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The Journal of Biological Chemistry Feb 1984The thy- mutator phenotype of Chinese hamster ovary cells is distinguished by increased intracellular levels of dCTP, auxotrophy for thymidine, and elevated spontaneous...
The thy- mutator phenotype of Chinese hamster ovary cells is distinguished by increased intracellular levels of dCTP, auxotrophy for thymidine, and elevated spontaneous mutational rates. To determine the biochemical lesion responsible for this complex phenotype, enzymes responsible for the synthesis of dCTP and dTTP were investigated. Levels of ribonucleotide reductase and dCMP deaminase were identical in mutant and wild type strains. In contrast, CTP synthetase activity in extracts from thy- strains was consistently altered in that 50% of enzyme activity was resistant to feedback inhibition by CTP. Additionally, thy- strains obtained by DNA transfection also had CTP-resistant CTP synthetase. Thy+ revertants lost the resistant enzyme, and total activity was reduced. CTP-resistant CTP synthetase was regained in thy- mutants reselected from thy+ revertants, but in these strains all activity was resistant. These experiments demonstrate that the thy- mutator phenotype is a consequence of a mutation of CTP synthetase and suggest that one pathway of reversion to the wild type state is by loss or inactivation of the mutant allele rendering the revertants hemizygous for the gene.
Topics: Animals; Cell Line; Cricetinae; Cricetulus; Deoxyribonucleotides; Feedback; Female; Genes; Mutation; Ovary; Phenotype; Ribonucleotides; Thymidine; Thymine
PubMed: 6698969
DOI: No ID Found -
European Journal of Biochemistry Dec 1983Hypotheses about the interactions of effectors with conformations of allosteric enzymes having co-operative kinetics can be tested simply and exactly without knowledge...
Hypotheses about the interactions of effectors with conformations of allosteric enzymes having co-operative kinetics can be tested simply and exactly without knowledge of the nature of the intersubunit co-operativity by using a linkage approach to the analysis of steady-state kinetics. Applying this approach to competition for substrate sites in the allosteric enzyme donkey spleen dCMP aminohydrolase, we show that the kinetics are consistent with the hypothesis that the substrate dCMP and the competitor dAMP, as well as the allosteric activator dCTP, bind exclusively to the same conformation of the enzyme subunits. The linkage test can be applied in the presence of other effectors without knowledge of how these interact with the enzyme. Our tests showed that dCMP and DAMP are still bound exclusively to this same conformation in the presence of the product dUMP or of the allosteric inhibitor dTTP. We give evidence that dUMP binds to the same conformation as dCMP, but that it is also bound to other conformation(s). The advantages of the linkage approach, and some general problems in steady-state kinetics of allosteric enzymes, are discussed.
Topics: Allosteric Site; Animals; Binding, Competitive; DCMP Deaminase; Deoxyadenine Nucleotides; Deoxycytosine Nucleotides; Deoxyuracil Nucleotides; Kinetics; Models, Biological; Nucleotide Deaminases; Perissodactyla; Protein Conformation; Spleen; Substrate Specificity; Thymine Nucleotides
PubMed: 6662104
DOI: 10.1111/j.1432-1033.1983.tb07845.x -
Journal of Bacteriology Dec 1983Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They...
Cell-free extracts of Mycoplasma mycoides subsp. mycoides were assayed for enzymes associated with the salvage synthesis of pyrimidine deoxyribonucleotides. They possessed kinases for deoxycytidine, (d)CMP, thymidine (deoxyuridine), dTMP, and nucleoside diphosphates; dCTPase and dUTPase; dCMP deaminase; thymidine (deoxyuridine) phosphorylase; and dUMP (dTMP) phosphatase. The existence of these enzymic activities together with ribonucleoside diphosphate reductase explains the capacity of cytidine to provide M. mycoides with deoxyribose for the synthesis of thymidine nucleotides from thymine.
Topics: DCMP Deaminase; Deoxycytidine Kinase; Mycoplasma mycoides; Nucleoside-Diphosphate Kinase; Nucleoside-Phosphate Kinase; Phosphoric Monoester Hydrolases; Pyrimidine Nucleotides; Pyrophosphatases; Thymidine Kinase; Thymidine Phosphorylase
PubMed: 6139361
DOI: 10.1128/jb.156.3.1001-1005.1983 -
The Journal of Biological Chemistry Jul 1983The amino acid sequence of deoxycytidylate deaminase isolated from T2 phage-infected Escherichia coli has been determined. The enzyme is a hexamer, consisting of...
The amino acid sequence of deoxycytidylate deaminase isolated from T2 phage-infected Escherichia coli has been determined. The enzyme is a hexamer, consisting of identical polypeptide subunits, each composed of 188 amino acids with a calculated Mr = 20,560. The primary structure was established by automatic Edman degradation of the intact carboxymethylated protein and of peptides derived from the protein by cleavage with cyanogen bromide, trypsin, chymotrypsin, the Staphylococcus aureus V8 protease, and 2-(2-nitrophenylsulfenyl)-3-methyl-3-bromoindolenine. Knowledge of the primary structure of deoxycytidylate deaminase should aid in determining the allosteric binding site of the negative effector, dTTP, recently reported (Maley, F., and Maley, G.F. (1982) J. Biol. Chem. 257, 11876-11878), and eventually that of the enzyme's positive regulator, dCTP, as well as its substrate. The deaminase has been crystallized through the use of polyethylene glycol; a scanning electron micrograph is presented.
Topics: Allosteric Regulation; Amino Acid Sequence; Cyanogen Bromide; DCMP Deaminase; Escherichia coli; Macromolecular Substances; Nucleotide Deaminases; Peptide Fragments; T-Phages; Trypsin
PubMed: 6345541
DOI: No ID Found -
Antimicrobial Agents and Chemotherapy Mar 1983The incorporation into DNA of 5-bromocytosine and 5-iodocytosine, derived from their respective administered deoxyribonucleoside analogs, has been demonstrated in...
The incorporation into DNA of 5-bromocytosine and 5-iodocytosine, derived from their respective administered deoxyribonucleoside analogs, has been demonstrated in studies with cells infected with herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) and in cells transformed with the thymidine kinase gene of HSV-1. No significant incorporation of iodocytosine or iodouracil occurred in the DNA of uninfected or nontransformed cells when the deaminating enzymes were inhibited, in accord with past studies in our laboratory with 5-bromodeoxycytidine and tetrahydrouridine. When 2'-deoxytetrahydrouridine, a potent inhibitor of cytidine deaminase and dCMP deaminase, was utilized, all the counts in DNA that were derived from [(125)I]iododeoxycytidine appeared as iodocytosine in HSV-infected cells. In the absence of a deaminase inhibitor, 32 to 45% of the counts associated with DNA pyrimidines appeared as iodocytosine, and 55 to 68% appeared as iodouracil in HSV-infected cells. Substantial incorporation of iodocytosine (16%) occurred in cells transformed with the HSV thymidine kinase gene, suggesting the importance of the specificity of cellular nucleoside kinases and the activity of the deaminases in presenting unmodified bases to an undiscriminating polymerase. Incorporation into DNA of bromocytosine derived from [(3)H]bromodeoxycytidine was demonstrated in HSV-2 infected cells; very little incorporation of bromocytosine compared with bromouracil could be demonstrated in these cells in the absence of inhibition of the deaminases (19% of the total counts associated with pyrimidines with deaminase inhibition and 1.5% without). Limited studies with 5-methyl[5-(3)H]deoxycytidine indicated essentially no (or very little) incorporation of this analog as such in the DNA of HSV-1- and HSV-2-infected and -transformed cells. This suggests an exclusion or repair mechanism preventing inappropriate methylcytosine incorporation in DNA. The addition of nucleoside and deoxyribonucleoside deaminase inhibitors, which leads to the incorporation of 5-halogenated analogs of deoxycytidine into DNA as such, does not impair their antiviral activity. We infer from studies with 4-N-alkyl (ethyl and isopropyl)-substituted analogs of iododeoxycytidine that they are incorporated as such into DNA without deamination and effectively inhibit the virus at concentrations that are marginally toxic. Among the several reasons presented for the heightened potential efficacy of analogs of deoxycytidine compared with those of deoxyuridine is that the former, as analogs of 5-methyldeoxycytidine, may impair viral replication by perturbing processes involving methylation and changes in the methylation of deoxycytidine in DNA which appear to be important for the process of HSV maturation. In addition, this capacity to perturb methylation may, in turn, be the key to their potential as agents affecting entry into or emergence from latency, a process in which dramatic changes in the postpolymer 5-methylation of deoxycytidine occur in the DNA of herpesviruses.
Topics: Animals; Antiviral Agents; Bromodeoxycytidine; Cell Transformation, Viral; Cytidine Deaminase; DNA, Viral; Deamination; Deoxycytidine; Kinetics; Simplexvirus; Structure-Activity Relationship; Thymidine Kinase; Tritium
PubMed: 6303214
DOI: 10.1128/AAC.23.3.465 -
The Biochemical Journal Feb 1983Bone-marrow macrophages from both rat and mouse release deoxycytidine derived from phagocytosed nuclei. Mouse plasma contains no detectable deoxycytidine (less than 0.1... (Comparative Study)
Comparative Study
Bone-marrow macrophages from both rat and mouse release deoxycytidine derived from phagocytosed nuclei. Mouse plasma contains no detectable deoxycytidine (less than 0.1 microM), whereas the concentration in rat plasma is 18 microM. Enzyme assays of tissue extracts show that both mouse and rat spleen contain high deoxycytidine kinase activity. Mouse organs, including kidney, liver and lung, also have deoxycytidine deaminase activity. In contrast, rat tissues have virtually no deoxycytidine deaminase activity. Lack of deaminase provides an explanation for the presence of deoxycytidine in rat plasma. Cytotoxicity assays show that cultured mouse lymphoid cells grown in undialysed rat serum are more resistant to cytotoxic effects of deoxyadenosine than are those cells grown in dialysed rat serum. The results suggest that a major difference in deoxycytidine metabolism between mouse and rat may account for discrepancies in the pharmacological response of the two animals to certain nucleoside compounds.
Topics: Animals; Bone Marrow; Cell Line; Cell Survival; Cells, Cultured; DCMP Deaminase; Deoxyadenosines; Deoxycytidine; Deoxycytidine Kinase; Macrophages; Mice; Mice, Inbred C57BL; Rats; Rats, Inbred Strains; Species Specificity; Tissue Distribution
PubMed: 6602609
DOI: 10.1042/bj2100367 -
The Journal of Biological Chemistry Dec 1982A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at...
A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.
Topics: Centrifugation, Density Gradient; Cytosol; DNA Replication; Deoxyribonucleotides; Escherichia coli; Kinetics; Multienzyme Complexes; T-Phages; Virus Replication
PubMed: 6757252
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1982Thymidine triphosphate, a negative regulator of deoxycytidylate deaminase, was found to bind covalently to this enzyme on exposure to UV light at 254 nM. The rate of...
Thymidine triphosphate, a negative regulator of deoxycytidylate deaminase, was found to bind covalently to this enzyme on exposure to UV light at 254 nM. The rate of half-maximal fixation was extremely rapid, occurring within 30 s and probably attaining a maximum of about 1 mol of dTTP fixed/mol of enzyme subunit. In contrast to the case of ribonucleotide reductase (Ericksson, S., Caras, I. W., and Martin, D. W., Jr. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 81-85) where the fixation of dTTP inactivated this enzyme, the activity of the deaminase was unaffected. The bound nucleotide could be released on exposure to UV 254 nm light in the presence of dCTP or dTTP but not dATP or dGTP. The enzyme-fixed nucleotide was found to remain with the larger of the two peptides released as a result of CNBr treatment of the labeled enzyme. Studies are in progress to define the location of this nucleotide, which will be aided greatly by our recent clarification of the complete amino acid sequence of T2-deoxycytidylate deaminase.
Topics: Binding Sites; DCMP Deaminase; Deoxyribonucleotides; Escherichia coli; Macromolecular Substances; Nucleotide Deaminases; Photochemistry; Thymine Nucleotides; Ultraviolet Rays
PubMed: 6749849
DOI: No ID Found -
The Journal of Biological Chemistry Jun 1981
Topics: DCMP Deaminase; Deoxycytidine Monophosphate; Deoxycytosine Nucleotides; Enzyme Activation; Humans; Hydrogen-Ion Concentration; Molecular Weight; Nucleotide Deaminases; Spleen; Thymine Nucleotides
PubMed: 7240207
DOI: No ID Found -
Proceedings of the National Academy of... Apr 1981Recent studies of in vitro DNA synthesis have shown that fidelity of replication is influenced by the relative concentrations of deoxyribonucleoside triphosphates...
Recent studies of in vitro DNA synthesis have shown that fidelity of replication is influenced by the relative concentrations of deoxyribonucleoside triphosphates (dNTPs). Several investigators have used reconstituted prokaryotic replication systems to copy defined natural templates and have shown that specific incorporation errors can be induced by an appropriate bias of the precursor pools. The recent demonstration of mutator phenotypes among mutant Chinese hamster ovary cell lines with altered intracellular dNTP pools has allowed extension of the in vitro observations to eukaryotic replication and repair mechanisms. We describe here three mutant murine T-lymphosarcoma cell lines with altered dNTP pools and increased rates of spontaneous mutation to dexamethasone resistance and 6-thioguanine resistance. Unlike previously described mammalian cells with mutator phenotypes, these three lines have demonstrable defects in known structural gene products. Two of these cell lines are heterozygous for mutations affecting the M1 subunit of ribonucleoside diphosphate reductase; the other mutant is deficient in deoxycytidylate deaminase. In each cell line these mutations result in deranged endogenous dNTP pools and increased rates of spontaneous mutation, which are shown to be characteristic of the cell line and independent of the two genetic markers examined. Furthermore, normalization of the dNTP pools of the deaminase-deficient cells suppresses its mutator phenotype. Thus, abnormal dNTP pools seem to cause enhanced mutagenesis in mammalian cells.
Topics: Animals; Cell Line; DCMP Deaminase; Deoxyribonucleotides; Lymphoma; Mice; Mutation; Phenotype; Ribonucleotide Reductases
PubMed: 7017732
DOI: 10.1073/pnas.78.4.2447