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C K Mathews

Publications and source records attributed to C K Mathews.

At least 37 records · Page 2Linked to original sources
PubMed1994

Allosteric effectors are required for subunit association in T4 phage ribonucleotide reductase.

Bacteriophage T4 encodes its own aerobic ribonucleotide reductase (RNR), which reduces ribonucleoside diphosphates to the corresponding deoxyribonucleoside diphosphates. T4 RNR is composed of homodimeric large (R1) and small (R2) subunits. Intricate regulation of enzymatic activity is accomplished by the binding of nucleotide effectors to R1. Berglund (Berglund, O. (1972) J. Biol. Chem. 247, 7270-7275) described similarities between T4 RNR and the corresponding enzyme from aerobic Escherichia coli. An important difference, however, is that T4 RNR forms a tight R1.R2 complex, while the E. coli R1 and R2 more readily dissociate. In this study we purified the phage R2 subunit from an overexpression vector constructed by Tseng et al. (Tseng, M., Hilfinger, J., He, P., and Greenberg, R. (1992) J. Bacteriol. 174, 5740-5744) and used this as an immunogen to generate polyclonal antiserum. Using co-immunoprecipitation techniques, we probed in vitro for interactions between the phage-induced R1 and R2 subunits. Our studies indicate that tight binding of the phage RNR subunits is completely dependent upon the known allosteric effectors of the enzyme. Once the R1.R2 holoenzyme has been formed it appears to be remarkably stable when in the presence of dATP. However, if dATP is removed, the R1.R2 complex readily dissociates.

Adenosine Triphosphate↗
PubMed1994

Ribonucleotide reductase: evidence for specific association with HeLa cell mitochondria.

Mammalian mitochondria contain pools of deoxyribonucleoside 5'-triphosphates that behave differently from the much larger whole-cell pools. To investigate the origins of these pools, we analyzed HeLa cell mitochondria for ribonucleotide reductase activity. Three findings suggest specific association of a reductase with mitochondria: (1) enzyme activity in extracts of washed mitochondria, (2) stimulation of that activity by dATP at levels inhibitory to the major cellular activity, and (3) association of immunoreactive material with washed and fractionated mitochondria.

Cell Fractionation↗
PubMed1994

Effect of DNA cytosine methylation upon deamination-induced mutagenesis in a natural target sequence in duplex DNA.

Are 5-methylcytosine residues in DNA hot spots for transition mutagenesis? Numerous studies identify 1) structural changes induced by DNA methylation, 2) high percentages of human mutations that result from GC to AT transition pathways, and 3) differences between G.C and G.mC base pairs in susceptibility to nonenzymatic deamination. However, investigations of chemical stability necessarily involve non-physiological conditions for chemical analysis of deamination. Here we describe an experiment that compares rates of deamination-induced mutagenesis between a G.C and G.mC base pair, when both are present in duplex DNA, incubated at 37 degrees C and pH 7.4, within identical sequence contexts, in a natural mutational target (the Escherichia coli lacZ alpha gene) that selects for mutagenesis at the specific site under investigation. Under these conditions the rate of spontaneous deamination at G.mC exceeds that at G.C by more than 21-fold. Our data implicate differences in chemical stability toward deamination as a major causal factor releasing DNA cytosine methylation to spontaneous mutagenesis.

5-Methylcytosine↗
PubMed1994

International Commission for Protection Against Environmental Mutagens and Carcinogens. Deoxyribonucleoside triphosphate levels: a critical factor in the maintenance of genetic stability.

DNA precursor pool imbalances can elicit a variety of genetic effects and modulate the genotoxicity of certain DNA-damaging agents. These and other observations indicate that the control of DNA precursor concentrations is essential for the maintenance of genetic stability, and suggest that factors which offset this control may contribute to environmental mutagenesis and carcinogenesis. In this article, we review the biochemical and genetic mechanisms responsible for regulating the production and relative amounts of intracellular DNA precursors, describe the many outcomes of perturbations in DNA precursor levels, and discuss implications of such imbalances for sensitivity to DNA-damaging agents, population monitoring, and human diseases.

Animals↗
PubMed1993

Vaccinia virus ribonucleotide reductase expression and isolation of the recombinant large subunit.

The vaccinia virus gene encoding the 87-kDa protein that comprises the large subunit of ribonucleotide reductase (vvR1) was cloned into a bacterial expression vector under the control of an inducible promoter. Culture of Escherichia coli cells harboring the recombinant plasmid under standard induction conditions (0.4 mM isopropyl beta-D-thiogalactopyranoside, 37 degrees C) resulted in synthesis of a completely insoluble product. Production of soluble vvR1 was achieved by growing bacteria at low temperature (15 degrees C) during the induction period, initiating induction at low cell density, and using a low concentration (0.05 mM) of the inducer isopropyl beta-D-thiogalactopyranoside. Hydroxyurea, an inhibitor of ribonucleotide reductase, increased production of soluble vvR1 in a dose-dependent manner. Recombinant vvR1 was purified from a high salt extract of the E. coli lysate in four steps, the last utilizing an affinity column consisting of the carboxyl-terminal seven amino acids of the vvR2 protein linked to an insoluble resin. Using purified recombinant vvR2 to reconstitute active enzyme, we determined that maximizing the rate of CDP reduction required pH 8.0-8.8, 50 mM dithiothreitol, and 2 mM ATP. Specific activity of purified vvR1 was 122 nmol/min/mg. Limited proteolysis of the vvR1 protein revealed protease-resistant fragments approximately 30 and 58 kDa in size. To our knowledge, this study represents the first expression, solubilization, and isolation of a recombinant "eukaryotic" form of ribonucleotide reductase large subunit.

Base Sequence↗
PubMed1993

Vaccinia virus ribonucleotide reductase. Correlation between deoxyribonucleotide supply and demand.

Ribonucleotide reductase has been suggested as a rate-limiting enzyme in DNA synthesis, partly because activities of the enzyme in cell-free preparations are low relative to rates needed to sustain DNA replication at observed rates. Vaccinia virus, with a large duplex DNA genome, encodes both subunits of a specific ribonucleoside diphosphate reductase. In this report, we describe quantitative analysis of ribonucleotide reductase protein levels and DNA accumulation in vaccinia virus-infected cell extracts, to correlate the supply of deoxyribonucleotides with the demand for these precursors in viral DNA synthesis. To do this, we generated polyclonal antisera to TrpE fusion proteins constructed from the carboxyl termini of both subunits of viral ribonucleotide reductase. We used S1 nuclease and immunoprecipitation analysis to determine the transcriptional and translational kinetics of vaccinia virus ribonucleotide reductase expression. Enzyme activity and ribonucleotide reductase protein stability were also assayed during the time course of viral infection. Enzyme-linked immunoassays were used to quantitate protein levels, and filter hybridizations were used to measure the accumulation of viral DNA. We show that ribonucleotide reductase activity in vaccinia virus-infected cells is severalfold higher than needed to provide deoxyribonucleotides at rates commensurate with DNA synthesis. Thus, while the enzyme is important as catalyst for the first committed reaction in DNA replication, it is not rate-limiting for this process.

Animals↗
PubMed1993

Acidic C terminus of vaccinia virus DNA-binding protein interacts with ribonucleotide reductase.

Evidence from prokaryotic systems suggests that enzymes of dNTP synthesis are organized near the DNA replication apparatus, allowing direct utilization of dNTPs at their sites of synthesis. To investigate whether similar interactions exist within a eukaryotic environment, we have prepared anti-idiotypic antibodies to the small subunit of vaccinia virus ribonucleotide reductase, and we used these antibodies to search for proteins that interact with this enzyme. This approach identified a 34-kDa viral phosphoprotein, which, like ribonucleotide reductase itself, is localized within infected cells at DNA replication sites. After expression of its structural gene in Escherichia coli, the recombinant protein was purified and found (i) to bind tightly to single-stranded DNA and (ii) to stimulate enzymatic activity of vaccinia ribonucleotide reductase. These observations suggest a physical association between dNTP synthesis and DNA replication in this viral system.

Animals↗
PubMed1993

A forward mutation assay in phage T4: application to gene 42 mutator mutations.

A forward mutation assay was developed to study mutagenic specificity induced by temperature-sensitive alleles of bacteriophage T4 gene 42, which encodes a thermolabile deoxycytidylate hydroxymethylase. Thymidine kinase (tk) mutations induced by T4 ts B3 at a semi-permissive temperature (34 degrees C) were selected under near-ultraviolet light on synthetic agar plates containing bromodeoxyuridine, and sequenced after PCR amplification of the tk gene. 21 of 23 tk- mutations identified were C-->T transitions, while the remainder were C-->A transversions. Analyses of the DNA sequence around each mutant site suggest that the mispairing of thymine with guanine in the template is suppressed when the next nucleotide is dGTP. The 5' neighbor nucleotide of the mismatch may influence mutation frequency as well; no mutations with dAMP residues on the upstream side were seen. Our observations with the forward mutation assay here are consistent with previous results from an rII reversion assay, supporting our model that the mutator phenotype displayed by tsLB3 is a consequence of perturbation of dNTP supplies to replication sites due to partial impairment of thermolabile deoxycytidylate hydroxymethylase at a semi-permissive temperature. The forward mutation assay described here is readily adapted for other studies of mutagenesis in T4 phage.

Bacteriophage T4↗
PubMed1992

Interactions between T4 phage-coded deoxycytidylate hydroxymethylase and thymidylate synthase as revealed with an anti-idiotypic antibody.

Anti-idiotypic antibodies were used to mimic the binding surface of the T4 bacteriophage deoxycytidylate hydroxymethylase enzyme, providing an immunological probe for protein-protein interactions involving this enzyme. Polyclonal dCMP hydroxymethylase antibodies were affinity-purified and used to generate anti-idiotypic antibodies. The anti-idiotypic serum immunoprecipitated two native viral proteins, deoxycytidylate hydroxymethylase (EC 2.1.2.8) and thymidylate synthase (EC 2.1.1.45), from a sonicated detergent-treated extract of T4-infected Escherichia coli. The anti-anti-dCMP hydroxymethylase antibody was found to be specific in binding to the T4 dTMP synthase, with no detectable affinity for the host dTMP synthase. Previous work in our laboratory has demonstrated the viral dCMP hydroxymethylase and dTMP synthase to be associated in a deoxyribonucleotide synthetase enzyme complex. Our current approach, using anti-idiotypic antibodies as probes for protein-protein interactions, and complementary studies involving dCMP hydroxymethylase enzyme affinity columns indicate a direct association between bacteriophage T4 dCMP hydroxymethylase and dTMP synthase.

Antibodies, Anti-Idiotypic↗
PubMed1992

Specific associations of T4 bacteriophage proteins with immobilized deoxycytidylate hydroxymethylase.

Is the enzymatic machinery for DNA precursor biosynthesis linked to the DNA replication apparatus? To identify intermolecular associations among deoxyribonucleotide biosynthetic enzymes and to ask whether these enzymes are linked to replication proteins, we analyzed radiolabeled T4 bacteriophage proteins that bind specifically to a column of immobilized T4 deoxycytidylate hydroxymethylase. More than a dozen T4 proteins and a few Escherichia coli proteins are adsorbed specifically by this column. Several of the T4 proteins were identified by two-dimensional gel electrophoresis and radioautography. These include five enzymes involved in DNA precursor biosynthesis, dCMP hydroxymethylase, thymidylate synthase, dihydrofolate reductase, dCTPase-dUTPase, and ribonucleotide reductase large and small subunits, plus several proteins of DNA metabolism and replication. Analysis of extracts of cells infected with phage amber mutants defective in specific proteins suggested a specific association involving thymidylate synthase and the gene 32 single-strand DNA-binding protein.

Autoradiography↗
PubMed1992

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.

Base Sequence↗
PubMed1992

DNA precursor asymmetries, replication fidelity, and variable genome evolution.

Balanced pools of deoxyribonucleoside triphosphates (dNTPs) are essential for DNA replication to occur with maximum fidelity. Conditions that create biased dNTP pools stimulate mutagenesis, as well as other phenomena, such as recombination or cell death. In this essay we consider the effective dNTP concentrations at replication sites under normal conditions, and we ask how maintenance of these levels contributes toward the natural fidelity of DNA replication. We focus upon two questions. (1) In prokaryotic systems, evidence suggests that replication is driven by small, localized, rapidly replenished dNTP pools that do not equilibrate with the bulk dNTP pools in the cell. Since these pools cannot be analyzed directly, what indirect approaches can illuminate the nature of these replication-active pools? (2) In eukaryotic cells, the normal dNTP pools are highly asymmetric, with dGTP being the least abundant nucleotide. Moreover, the composition of the dNTP pools changes as cells progress through the cell cycle. To what extent might these natural asymmetries contribute toward a recently described phenomenon, the differential rate of evolution of different genes in the same genome?

DNA Replication↗
PubMed1991

T4 phage ribonucleotide reductase. Allosteric regulation in vivo by thymidine triphosphate.

Based upon analyses of purified enzyme preparations, T4 bacteriophage-coded ribonucleotide reductase is considered to be relatively insensitive to control by allosteric inhibition. However, two factors suggest that CDP reduction to dCDP is feedback-controlled by dTTP in infected cells. First, the pool of 5-hydroxymethyldeoxycytidine triphosphate, which expands manyfold upon infection by a dCMP deaminase-deficient T4 mutant, shrinks to near-normal levels as a consequence of dTTP accumulation, and ribonucleotide reductase is the only apparent control point. Second, analysis of mutagenesis by 5-bromodeoxyuridine suggests that most induced mutations result from localized pool depletion of 5-hydroxymethyl-dCTP at replication sites, as if 5-bromo-dUTP were behaving like dTTP in inhibiting the CDP reductase activity of the phage enzyme. We found that CDP reductase activity in crude extracts of T4 phage-infected bacteria is sensitive to inhibition by either dTTP or 5-bromo-dUTP, at concentrations as low as 0.01 mM. However, in partially purified enzyme preparations that sensitivity is lost. Although we don't know the basis for this loss of feedback sensitivity, the results suggest that kinetic properties of enzymes in intact cells are determined by the cellular milieu in ways not apparent from analysis of purified enzymes.

Allosteric Regulation↗