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The Journal of Biological Chemistry Feb 1993Deoxycytidylate (dCMP) deaminase, a hexameric allosteric enzyme induced on infection of Escherichia coli by bacteriophage T4, was shown to contain two atoms of zinc per...
Deoxycytidylate (dCMP) deaminase, a hexameric allosteric enzyme induced on infection of Escherichia coli by bacteriophage T4, was shown to contain two atoms of zinc per subunit by atomic absorption spectroscopy. One zinc appears to be involved in catalysis, as described for adenosine deaminase (Sharaff, A. J., Wilson, D. K., Chang, Z., and Quiocho, F. A. (1992) J. Mol. Biol. 226, 917-921) and cytidine deaminase (Yang, C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). This thesis is supported by the finding that the enzyme loses about 80% of its activity in the presence of o-phenanthroline. It has also been found that zinc is released when the enzyme is denatured in the presence of the metallochromic indicator, 4-(2-pyridylazo)resorcinol. Renaturation of the deaminase to an active form occurred in the presence but not in the absence of zinc. The second atom of zinc is proposed to be located in a region of T4-dCMP deaminase that resembles a zinc finger. This region, which has the sequence His-X3-Cys-X14-His-X3-His, would represent a zinc-binding motif that has not been described previously.
Topics: Amino Acid Sequence; Binding Sites; DCMP Deaminase; Metalloproteins; Molecular Sequence Data; Phenanthrolines; Protein Denaturation; Spectrophotometry, Atomic; T-Phages; Viral Proteins; Zinc; Zinc Fingers
PubMed: 8428902
DOI: No ID Found -
The Journal of Biological Chemistry Sep 1991Based upon analyses of purified enzyme preparations, T4 bacteriophage-coded ribonucleotide reductase is considered to be relatively insensitive to control by allosteric...
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.
Topics: Allosteric Regulation; Base Sequence; Cytidine Diphosphate; DNA; Molecular Sequence Data; Mutagenesis; Nucleotides; Oxidation-Reduction; Ribonucleotide Reductases; T-Phages; Thymine Nucleotides
PubMed: 1885561
DOI: No ID Found -
Journal of Bacteriology Aug 1991Thymidylate biosynthesis via the methylation of dUMP is required for DNA replication in Rickettsia prowazekii, an obligate intracytoplasmic bacterium. In theory, dUMP...
Thymidylate biosynthesis via the methylation of dUMP is required for DNA replication in Rickettsia prowazekii, an obligate intracytoplasmic bacterium. In theory, dUMP synthesis could occur either by the deamination of deoxycytidine nucleotides or by the reduction of uridine nucleotides. Accordingly, the incorporation of both radiolabeled cytidine and uridine into the thymidylate of R. prowazekii was examined. After DNA hydrolysis and high-performance liquid chromatography, it was determined that 85% of the rickettsial thymidylate was derived from cytidine and the remaining 15% was derived from uridine. These findings were supported by the identification of a dCTP deaminase activity in extracts of R. prowazekii. Extracts of R. prowazekii deaminated 1.7 +/- 0.3 nmol of dCTP/min/mg of protein (a value calculated to suffice for rickettsial growth), and no measurable activity was observed with dCMP as the substrate.
Topics: Cytidine; Cytidine Deaminase; Cytoplasm; Deoxycytosine Nucleotides; Nucleoside Deaminases; Rickettsia prowazekii; Uridine
PubMed: 1906875
DOI: 10.1128/jb.173.15.4902-4903.1991 -
The Journal of Biological Chemistry Jan 1990The cd gene of bacteriophage T4, which encodes the enzyme deoxycytidylate deaminase, was isolated as a 1.9-kilobase DNA fragment and completely sequenced. The deduced... (Comparative Study)
Comparative Study
The cd gene of bacteriophage T4, which encodes the enzyme deoxycytidylate deaminase, was isolated as a 1.9-kilobase DNA fragment and completely sequenced. The deduced amino acid sequence was found to be 193 residues long compared with 188 for the corresponding enzyme from bacteriophage T2. There were nine amino acid differences between the two enzymes in addition to a 5-residue insert near the carboxyl terminus of the T4 deaminase which was not present in the T2 deaminase. The cd-containing fragment also contained all of gene 31 (Nivinskas, R., and Black, L. W. (1988) Gene (Amst.) 73, 251-257) and thus precisely locates the two genes relative to one another within the T4 phage genomic map. Attempts to place the cd gene within a high expression vector have not been successful so far due to possible toxic effects of the gene product. However, placement of the gene within pUC18 resulted in a degree of expression which is about 10-20 times that found in T4-infected Escherichia coli. The enzyme was purified to homogeneity and found to possess properties similar to T2 phage deoxycytidylate deaminase.
Topics: Amino Acid Sequence; Bacterial Proteins; Bacteriophage lambda; Base Sequence; Cloning, Molecular; DCMP Deaminase; DNA, Viral; Deoxyribonuclease EcoRI; Deoxyribonucleases, Type II Site-Specific; Escherichia coli; Gene Expression; Molecular Sequence Data; Nucleotide Deaminases; Restriction Mapping; Sequence Homology, Nucleic Acid; T-Phages
PubMed: 2136740
DOI: No ID Found -
Molecular and Cellular Biology Dec 1987A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles...
A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.
Topics: Animals; Cell Division; Cell Line; Cricetinae; Cytidine Diphosphate; DCMP Deaminase; DNA; Deoxycytidine; Deoxycytidine Monophosphate; Deoxycytosine Nucleotides; Deoxyribonucleotides; Deoxyuridine; Nucleotide Deaminases; Pyrimidines; Thymidine; Thymidine Monophosphate; Thymine Nucleotides; Uridine Diphosphate
PubMed: 3437888
DOI: 10.1128/mcb.7.12.4218-4224.1987 -
FEBS Letters Nov 1987The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea...
The organization of specific pyrimidine pathways to channel various nucleoside precursors into DNA is poorly understood. We show that concanavalin A-stimulated guinea pig lymphocytes incorporate [3H]dThd, [3H]dCyd, [3H]dUrd, [3H]Cyd and [3H]Urd into DNA-thymines and DNA-cytosines in a highly conserved distribution pattern. DNA-thymines were labeled only by dThd and dUrd, while DNA-cytosines were labeled only by dCyd, Cyd and Urd. The kinetics for the incorporation of the [3H]nucleosides were essentially identical, indicating equivalent abilities to measure DNA synthesis. Pyrazofurin inhibition of the pyrimidine de novo synthetic pathway inhibited cell proliferation and the levels of [3H]nucleoside incorporation by approx. 50%, but did not alter restricted distribution of the [3H]nucleosides among DNA-thymines and DNA-cytosines. These findings indicate the absence of Cyd and dCMP deaminase salvage pathways and suggest either subcellular compartmentalization or differential regulation of ribonucleoside diphosphoreductase which permits reduction of CDP but not UDP.
Topics: Amides; Animals; Cell Division; Concanavalin A; Cytidine Deaminase; DCMP Deaminase; DNA Replication; Guinea Pigs; Lymphocyte Activation; Male; Models, Biological; Pyrazoles; Pyrimidines; Ribonucleosides; Ribose; T-Lymphocytes
PubMed: 3500072
DOI: 10.1016/0014-5793(87)80422-2 -
The Journal of Biological Chemistry Apr 1987DNA precursor imbalances are known to be mutagenic in both eukaryotic and prokaryotic systems. Almost certainly, such mutagenesis involves competition between correctly...
DNA precursor imbalances are known to be mutagenic in both eukaryotic and prokaryotic systems. Almost certainly, such mutagenesis involves competition between correctly and incorrectly base-paired precursors at replication sites. Since other factors may be involved, it is important to identify specific mutations induced by specific pool imbalances. Using bacteriophage T4, we have developed a system for such analysis. We prepare double mutants of T4; one mutation affects a phage-coded enzyme of deoxyribonucleoside triphosphate (dNTP) metabolism, while the second is an rII mutation known to revert along a specific pathway. We determine dNTP pools in infection by such a mutant and measure both the spontaneous reversion rate of the rII mutation and, in some cases, the nucleotide sequence at the mutant site. In this paper we analyze mutations induced by a deficiency of T4-encoded deoxycytidylate deaminase. This causes pools of 5-hydroxymethyl-dCTP to expand some 30-fold, while dTTP pools contract. This specifically stimulates AT-to-GC reversion. One of the four AT-to-GC reverters tested, rIIUV215, increases its reversion rate at least 1000-fold under these pool-imbalance conditions, while the other mutants tested show increases of only about 10-fold. Therefore, factors other than dNTP competition, including local DNA sequence environment, must be invoked to fully explain mechanisms of dNTP pool imbalance-induced mutagenesis. We discuss models for this, and we also report unexpected effects of the dCMP deaminase deficiency upon pools of ribonucleoside triphosphates.
Topics: Base Sequence; DCMP Deaminase; Deoxyribonucleotides; Escherichia coli; Genes; Genes, Viral; Mutation; Nucleotide Deaminases; T-Phages
PubMed: 3553179
DOI: No ID Found -
Molecular and Cellular Biology May 1986The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1...
The dCMP deaminase gene (DCD1) of Saccharomyces cerevisiae has been isolated by screening a Sau3A clone bank for complementation of the dUMP auxotrophy exhibited by dcd1 dmp1 haploids. Plasmid pDC3, containing a 7-kilobase (kb) Sau3A insert, restores dCMP deaminase activity to dcd1 mutants and leads to an average 17.5-fold overproduction of the enzyme in wild-type cells. The complementing activity of the plasmid was localized to a 4.2-kb PvuII restriction fragment within the Sau3A insert. Subcloning experiments demonstrated that a single HindIII restriction site within this fragment lies within the DCD1 gene. Subsequent DNA sequence analysis revealed a 936-nucleotide open reading frame encompassing this HindIII site. Disruption of the open reading frame by integrative transformation led to a loss of enzyme activity and confirmed that this region constitutes the dCMP deaminase gene. Northern analysis indicated that the DCD1 mRNA is a 1.15-kb poly(A)+ transcript. The 5' end of the transcript was mapped by primer extension and appears to exhibit heterogeneous termini. Comparison of the amino acid sequence of the T2 bacteriophage dCMP deaminase with that deduced for the yeast enzyme revealed a limited degree of homology which extends over the entire length of the phage polypeptide (188 amino acids) but is confined to the carboxy-terminal half of the yeast protein (312 amino acids). A potential dTTP-binding site in the yeast and phage enzymes was identified by comparison of homologous regions with the amino acid sequences of a variety of other dTTP-binding enzymes. Despite the role of dCMP deaminase in dTTP biosynthesis, Northern analysis revealed that the DCD1 gene is not subject to the same cell cycle-dependent pattern of transcription recently found for the yeast thymidylate synthetase gene (TMP1).
Topics: Amino Acid Sequence; Base Sequence; DCMP Deaminase; DNA Restriction Enzymes; Genes; Genes, Fungal; Nucleic Acid Hybridization; Nucleotide Deaminases; Plasmids; Saccharomyces cerevisiae; Sequence Homology, Nucleic Acid; Transcription, Genetic
PubMed: 3023902
DOI: 10.1128/mcb.6.5.1711-1721.1986 -
Biochemical Genetics Aug 1984Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine,...
Following chemical mutagenesis and multiple-step indirect selection, four clones of Chinese hamster V79 cells were isolated which exhibited auxotrophy for thymidine, deoxycytidine, or deoxyuridine but not for cytidine or uridine. All were resistant to uridine, 3-deazauridine, 5-fluorouridine, thymidine, and cytosine arabinoside at concentrations that were toxic to wild-type V79 cells. The cytidine 5'-triphosphate (CTP) and deoxycytidine 5'-triphosphate (dCTP) pools in the mutants were expanded, but the uridine 5'-triphosphate (UTP) pool either decreased or remained unchanged relative to the wild-type level. Furthermore, since the parental cells appear to be deficient in dCMP deaminase activity and CTP (or one of its metabolites) has been shown to inhibit uridine 5'-diphosphate (UDP) reduction, an elevated CTP level should lead to the observed thymidine auxotrophy. It also explains the joint resistance of mutant clones to thymidine and cytosine arabinoside. The change in the ratio of intracellular dCTP to thymidine 5'-triphosphate (dTTP) may be responsible for the elevation in the rates of spontaneous mutations in these mutants.
Topics: 3-Deazauridine; Animals; Carbon-Nitrogen Ligases; Cell Line; Cell Survival; Cricetinae; Cricetulus; Kinetics; Ligases; Lung; Mutagens; Mutation; Nucleosides
PubMed: 6497832
DOI: 10.1007/BF00485854 -
Journal of Bacteriology May 1984Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of...
Deoxycytidylate deaminase activity in Saccharomyces cerevisiae has been partially characterized. The yeast enzyme was found to exhibit properties similar to those of dCMP deaminases isolated from higher eucaryotes. A mutant strain completely deficient in dCMP deaminase activity was isolated by selection for resistance to 5-fluoro-2'-deoxycytidylate followed by screening for cross sensitivity to 5-fluoro-2'-deoxyuridylate, a potent inhibitor of the yeast thymidylate synthetase. We have designated this new allele dcd1 . A strain exhibiting an auxotrophic requirement for dUMP was isolated after mutagenesis of a dcd1 tup7 haploid. Genetic analysis revealed that this auxotrophic phenotype resulted from a combination of the dcd1 allele and a second, unlinked, nuclear mutation that we designated dmp1 . This allele, which by itself conveys no readily discernible phenotype, presumably impairs efficient synthesis of dUMP from UDP. The auxotrophic requirement of dcd1 dmp1 tup7 strains also can be satisfied by exogenous dTMP but not deoxyuridine.
Topics: Alleles; DCMP Deaminase; Deoxycytidine Monophosphate; Deoxyuracil Nucleotides; Genes, Fungal; Mutation; Nucleotide Deaminases; Phenotype; Saccharomyces cerevisiae; Thymidine Monophosphate
PubMed: 6373725
DOI: 10.1128/jb.158.2.644-649.1984