependent zinc protease HflB (FtsH) is involved in the degradation of four unstable derivatives of the amino-terminal domain of the lambdacI repressor: three with nonpolar pentapeptide tails (cI104, cI105, cI108) and one with the SsrA tag (cI-SsrA). cI105 and cI-SsrA are also degraded by the ClpP-dependent proteases. Loss of ClpP can be compensated for by overproducing HflB. In an in vitro system, cI108 and cI-SsrA are degraded by HflB in an energy-dependent reaction, indicating that HflB itself recognizes the carboxyl terminus. These results establish a tail-specific pathway for removing abnormal cytoplasmic proteins via the HflB and Clp proteases.

ATP-Dependent Proteases↗
PubMed1998

The Rhizobium meliloti ExoK and ExsH glycanases specifically depolymerize nascent succinoglycan chains.

The Rhizobium meliloti ExoK and ExsH glycanases have been proposed to contribute to production of low molecular weight (LMW) succinoglycan by depolymerizing high molecular weight succinoglycan chains in R. meliloti cultures. We expressed and purified ExoK and ExsH and determined that neither enzyme can extensively cleave succinoglycan prepared from R. meliloti cultures, although neutral/heat treatment and acid/heat treatment convert succinoglycan to forms that can be cleaved efficiently by both enzymes. These results were somewhat surprising, given that the exoK+ and exsH+ genes play a crucial role in production of LMW succinoglycan in R. meliloti cultures. We demonstrated by Western blot analyses that R. meliloti expresses ExoK and ExsH, that both proteins can be detected extracellularly, and that ExsH secretion depends on the prsD+/prsE+ genes, consistent with previous predictions based on mutant analyses. Furthermore, we determined that the depolymerization activities associated with purified ExoK and ExsH are comparable with exoK+ and exsH+-dependent depolymerization activities expressed in R. meliloti cultures. We resolved the apparent contradiction between the results of our previous genetic analyses and depolymerization assays by determining that ExoK and ExsH can cleave high molecular weight succinoglycan that is being produced actively by R. meliloti, but not succinoglycan that has accumulated in cultures, to yield LMW succinoglycan. We propose that ExoK and ExsH dynamically regulate the molecular weight distribution of succinoglycan by cleaving nascent succinoglycan only during a limited period after its synthesis, perhaps before it undergoes a time-dependent change in its conformation or aggregation state.

Bacterial Proteins↗