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C K Mathews

Publications and source records attributed to C K Mathews.

At least 19 recordsLinked to original sources
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↗
PubMed1991

Analysis of mutagenesis induced by a thermolabile T4 phage deoxycytidylate hydroxymethylase suggests localized deoxyribonucleotide pool imbalance.

To understand the molecular basis of mutation stimulated by deoxyribonucleotide pool imbalance, we studied a temperature-sensitive T4 phage gene 42 mutant (LB3), which specifies a thermolabile deoxycytidylate hydroxymethylase. Analysis of rII mutations, revertible to wild type along either GC-to-AT or AT-to-GC transition pathways, showed 8- to 80-fold stimulation of GC-to-AT mutations at a semi-permissive temperature (34 degrees C). One such marker, rII SN103, which showed the highest stimulation at 34 degrees C, was sequenced after amplification of the template by polymerase chain reaction. The mutant site in rII SN103 was identified at nucleotide position 265 from the rII B translational start as an AT-to-GC transition, which changes TCA to CCA. Sequence analysis of revertants and pseudorevertants generated at 34 degrees C showed that both cytosines within this triplet can undergo change to either thymine or adenine, consistent with the hypothesis that hydroxymethyldeoxycytidine triphosphate pools are depleted at replication sites. However, dNTP pool measurements in extracts of 34 degrees C cultures showed no significant deviations from values obtained at 30 degrees C, suggesting that pool imbalances occur only locally, close to replication forks. Our studies support the hypothesis that the mutator phenotype displayed by ts LB3 at semi-permissive temperature is a consequence of perturbation of the flow of nucleotide precursors into the DNA replication machinery. A putative localized depletion of hm-dCTP presumably enlarges effective dTTP/hm-dCTP and dATP/hm-dCTP pool ratios, resulting in the observed C-to-T transition and C-to-A transversion mutations.

Base Sequence↗
PubMed1991

Deoxyadenosine reverses hydroxyurea inhibition of vaccinia virus growth.

Hydroxyurea, an inhibitor of ribonucleotide reductase, blocks replication of vaccinia virus. However, when medium containing hydroxyurea and dialyzed serum was supplemented with deoxyadenosine, the block to viral reproduction was circumvented, provided that an inhibitor of adenosine deaminase was also present. Deoxyguanosine, deoxycytidine, and deoxythymidine were ineffective alone and did not augment the deoxyadenosine effect. In fact, increasing concentrations of deoxyguanosine and deoxythymidine, but not deoxycytidine, eliminated the deoxyadenosine rescue, an effect that was reversed by the addition of low concentrations of deoxycytidine. These results suggested that the inhibition of viral replication by hydroxyurea was primarily due to a deficiency of dATP. Deoxyribonucleoside triphosphate pools in vaccinia virus-infected cells were measured at the height of viral DNA synthesis after a synchronous infection. With 0.5 mM hydroxyurea, the dATP pool was greater than 90% depleted, the dCTP and dGTP pools were 40 to 50% reduced, and the dTTP pool was increased. Assay of ribonucleotide reductase activity in intact virus-infected cells suggested that hydroxyurea may differentially affect reduction of the various substrates of the enzyme.

Adenine↗
PubMed1991

Cell cycle-dependent variations in deoxyribonucleotide metabolism among Chinese hamster cell lines bearing the Thy- mutator phenotype.

Deoxyribonucleotide pool imbalances are frequently mutagenic. We have studied two Chinese hamster ovary cell lines, Thy- 49 and Thy- 303, that were originally characterized by M. Meuth (Mol. Cell. Biol. 1:652-660, 1981). In comparison with wild-type CHO cells, both lines have elevated dCTP/dTTP ratios, resulting from loss of feedback control of CTP synthetase. While asynchronous cultures of both cell lines contain nearly identical deoxyribonucleoside triphosphate (dNTP) pools and both display elevated spontaneous mutation frequencies, the mutation frequencies between the two cell lines differ by as much as 10-fold. We asked whether differences in dNTP pools could be seen in extracts of rapidly isolated nuclei. Small differences, probably not large enough to account for the differences in mutation frequencies, were seen. However, when synchronized S-phase-enriched cell populations were examined, substantial differences were seen, both in whole-cell extracts and in nuclear extracts. Thy- 303 cells, which have higher mutation frequencies than do Thy- 49 cells, also showed the more aberrant dNTP pools. These data indicate that the Thy- 303 line contains a second mutation in addition to the mutation affecting CTP synthetase control. Evidence suggests that this putative second mutation affects an allosteric regulatory site of ribonucleotide reductase. The data on intranuclear dNTP pools in synchronized S-phase cells indicate that higher proportions of cellular dATP and dGTP are found in the nucleus than are corresponding amounts of dCTP and dGTP. Thus, despite the porous nature of the nuclear membrane, there are conditions under which the distributions of deoxyribonucleotides across this membrane are not random.

Animals↗
PubMed1990

Temperature-sensitive DNA mutant of Chinese hamster ovary cells with a thermolabile ribonucleotide reductase activity.

JB3-B is a Chinese hamster ovary cell mutant previously shown to be temperature sensitive for DNA replication (J. J. Dermody, B. E. Wojcik, H. Du, and H. L. Ozer, Mol. Cell. Biol. 6:4594-4601, 1986). It was chosen for detailed study because of its novel property of inhibiting both polyomavirus and adenovirus DNA synthesis in a temperature-dependent manner. Pulse-labeling studies demonstrated a defect in the rate of adenovirus DNA synthesis. Measurement of deoxyribonucleoside triphosphate (dNTP) pools as a function of time after shift of uninfected cultures from 33 to 39 degrees C revealed that all four dNTP pools declined at similar rates in extracts prepared either from whole cells or from rapidly isolated nuclei. Ribonucleoside triphosphate pools were unaffected by a temperature shift, ruling out the possibility that the mutation affects nucleoside diphosphokinase. However, ribonucleotide reductase activity, as measured in extracts, declined after cell cultures underwent a temperature shift, in parallel with the decline in dNTP pool sizes. Moreover, the activity of cell extracts was thermolabile in vitro, consistent with the model that the JB3-B mutation affects the structural gene for one of the ribonucleotide reductase subunits. The kinetics of dNTP pool size changes after temperature shift are quite distinct from those reported after inhibition of ribonucleotide reductase with hydroxyurea. An indirect effect on ribonucleotide reductase activity in JB3-B has not been excluded since human sequences other than those encoding the enzyme subunits can correct the temperature-sensitive growth defect in the mutant.

Animals↗
PubMed1989

On the inhibition of deoxycytidylate hydroxymethylase by 5-fluoro-2'-deoxycytidine 5'-monophosphate.

Studies were performed to determine whether 5-fluoro-2'-deoxycytidine 5'-monophosphate (FdCMP) is an inhibitor of deoxycytidylate hydroxymethylase and whether it could form an isolable covalent complex with the enzyme and the cofactor, 5,10-methyl-enetetrahydrofolate. The results showed that although FdCMP is a competitive inhibitor of dCMP hydroxymethylase, it does not cause time-dependent inhibition of the enzyme in the presence of cofactor. Further, although uv difference spectral evidence was found for FdCMP-cofactor-enzyme complex, the complex was not sufficiently stable to isolate on nitrocellulose filters. We conclude that FdCMP is not a mechanism-based inhibitor of dCMP hydroxymethylase.

Cytidine Monophosphate↗
PubMed1989

Essential role of T4 phage deoxycytidylate hydroxymethylase in a multienzyme complex for deoxyribonucleotide synthesis.

Several enzymes of deoxyribonucleoside triphosphate (dNTP) biosynthesis interact in T4 phage-infected Escherichia coli to form a multienzyme aggregate, the T4 dNTP synthetase complex. To test the specificity of enzyme interactions seen in vitro with this complex, we analyzed bacteria infected with four T4 gene 42 amber mutants, which specify truncated forms of dCMP hydroxymethylase, one of the constituent enzymes in the complex. Mutants that specify a nearly full length gene 42 product can form an intact complex, as revealed by two criteria: kinetic coupling among constituent enzymes in crude extracts of infected bacteria, and co-elution of enzyme activities from a gel filtration column. By these criteria, mutations that specify truncated proteins less than half the size of the full length protein cause disruption of the complex. These findings suggest that an enzymatically inactive form of dCMP hydroxymethylase can contribute toward assembly of an intact complex, so long as the incomplete protein is of sufficient size to fold normally, allowing interaction with other proteins in the complex.

Chromatography, Gel↗
PubMed1989

Amplification of the ribonucleotide reductase small subunit gene: analysis of novel joints and the mechanism of gene duplication in vaccinia virus.

Amplification of the M2 gene encoding the small subunit of ribonucleotide reductase (EC 1.17.4.1) was analyzed in a collection of vaccinia virus (VV) isolates selected for resistance to 5 mM hydroxyurea (HU). Most of the mutants harbored tandem direct repeat arrays of the M2 gene, but several had duplicated M2 as an inverted repeat by genomic rearrangements involving the chromosomal termini. Novel joints formed by direct repeats were mapped, amplified in vitro, and sequenced. The junctions were simple fusions between DNA downstream and upstream of the M2 gene. Lack of sequence homology at the breakpoints indicated that the initial genomic rearrangements leading to gene amplification were due to nonhomologous recombination events.

Base Sequence↗
PubMed1989

Ribonucleotide reductase: a determinant of 5-bromodeoxyuridine mutagenesis in phage T4.

Mutagenesis by 5-bromodeoxyuridine (BrdUrd) can result from base-pairing errors either during replication of a BrdUrd-containing template or at the nucleotide incorporation step. Replication errors give rise predominantly to AT-to-GC transitions, while incorporation errors, in which 5-bromo-dUTP competes with dCTP at a template guanine site, should give rise to GC-to-AT transitions. The latter pathway should be sensitive to deoxyribonucleoside triphosphate (dNTP) pool fluctuations. Since dNTP pools are regulated through allosteric control of ribonucleotide reductase, the control of this enzyme should be a determinant of BrdUrd mutagenesis--if mutagenesis results largely from incorporation errors. Since T4 phage-encoded ribonucleotide reductase is insensitive to feedback inhibition, we established conditions under which phage DNA replication is dependent upon ribonucleotide reductase of the host, Escherichia coli. We examined BrdUrd mutagenesis of rII mutants known to revert to wild type either by AT-to-GC or GC-to-AT transition pathways. While both reversion pathways were stimulated under all conditions analyzed, the AT-to-GC pathway was stimulated more when the E. coli reductase was functioning, while the GC-to-AT pathway was more specifically enhanced when the T4 reductase was active. These results confirm that ribonucleotide reductase is a determinant of BrdUrd mutagenesis, but our observations, plus experiments showing that BrdUrd has relatively small effects upon dNTP pool sizes, indicate that the relationship between deoxyribonucleotide metabolism and BrdUrd mutagenesis is more complex than anticipated.

Bromodeoxyuridine↗
PubMed1989

Analysis of T4 bacteriophage deletion mutants that lack td and frd genes.

The roles of bacteriophage T4-encoded thymidylate synthase and dihydrofolate reductase as virion structural components have been further investigated. Two mutants, del(63-32)7 and del(63-32)9, bearing deletions in the gene 63 to 32 region of the T4 genome, were characterized by Southern blotting analysis, as well as by enzyme and immunological assays. Our results have confirmed the original report of Homyk and Weil (Virology 61:505-523, 1974) that del7 and del9 each carries a deletion of about 4.0 kilobases, which totally eliminates the frd gene, encoding dihydrofolate reductase, and the td gene, encoding thymidylate synthase. With the well-characterized deletion mutants, along with newly prepared antisera against T4-encoded thymidylate synthase and dihydrofolate reductase, we have reevaluated the experimental results supporting the idea that T4-induced dihydrofolate reductase and thymidylate synthase are essential T4 baseplate components and antigenic determinants of phage particles. These deletion mutant phages are not targets for neutralization by antisera against either dihydrofolate reductase or thymidylate synthase purified from cloned genes. Furthermore, these newly prepared antisera also cannot neutralize the infectivity of T4D. Those results suggest that the phage-neutralizing components in the old antisera used in the earlier studies were not antibodies against either dihydrofolate reductase or thymidylate synthase but were antibodies against minor components of the purified enzyme preparations. Study of the biological properties of the deletion mutants indicates that T4-induced thymidylate synthase and dihydrofolate reductase play significant roles in growth of the phage beyond their known roles in nucleotide biosynthesis, even though they are apparently not essential for phage viability. The deletion mutants should be useful in defining these roles.

Blotting, Southern↗
PubMed1988

Deoxycytidylate hydroxymethylase gene of bacteriophage T4. Nucleotide sequence determination and over-expression of the gene.

We describe two approaches to cloning and over-expressing gene 42 of bacteriophage T4, which encodes the early enzyme deoxycytidylate hydroxymethylase. In Bochum a library of sonicated fragments of wild-type phage DNA cloned into M13mp18 was screened with clones known to contain parts of gene 42. Two overlapping fragments, each of which contained one end of the gene, were cleaved at a HincII site and joined, to give a fragment containing the entire gene. In Corvallis a 1.8-kb fragment of cytosine-substituted DNA, believed to contain the entire gene, was cloned into pUC18 and shown to express the enzyme at low level. The cloned fragment bore an amber mutation in gene 42. From the DNA sequence of gene 42, the cloned gene was converted to the wild-type allele by site-directed mutagenesis. Both gene-42-containing fragments were cloned into the pT7 expression system and found to be substantially overexpressed. dCMP hydroxymethylase purified from one of the over-expressing strains had a turnover number similar to that of the enzyme isolated earlier from infected cells. In addition, the N-terminal 20 amino acid residues matched precisely the sequence predicted from the gene sequence. The amino acid sequence of gp42 bears considerable homology with that of thymidylate synthase of either host or T4 origin. The gene 42 nucleotide sequences of bacteriophages T2 and T6 were determined and found to code for amino acid sequences nearly identical to that of T4 gp42.

Base Sequence↗