-
Molecular and Cellular Biology Nov 2012Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside...
Ribonucleotide reductase (RNR) and deoxycytidylate deaminase (dCMP deaminase) are pivotal allosteric enzymes required to maintain adequate pools of deoxyribonucleoside triphosphates (dNTPs) for DNA synthesis and repair. Whereas RNR inhibition slows DNA replication and activates checkpoint responses, the effect of dCMP deaminase deficiency is largely unknown. Here, we report that deleting the Schizosaccharomyces pombe dcd1(+) dCMP deaminase gene (SPBC2G2.13c) increases dCTP ∼30-fold and decreases dTTP ∼4-fold. In contrast to the robust growth of a Saccharomyces cerevisiae dcd1Δ mutant, fission yeast dcd1Δ cells delay cell cycle progression in early S phase and are sensitive to multiple DNA-damaging agents, indicating impaired DNA replication and repair. DNA content profiling of dcd1Δ cells differs from an RNR-deficient mutant. Dcd1 deficiency activates genome integrity checkpoints enforced by Rad3 (ATR), Cds1 (Chk2), and Chk1 and creates critical requirements for proteins involved in recovery from replication fork collapse, including the γH2AX-binding protein Brc1 and Mus81 Holliday junction resolvase. These effects correlate with increased nuclear foci of the single-stranded DNA binding protein RPA and the homologous recombination repair protein Rad52. Moreover, Brc1 suppresses spontaneous mutagenesis in dcd1Δ cells. We propose that replication forks stall and collapse in dcd1Δ cells, burdening DNA damage and checkpoint responses to maintain genome integrity.
Topics: Cell Cycle; Checkpoint Kinase 1; DCMP Deaminase; DNA Damage; DNA Helicases; DNA Repair; DNA Replication; Deoxycytosine Nucleotides; Genomic Instability; Nucleotidyltransferases; Protein Kinases; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Schizosaccharomyces; Schizosaccharomyces pombe Proteins; Thymine Nucleotides
PubMed: 22927644
DOI: 10.1128/MCB.01062-12 -
British Journal of Pharmacology Oct 2009The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and... (Review)
Review
The metabolism and efficacy of 5-fluorouracil (FUra) and other fluorinated pyrimidine (FP) derivatives have been intensively investigated for over fifty years. FUra and its antimetabolites can be incorporated at RNA- and DNA-levels, with RNA level incorporation provoking toxic responses in human normal tissue, and DNA-level antimetabolite formation and incorporation believed primarily responsible for tumour-selective responses. Attempts to direct FUra into DNA-level antimetabolites, based on mechanism-of-action studies, have led to gradual improvements in tumour therapy. These include the use of leukovorin to stabilize the inhibitory thymidylate synthase-5-fluoro-2'-deoxyuridine 5' monophoshate (FdUMP)-5,10-methylene tetrahydrofolate (5,10-CH(2)FH(4)) trimeric complex. FUra incorporated into DNA also contributes to antitumour activity in preclinical and clinical studies. This review examines our current state of knowledge regarding the mechanistic aspects of FUra:Gua lesion detection by DNA mismatch repair (MMR) machinery that ultimately results in lethality. MMR-dependent direct cell death signalling or futile cycle responses will be discussed. As 10-30% of sporadic colon and endometrial tumours display MMR defects as a result of human MutL homologue-1 (hMLH1) promoter hypermethylation, we discuss the use and manipulation of the hypomethylating agent, 5-fluorodeoxycytidine (FdCyd), and our ability to manipulate its metabolism using the cytidine or deoxycytidylate (dCMP) deaminase inhibitors, tetrahydrouridine or deoxytetrahydrouridine, respectively, as a method for re-expression of hMLH1 and re-sensitization of tumours to FP therapy.
Topics: Antimetabolites, Antineoplastic; Cell Line, Tumor; DNA Mismatch Repair; Fluorouracil; Humans; Models, Biological; Neoplasms; Signal Transduction
PubMed: 19775280
DOI: 10.1111/j.1476-5381.2009.00423.x -
Cell Biology International Reports Oct 1983The enzyme activity of dCMP deaminase (EC 3.5.4.12) In Acetabularia mediterranea is substantially increased at the beginning of cyst formation. A similar albeit advanced...
The enzyme activity of dCMP deaminase (EC 3.5.4.12) In Acetabularia mediterranea is substantially increased at the beginning of cyst formation. A similar albeit advanced increase is observed in anucleate cells. The advance in the anucleate cells is not due to non-specific effects of cell surgery since nucleate grafts which are also subjected to surgery exhibit a delayed regulation characteristic for nucleate cells. Moreover, the nuclear effect on the regulation is demonstrated by experiments in which the flow of information from nucleus to cytoplasm is interrupted by treatment with actinomycin D or by tying off the rhizoid for five days. In these cells not only is the time of the increase advanced but also cyst formation is accelerated.
Topics: Cell Nucleus; Chlorophyta; DCMP Deaminase; Nucleotide Deaminases
PubMed: 6640680
DOI: 10.1016/0309-1651(83)90190-x -
Bulletin Du Cancer Aug 2002The drugs concerned by this review are cytarabine (ara-C), gemcitabine and fludarabine. Seventy-eighty per cent of a dose of ara-C are excreted under the form of ara-U... (Review)
Review
The drugs concerned by this review are cytarabine (ara-C), gemcitabine and fludarabine. Seventy-eighty per cent of a dose of ara-C are excreted under the form of ara-U (main metabolite). Plasma concentrations of ara-C are not related to drug pharmacodynamics (response to treatment) in contrast to intracellular levels of ara-CTP (active metabolite) which are associated with cytotoxic activity. Gemcitabine is able to autoactivate its own mechanism of action. Gemcitabine is characterized by a short half-life of elimination (15-20 min) and plasma pharmacokinetics of the drug are not linked to pharmacodynamics. Prolonged administration of gemcitabine is pharmacokinetically and pharmacologically justified and should deserve more intense clinical investigations. Total body clearance of F-ara-A (main circulating metabolite of fludarabine) is linked to creatinine clearance and drug-related neutropenia are more frequent in patients with creatinine clearance below 50 mL/min. So far there are no relationships between intracellular levels of F-ara-CTP and response to treatment.
Topics: Antimetabolites; Antimetabolites, Antineoplastic; Biotransformation; Cytarabine; Cytidine Deaminase; DCMP Deaminase; Deoxycytidine; Deoxycytidine Kinase; Female; Half-Life; Humans; Male; Metabolic Clearance Rate; Neoplasm Proteins; Phosphorylation; Prodrugs; Vidarabine; Gemcitabine
PubMed: 12449033
DOI: No ID Found -
The Journal of Biological Chemistry Nov 2015Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine...
Functional and deep sequencing studies have combined to demonstrate the involvement of APOBEC3B in cancer mutagenesis. APOBEC3B is a single-stranded DNA cytosine deaminase that functions normally as a nuclear-localized restriction factor of DNA-based pathogens. However, it is overexpressed in cancer cells and elicits an intrinsic preference for 5'-TC motifs in single-stranded DNA, which is the most frequently mutated dinucleotide in breast, head/neck, lung, bladder, cervical, and several other tumor types. In many cases, APOBEC3B mutagenesis accounts for the majority of both dispersed and clustered (kataegis) cytosine mutations. Here, we report the first structures of the APOBEC3B catalytic domain in multiple crystal forms. These structures reveal a tightly closed active site conformation and suggest that substrate accessibility is regulated by adjacent flexible loops. Residues important for catalysis are identified by mutation analyses, and the results provide insights into the mechanism of target site selection. We also report a nucleotide (dCMP)-bound crystal structure that informs a multistep model for binding single-stranded DNA. Overall, these high resolution crystal structures provide a framework for further mechanistic studies and the development of novel anti-cancer drugs to inhibit this enzyme, dampen tumor evolution, and minimize adverse outcomes such as drug resistance and metastasis.
Topics: Catalytic Domain; Crystallography, X-Ray; Cytidine Deaminase; Enzyme Stability; Humans; Minor Histocompatibility Antigens; Models, Molecular; Protein Conformation; Solubility
PubMed: 26416889
DOI: 10.1074/jbc.M115.679951 -
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 -
The Journal of Biological Chemistry Sep 1996The enzymes deoxycytidylate deaminase (EC) and thymidylate synthase (EC) are functionally associated with one another, since they catalyze sequential reactions. In T4...
The enzymes deoxycytidylate deaminase (EC) and thymidylate synthase (EC) are functionally associated with one another, since they catalyze sequential reactions. In T4 coliphage infection the two enzymes are found in dNTP synthetase, a multienzyme complex for deoxyribonucleotide biosynthesis. Protein-protein interactions involving the phage-coded forms of these two enzymes have been explored in three experiments that use the respective purified protein as an affinity ligand. First, an extract of radiolabeled T4 proteins was passed through a column of immobilized enzyme (either dTMP synthase or dCMP deaminase), and the specifically bound proteins were identified. Second, two mutant form of dCMP deaminase (H90N and H94N), altered in presumed zinc-binding sites, were analyzed similarly, with the results suggesting that some, but not all, interactions require normal structure near the catalytic site. Third, affinity chromatography using either enzyme as the immobilized ligand, revealed interactions between the two purified enzymes in the absence of other proteins. In these experiments we noted a significant effect of dCTP, an allosteric modifier of dCMP deaminase, upon the interactions.
Topics: Bacteriophage T4; DCMP Deaminase; Deoxycytosine Nucleotides; Enzymes, Immobilized; Models, Molecular; Multienzyme Complexes; Protein Binding; Thymidylate Synthase
PubMed: 8798492
DOI: 10.1074/jbc.271.38.23037 -
Molecular Pharmacology Jan 2003Deoxycytidylate deaminase, catalyzing the conversion of dCMP to dUMP, is an important enzyme in the de novo synthesis of thymidine nucleotides. It also may be involved...
Assessment of the effect of phosphorylated metabolites of anti-human immunodeficiency virus and anti-hepatitis B virus pyrimidine analogs on the behavior of human deoxycytidylate deaminase.
Deoxycytidylate deaminase, catalyzing the conversion of dCMP to dUMP, is an important enzyme in the de novo synthesis of thymidine nucleotides. It also may be involved in the action, as well as the metabolism of anticancer agents. Recently, several L- and D-configuration pyrimidine deoxynucleoside analogs were found to be potent antiviral and antitumor agents. Their interaction with dCMP deaminase as a monophosphate or a triphosphate metabolite is not clear. These include D-nucleoside analogs such as beta-D-2',3'-dideoxycytidine (ddC), beta-2'-fluoro-5-methyl-arabinofuranosyluracil (FMAU), 3'-azido-2',3'-dideoxythymidine (AZT), and 2',3'-didehydro-2',3'-dideoxythymidine (D4T) as well as L-nucleoside analogs such as beta-L-dioxolane-cytidine (L-OddC), beta-L-2',3'-dideoxy-3'-thiacytidine, beta-L-2',3'-dideoxy-5'-fluoro-3'-thia-cytidine (L-FSddC), beta-L-2',3'-dideoxy-2',3'-didehydro-5-fluorocytidine, and L-FMAU. None of the L-deoxycytidine analog monophosphates act as substrates or inhibitors. Among these pyrimidine deoxynucleoside analog monophosphates, D-FMAU monophosphate (MP) is the most potent competitive inhibitor, whereas L-FMAUMP has no inhibitory activity. Interestingly, AZTMP and D4TMP also have potent inhibitory activities on dCMP deaminase. Among the dCTP and TTP analogs examined, D- and L-FMAUTP were the most potent inhibitors and had the same extent of inhibitory effect. These results suggest that a chiral specificity for the substrate-binding site may exist, but there is no chiral specificity for the regulator-binding site. This is also supported by the observation that L-OddC and L-FSddC have inhibitory activities as triphosphates but not as monophosphates. None of the D- and L-dCTP analogs activated dCMP deaminase as dCTP. The biological activities of AZT and D4T could be partially attributable to their inhibitory activity against dCMP deaminase by their phosphorylated metabolites, whereas that of ddC and the L-deoxycytidine analogs may not involve dCMP deaminase directly.
Topics: Antiviral Agents; DCMP Deaminase; Deamination; Deoxycytidine Monophosphate; HIV; Hepatitis B virus; Humans; Pyrimidines; Substrate Specificity; Tumor Cells, Cultured
PubMed: 12488542
DOI: 10.1124/mol.63.1.105 -
Experimental Cell Research Nov 1984The size of the dCTP pool has been implicated as a possible regulator of DNA synthesis. In this investigation we correlate large intracellular variations in...
The size of the dCTP pool has been implicated as a possible regulator of DNA synthesis. In this investigation we correlate large intracellular variations in deoxyribonucleoside triphosphate levels to the growth rates and cell-cycle kinetics of mouse S49 T-lymphoma cells. Wild-type and a mutant line AzidoC-100-5, lacking dCMP-deaminase activity resulting in a 10-fold expanded dCTP pool were studied and compared using flow cytometry, centrifugal elutriation and nucleoside triphosphate determinations. An increase in the dCTP pool was closely correlated to the passage of cells from G1 to S phase in both cell types. Addition of thymidine to wild-type and mutant cells resulted in an accumulation of cells in early S phase, concomitant with a decreased dCTP level. Mutant cells excreted large amounts of deoxycytidine into the medium which partially protected the cells from thymidine inhibition. The doubling times for the mutant and wild-type cells were very similar but the mutant had a somewhat prolonged S phase and shortened G1 phase compared with the wild-type cells. Large changes in the DNA precursor levels were produced by addition of thymidine to mutant cultures. This gave no change in the growth rate but a somewhat shortened S phase and prolonged G1. The biochemical background for these effects is discussed.
Topics: Animals; Cell Cycle; Cell Line; Cells, Cultured; DCMP Deaminase; Deoxyribonucleotides; Flow Cytometry; Lymphoma; Mice; Nucleotide Deaminases; Thymidine
PubMed: 6489455
DOI: 10.1016/0014-4827(84)90774-2 -
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