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The Journal of Biological Chemistry Aug 1980This laboratory has described a multienzyme aggregate from T4 phage-infected Escherichia coli which seems to participate in deoxyribonucleotide biosynthesis and...
This laboratory has described a multienzyme aggregate from T4 phage-infected Escherichia coli which seems to participate in deoxyribonucleotide biosynthesis and efficient delivery of DNA precursors to the replication apparatus. This paper describes improved methodology for isolation of this aggregate, and we present three lines of evidence supporting a role for ribonucleoside diphosphate reductase in functioning of the presumed complex. 1) Ribonucleoside diphosphates are readily incorporated into DNA as deoxyribonucleotides in an in situ DNA-synthesizing system from T4 phage-infected cells. 2)Ribonucleotide reductase is associated with the complex, as shown by co-sedimentation of reductase activity with other activities in the multienzyme aggregate we have described. 3)Ribonucleotide reductase is kinetically coupled to at least four other enzymes involved in a sequential pathway. The aggregated enzymes catalyze the five-step conversion of uridine diphosphate to deoxythymidine triphosphate with but a brief lag before dTTP production reaches its maximal rate. These studies have also confirmed the existence of dCTPase-dUTPase and dCMP deaminase activities in the putative complex.
Topics: DNA, Bacterial; Deoxycytosine Nucleotides; Deoxyribonucleotides; Escherichia coli; Kinetics; Multienzyme Complexes; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Sucrose; T-Phages; Thymidylate Synthase; Ultracentrifugation
PubMed: 6995453
DOI: No ID Found -
The Journal of Biological Chemistry Jan 1980
The modulation of the thymidine triphosphate pool of Chinese hamster cells by dCMP deaminase and UDP reductase. Thymidine auxotrophy induced by CTP in dCMP deaminase-deficient lines.
Topics: Animals; Carcinoma; Cell Line; Cell Survival; Cricetinae; Cytidine; DCMP Deaminase; Humans; Lung; Mouth Neoplasms; Nucleotide Deaminases; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Thymidine; Thymine Nucleotides; Uridine Diphosphate
PubMed: 7350151
DOI: No ID Found -
Journal of Virology Jun 1978Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of...
Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.
Topics: Animals; Cell Line; Cell-Free System; Cricetinae; Cricetulus; DCMP Deaminase; Mutation; Nucleotide Deaminases; Ribonucleoside Diphosphate Reductase; Ribonucleotide Reductases; Simplexvirus; Thymidine Kinase; Virus Replication
PubMed: 209209
DOI: 10.1128/JVI.26.3.547-553.1978 -
The Journal of Biological Chemistry May 1978dCMP deaminase from Bacillus subtilis has been purified 700-fold. In addition to the substrate, dCMP, the enzyme requires dCTP, Zn2+, and 2-mercaptoethanol, Mg2+ cannot...
dCMP deaminase from Bacillus subtilis has been purified 700-fold. In addition to the substrate, dCMP, the enzyme requires dCTP, Zn2+, and 2-mercaptoethanol, Mg2+ cannot substitute for Zn2+. The dCMP saturation curve is hyperbolic in the presence of saturating concentrations of dCTP and Zn2+. The dCTP saturation curve is sigmoidal, the sigmoidicity being dependent on the Zn2+ and dCMP concentrations. The molecular weight as determined by gel filtration is 170,000 both in the presence and in the absence of dCTP and Zn2+. In the absence of thiols, the enzyme is highly unstable. At 0 degrees, the half-life of the enzyme activity is 30 min. Addition of Zn2+ and dCTP protects against this inactivation. In the presence of a thiol, dCTP and Zn2+ protect the enzyme against heat inactivation at 50 degrees. A mutant lacking dCMP deaminase (dcd) was isolated. Labeling of the pyrimidine nucleotide pools reveals that in the parent strain, 45% of the dTTP pool is derived via dCMP deamination, the residual 55% being derived via reduction of a uridine nucleotide. Since the dcd mutant grows with the same doubling time as the parent strain, we conclude that uridine nucleotide reduction alone is capable of supplying sufficient dUMP for normalthymidine nucleotide synthesis.
Topics: Bacillus subtilis; DCMP Deaminase; Deoxyribonucleotides; Drug Stability; Kinetics; Nucleotide Deaminases; Ribonucleotides
PubMed: 418064
DOI: No ID Found -
The Journal of Biological Chemistry Dec 1977
Topics: Coliphages; DCMP Deaminase; DNA Replication; Deoxyribonucleotides; Kinetics; Mutation; Nucleotide Deaminases; Proflavine; Species Specificity; Virus Replication
PubMed: 925015
DOI: No ID Found -
Journal of Virology Oct 1977[125I]deoxycytidine was a good substrate for herpes simplex virus type 1 thymidine kinase (TK), whereas [125I]deoxycytidine was a very poor substrate for cellular TK....
[125I]deoxycytidine was a good substrate for herpes simplex virus type 1 thymidine kinase (TK), whereas [125I]deoxycytidine was a very poor substrate for cellular TK. Simple, sensitive, and specific assays for viral TK could be carried out in vivo and in vitro even in the presence of cell TK. Autoradiographic detection of incorporated [125I]deoxycytidine provided a rapid and simple method for detection of and screening for viral TK in infected as well as viral TK-transformed cells.
Topics: Cell Line; DCMP Deaminase; Deoxycytidine; Methods; Simplexvirus; Substrate Specificity; Tetrahydrouridine; Thymidine Kinase
PubMed: 198581
DOI: 10.1128/JVI.24.1.314-318.1977 -
Proceedings of the National Academy of... Aug 1977A DIRECT APPROACH IS DESCRIBED TO THE QUESTION: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation...
A DIRECT APPROACH IS DESCRIBED TO THE QUESTION: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Escherichia coli. One-third to one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas an equivalent mixture of uncomplexed enzymes required nearly 20 min before dTTP synthesis reached its maximal rate. The effect of aggregation is evidently to decrease the volume into which intermediates are free to diffuse. Because there is reason to believe that intracellular concentration gradients of DNA precursors exist, the properties of this enzyme aggregate in vitro may help to explain how such gradients are maintained.
Topics: Coliphages; DNA Replication; DNA, Viral; DNA-Directed DNA Polymerase; Escherichia coli; Kinetics; Molecular Weight; Nucleoside-Diphosphate Kinase; Tetrahydrofolate Dehydrogenase; Thymidylate Synthase; Transferases
PubMed: 198773
DOI: 10.1073/pnas.74.8.3152 -
Journal of Bacteriology Jan 1977In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the...
In a Salmonella typhimurium strain made diploid for the thy region by introduction of the Escherichia coli episome, F'15, mutants resistant to trimethoprim in the presence of thymidine were selected. One was shown to be defective in deoxyuridine 5'-phosphate (dUMP) synthesis; it requires deoxyuridine or thymidine for growth and is sensitive to trimethoprim in the presence of deoxyuridine. Genetic studies showed that the mutant is mutated in two genes, dcd and dum, located at 70 and 18 min, respectively, on the Salmonella linkage map. The dcd gene cotransduces 95% with udk, the structural gene for uridine kinase. Both mutations are necessary to create a deoxyuridine requirement, providing evidence for the existence of two independent pathways for dUMP synthesis. Pool studies showed that a dum mutation by itself causes a small decrease in the deoxythymidine 5'-triphosphate (dTTP) pool of the cells, whereas a dcd mutation results in a much more marked decrease. The double mutant dcd dum, when incubated in the absence of deoxyuridine, contains barely detectable levels of dTTP. Enzyme analysis revealed that dcd encodes deoxycytidine 5'-triphosphate deaminase. The gene product of the dum gene has not yet been identified; it does not encode either subunit of ribonucleoside diphosphate reductase or deoxyuridine 5'-triphosphate pyrophosphatase. Mutants deleted for the dcd-udk region of the S. typhimurium chromosome were isolated.
Topics: Chromosome Mapping; Chromosomes, Bacterial; DCMP Deaminase; Deoxyribonucleotides; Genes; Mutation; Phenotype; Plasmids; Ribonucleoside Diphosphate Reductase; Salmonella typhimurium; Thymidine; Thymine Nucleotides; Transduction, Genetic; Uracil Nucleotides
PubMed: 318643
DOI: 10.1128/jb.129.1.305-316.1977 -
The Biochemical Journal Feb 1976Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel...
Increased entry of deoxy[3H]cytidine begins at about 12h after addition of phytohaemagglutinin to peripheral pig lymphocyte cultures, and is accompanied by a parallel stimulation of deoxycytidine kinase up to the beginning of DNA synthesis at 24h. The increased deoxycytidine uptake is characterized by an increase in Vmax. without alteration of the apparent Km (0.7 +/- 0.11 muM). Although the entries of both nucleosides are promoted at the same time, the stimulation of deoxycytidine uptake is less than that of thymidine, and the two nucleosides are transported by separate systems. In addition to deoxycytidien kinase, the synthesis of deoxycytidylate deaminase and thymidylate synthetase are stimulated after addition of phytohaemagglutinin, but to a lesser extent than that of thymidine kinase. The importance of the latter enzyme in forming dTMP, and of thymidylate kinase in providing dTTP, is discussed.
Topics: Animals; Biological Transport; DCMP Deaminase; DNA; Deoxycytidine; Deoxyribonucleotides; Kinetics; Lectins; Lymphocyte Activation; Lymphocytes; Phosphotransferases; Swine; Thymidylate Synthase; Thymine Nucleotides
PubMed: 938456
DOI: 10.1042/bj1540395 -
Journal of Virology Feb 1976The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP...
The role of the T4 bacteriophage regA gene in stabilizing early mRNA was investigated by assaying the level of functional mRNA from eight prereplicative genes (56 [dCMP hydroxymethylase], cd [dCMP deaminase], 1 [deoxynucleotide kinase], rIIA, rIIB, 46 [DNA arrest], and 45) during extended infection of Escherichia coli B with T4 regA-, 43- and T4 43- bacteriophage. The above gene-specific transcripts in RNA isolated from infected cells were quantitated by translation with an E. coli B cell-free system. Conditions were chosen to insure that the amount of gene product formed in vitro, measured either as an enzyme activity or as a radioactive band in acrylamide gel, was directly proportional to the level of mRNA present. The failure of T4 regA-, 43- phage to terminate prereplicative synthesis (Wiberg et al., 1973) resulted in an enhanced production of many early gene products over those formed during T4 43- infection. This increase did not appear to be associated with an increment in mRNA levels, since in the present study gene-specific early mRNA's were found to be only marginally elevated and slightly more stable in T4 regA-, 43-- than in T4 43--infected cells. Of interest was the observation that significant quantities of all of the mRNA's studied; with the exception of those from genes 45 and 46, could be isolated from T4 43--infected cells after synthesis of the respective gene products had ceased. On termination of normal prereplicative synthesis during infection with T4 43- phage, polyribosomes were found to be dissociated completely, a finding which suggests that the residual mRNA present in these cells is free in the cytoplasm. The persistence in T4 43--infected cells of translatable mRNA for many prereplicative genes after product synthesis ceased indicates that the impairment in protein synthesis is not due solely to regA-mediated messenger degradation or modification. Rather, the results suggest that the regA gene product may act either by interfering with early mRNA polypeptide chain initiation or by promoting prereplicative polysome dissociation.
Topics: Cell-Free System; Coliphages; DCMP Deaminase; DNA Viruses; Escherichia coli; Mutation; Phosphoric Monoester Hydrolases; Polyribosomes; Protein Biosynthesis; RNA, Messenger; RNA, Viral; Viral Proteins
PubMed: 176430
DOI: 10.1128/JVI.17.2.538-549.1976