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The Journal of Biological Chemistry Jun 1993The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase....
The cDNA encoding human dCMP deaminase was isolated from a lambda ZAPII expression library using an antibody generated against highly purified HeLa cell dCMP deaminase. The cloned cDNA consists of 1856 base pairs and encodes a protein of 178 amino acids with a calculated molecular mass of 19,985 daltons. The sequence of several cyanogen bromide-cleaved peptides derived from HeLa cell dCMP deaminase are all contained within the deduced amino acid sequence. A zinc binding region is present in the enzyme, similar to that reported for cytidine deaminase (Yang, E. C., Carlow, D., Wolfenden, R., and Short, S. A. (1992) Biochemistry 31, 4168-4174). Northern blot analysis revealed a predominant messenger RNA species of 1.9 kilobases. Expression of the active protein to about 10% of Escherichia coli's total protein was achieved by subcloning the open reading frame into a high expression system using the polymerase chain reaction. Polyacrylamide gel electrophoresis revealed a prominent protein band which comigrated with affinity purified HeLa dCMP deaminase, while Western blot analysis yielded an immunoreactive band which comigrated with the single immunoreactive affinity column purified dCMP deaminase band. The enzyme which possesses a kcat of 1.02 x 10(3) s-1 was purified to homogeneity in over 60% yield. The overexpression of dCMP deaminase should permit more exacting studies on the regulation of this important allosteric enzyme which provides substrate for DNA synthesis.
Topics: Amino Acid Sequence; Bacteriophage T4; Base Sequence; Blotting, Northern; Cloning, Molecular; DCMP Deaminase; DNA; Electrophoresis, Agar Gel; Electrophoresis, Polyacrylamide Gel; Escherichia coli; Gene Expression; HeLa Cells; Humans; Kinetics; Molecular Sequence Data; Molecular Weight; Poly A; RNA; RNA, Messenger; Recombinant Proteins; Saccharomyces cerevisiae; Sequence Homology, Amino Acid
PubMed: 7685356
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 -
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 -
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 -
Scientific Reports Sep 2017Malignant glioma is the most common brain cancer with dismal outcomes. Individual variation of the patients' survival times is remarkable. Here, we investigated the...
Malignant glioma is the most common brain cancer with dismal outcomes. Individual variation of the patients' survival times is remarkable. Here, we investigated the transcriptome and promoter methylation differences between patients of malignant glioma with short (less than one year) and the patients with long (more than three years) survival in CGGA (Chinese Glioma Genome Atlas), and validated the differences in TCGA (The Cancer Genome Atlas) to identify the genes whose expression levels showed high concordance with prognosis of glioma patients, as well as played an important role in malignant progression. The gene coding a key enzyme in genetic material synthesis, dCMP deaminase (DCTD), was found to be significantly correlated with overall survival and high level of DCTD mRNA indicated shorter survival of the patients with malignant glioma in different databases. Our finding revealed DCTD as an efficient prognostic factor for malignant glioma. As DCTD inhibitor gemcitabine has been proposed as an adjuvant therapy for malignant glioma, our finding also suggests a therapeutic value of gemcitabine for the patients with high expression level of DCTD.
Topics: Adolescent; Adult; Aged; Computational Biology; DCMP Deaminase; DNA Methylation; Epigenesis, Genetic; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Gene Ontology; Genomics; Glioma; Humans; Kaplan-Meier Estimate; Male; Middle Aged; Mutation; Neoplasm Grading; Prognosis; RNA, Messenger; Transcriptome; Young Adult
PubMed: 28912488
DOI: 10.1038/s41598-017-11962-y -
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 -
STUDIES ON NORMAL AND LEUKEMIC LEUKOCYTES. VI. THYMIDYLATE SYNTHETASE AND DEOXYCYTIDYLATE DEAMINASE.The Journal of Clinical Investigation Dec 1963
Topics: Adenosine Triphosphate; Aminohydrolases; Carbon Isotopes; Chromatography; Clinical Enzyme Tests; DCMP Deaminase; Enzyme Inhibitors; Floxuridine; Leukemia; Leukemia, Lymphoid; Leukemia, Myeloid; Leukocytes; Ligases; Lymphocytes; Nucleotides; Spectrophotometry; Thymidylate Synthase
PubMed: 14086778
DOI: 10.1172/JCI104877 -
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 -
Journal of Medicinal Chemistry Dec 2008We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is...
We report herein the application of the phosphoramidate ProTide technology to improve the metabolism of the DNA methytransferase inhibitor, zebularine (Z). Zebularine is a riboside that must undergo a complex metabolic transformation before reaching the critical 2'-deoxyzebularine 5'-triphosphate (dZTP). Because 2'-deoxyzebularine (dZ) is not phosphorylated and therefore inactive, the ProTide strategy was employed to bypass the lack of phosphorylation of dZ and the inefficient reduction of zebularine 5'-diphosphate by ribonucleotide-diphosphate reductase required for zebularine. Several compounds were identified as more potent inhibitors of DNA methylation and stronger inducers of p16 tumor suppressor gene than zebularine. However, their activity was dependent on the administration of thymidine to overcome the potent inhibition of thymidylate synthase (TS) and deoxycytidine monophosphate (dCMP) deaminase by dZMP, which deprives cells of essential levels of thymidine. Intriguingly, the activity of the ProTides was cell line-dependent, and activation of p16 was manifest only in Cf-Pac-1 pancreatic ductal adenocarcinoma cells.
Topics: Adenocarcinoma; Amides; Cytidine; DCMP Deaminase; DNA Methylation; Dose-Response Relationship, Drug; Gene Silencing; Genes, p16; Humans; Molecular Structure; Pancreatic Neoplasms; Phosphoric Acids; Reverse Transcriptase Polymerase Chain Reaction; Stereoisomerism; Thymidylate Synthase; Tumor Cells, Cultured
PubMed: 19006382
DOI: 10.1021/jm8005965 -
The American Journal of Pathology Jul 2003Biliary tract carcinoma carries a poor prognosis, and difficulties with clinical management in patients with advanced disease are often due to frequent late-stage...
Biliary tract carcinoma carries a poor prognosis, and difficulties with clinical management in patients with advanced disease are often due to frequent late-stage diagnosis, lack of serum markers, and limited information regarding biliary tumor pathogenesis. RNA-based global analyses of gene expression have led to the identification of a large number of up-regulated genes in several cancer types. We have used the recently developed Affymetrix U133A gene expression microarrays containing nearly 22,000 unique transcripts to obtain global gene expression profiles from normal biliary epithelial scrapings (n = 5), surgically resected biliary carcinomas (n = 11), and biliary cancer cell lines (n = 9). Microarray hybridization data were normalized using dCHIP (http://www.dCHIP.org) to identify differentially up-regulated genes in primary biliary cancers and biliary cancer cell lines and their expression profiles was compared to that of normal epithelial scrapings using the dCHIP software as well as Significance Analysis of Microarrays or SAM (http://www-stat.stanford.edu/ approximately tibs/SAM/). Comparison of the dCHIP and SAM datasets revealed an overlapping list of 282 genes expressed at greater than threefold levels in the cancers compared to normal epithelium (t-test P <0.1 in dCHIP, and median false discovery rate <10 in SAM). Several pathways integral to tumorigenesis were up-regulated in the biliary cancers, including proliferation and cell cycle antigens (eg, cyclins D2 and E2, cdc2/p34, and geminin), transcription factors (eg, homeobox B7 and islet-1), growth factors and growth factor receptors (eg, hepatocyte growth factor, amphiregulin, and insulin-like growth factor 1 receptor), and enzymes modulating sensitivity to chemotherapeutic agents (eg, cystathionine beta synthase, dCMP deaminase, and CTP synthase). In addition, we identified several "pathway" genes that are rapidly emerging as novel therapeutic targets in cancer (eg, cytosolic phospholipase A2, an upstream target of the cyclooxygenase pathway, and ribosomal protein S6 kinase and eukaryotic translation initiation factor 4E, two important downstream mediators of the mitogenic Akt/mTOR signaling pathway). Overexpression of selected up-regulated genes was confirmed in tissue microarrays of biliary cancers by immunohistochemical analysis (n = 4) or in situ hybridization (n = 1), and in biliary cancer cell lines by reverse transcriptase PCR (n = 2). The majority of genes identified in the present study has not been previously reported in biliary cancers, and represent novel potential screening and therapeutic targets of this cancer type.
Topics: Biliary Tract Neoplasms; Carcinoma; Gene Expression Profiling; Humans; Immunohistochemistry; In Situ Hybridization; Oligonucleotide Array Sequence Analysis; Phylogeny; Tumor Cells, Cultured; Up-Regulation
PubMed: 12819026
DOI: 10.1016/S0002-9440(10)63645-0