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Journal of Virology Jul 2007The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis....
The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP. Unexpectedly, however, the viral enzyme also has dCTP deaminase activity, producing dUTP. Typically, these two reactions are catalyzed by proteins in separate enzyme classes; to our knowledge, this is the first example of a protein having both deaminase activities. Kinetic experiments established that (i) the PBCV-1 enzyme has a higher affinity for dCTP than for dCMP, (ii) dCTP serves as a positive heterotropic effector for the dCMP deaminase activity and a positive homotropic effector for the dCTP deaminase activity, and (iii) the enzymatic efficiency of the dCMP deaminase activity is about four times higher than that of the dCTP deaminase activity. Inhibitor studies suggest that the same active site is involved in both dCMP and dCTP deaminations. The discovery that the PBCV-1 dCMP deaminase has two activities, together with a previous report that the virus also encodes a functional dUTP triphosphatase (Y. Zhang, H. Moriyama, K. Homma, and J. L. Van Etten, J. Virol. 79:9945-9953, 2005), means that PBCV-1 is the first virus to encode enzymes involved in all three known pathways to form dUMP.
Topics: Amino Acid Sequence; Base Sequence; Chlorella; Cloning, Molecular; DCMP Deaminase; DNA Primers; Kinetics; Molecular Sequence Data; Nucleotide Deaminases; Phycodnaviridae; Phylogeny; Sequence Homology, Amino Acid; Thymine Nucleotides
PubMed: 17475641
DOI: 10.1128/JVI.00186-07 -
Molecular Microbiology May 2020Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase...
Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase ComGA during the state of competence. We show that ComEB plays an important role during competence, possibly because it is necessary for the recruitment of GomGA to the cell pole. ComEB localizes to the cell poles even upon expression during exponential phase, indicating that it can serve as polar marker. ComEB is also a deoxycytidylate monophosphate (dCMP) deaminase, for the function of which a conserved cysteine residue is important. However, cysteine-mutant ComEB is still capable of natural transformation, while a comEB deletion strain is highly impaired in competence, indicating that ComEB confers two independent functions. Single-molecule tracking (SMT) reveals that both proteins exchange at the cell poles between bound and unbound in a time scale of a few milliseconds, but turnover of ComGA increases during DNA uptake, whereas the mobility of ComEB is not affected. Our data reveal a highly dynamic role of ComGA during DNA uptake and an unusual role for ComEB as a mediator of polar localization, localizing by diffusion-capture on an extremely rapid time scale and functioning as a moonlighting enzyme.
Topics: Adenosine Triphosphatases; Bacillus subtilis; Bacterial Proteins; Cell Polarity; DCMP Deaminase; DNA, Bacterial; DNA-Binding Proteins; Green Fluorescent Proteins; Mutation; Protein Binding; Recombinant Fusion Proteins; Single Molecule Imaging; Transformation, Bacterial
PubMed: 31954084
DOI: 10.1111/mmi.14457 -
Journal of Experimental Botany Dec 2017Deoxycytidine monophosphate deaminase (dCMP deaminase, DCD) is crucial to the production of dTTP needed for DNA replication and damage repair. However, the effect of DCD...
Deoxycytidine monophosphate deaminase (dCMP deaminase, DCD) is crucial to the production of dTTP needed for DNA replication and damage repair. However, the effect of DCD deficiency and its molecular mechanism are poorly understood in plants. Here, we isolated and characterized a rice albinic leaf and growth retardation (alr) mutant that is manifested by albinic leaves, dwarf stature and necrotic lesions. Map-based cloning and complementation revealed that ALR encodes a DCD protein. OsDCD was expressed ubiquitously in all tissues. Enzyme activity assays showed that OsDCD catalyses conversion of dCMP to dUMP, and the ΔDCD protein in the alr mutant is a loss-of-function protein that lacks binding ability. We report that alr plants have typical DCD-mediated imbalanced dNTP pools with decreased dTTP; exogenous dTTP recovers the wild-type phenotype. A comet assay and Trypan Blue staining showed that OsDCD deficiency causes accumulation of DNA damage in the alr mutant, sometimes leading to cell apoptosis. Moreover, OsDCD deficiency triggered cell cycle checkpoints and arrested cell progression at the G1/S-phase. The expression of nuclear and plastid genome replication genes was down-regulated under decreased dTTP, and together with decreased cell proliferation and defective chloroplast development in the alr mutant this demonstrated the molecular and physiological roles of DCD-mediated dNTP pool balance in plant development.
Topics: Cell Cycle; DCMP Deaminase; DNA Repair; Deoxyribonucleotides; Gene Expression Regulation; Mutation; Oryza; Plant Leaves; Plant Proteins
PubMed: 29186482
DOI: 10.1093/jxb/erx380 -
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 -
Archives of Biochemistry and Biophysics Sep 20222-Deoxycytidylate deaminase (dCD) is a member of the zinc-dependent cytidine deaminase family features in its allosterically regulated mechanism by dCTP and dTTP. The...
2-Deoxycytidylate deaminase (dCD) is a member of the zinc-dependent cytidine deaminase family features in its allosterically regulated mechanism by dCTP and dTTP. The large double-stranded DNA-containing chlorovirus PBCV-1 encodes a dCD family enzyme PBCV1dCD that was reported to be able to deaminize both dCMP and dCTP, which makes PBCV1dCD unique in the dCD family proteins. In this study, we report the crystal structure of PBCV1dCD in complex with dCTP/dCMP and dTTP/dTMP, respectively. We further proved the ability of PBCV1dCD in the deamination of dCDP, which makes PBCV1dCD a multi-functional deaminase. The structural basis for the versatility of PBCV1dCD is analyzed and discussed, with the finding of a unique Trp121 residue key to the deamination and substrate binding ability. Our findings may broaden the understanding of dCD family proteins and provide novel insights into the multi-functional enzyme.
Topics: Crystallography, X-Ray; DCMP Deaminase; Deoxycytidine Monophosphate; Substrate Specificity
PubMed: 35764100
DOI: 10.1016/j.abb.2022.109339 -
The EMBO Journal May 2024The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC...
The nucleoside analogue decitabine (or 5-aza-dC) is used to treat several haematological cancers. Upon its triphosphorylation and incorporation into DNA, 5-aza-dC induces covalent DNA methyltransferase 1 DNA-protein crosslinks (DNMT1-DPCs), leading to DNA hypomethylation. However, 5-aza-dC's clinical outcomes vary, and relapse is common. Using genome-scale CRISPR/Cas9 screens, we map factors determining 5-aza-dC sensitivity. Unexpectedly, we find that loss of the dCMP deaminase DCTD causes 5-aza-dC resistance, suggesting that 5-aza-dUMP generation is cytotoxic. Combining results from a subsequent genetic screen in DCTD-deficient cells with the identification of the DNMT1-DPC-proximal proteome, we uncover the ubiquitin and SUMO1 E3 ligase, TOPORS, as a new DPC repair factor. TOPORS is recruited to SUMOylated DNMT1-DPCs and promotes their degradation. Our study suggests that 5-aza-dC-induced DPCs cause cytotoxicity when DPC repair is compromised, while cytotoxicity in wild-type cells arises from perturbed nucleotide metabolism, potentially laying the foundations for future identification of predictive biomarkers for decitabine treatment.
PubMed: 38760575
DOI: 10.1038/s44318-024-00108-2 -
Advances in Experimental Medicine and... 1986
Topics: Animals; Azacitidine; Cell Line; Cell Survival; DCMP Deaminase; DNA, Neoplasm; Decitabine; Deoxycytosine Nucleotides; Drug Synergism; Humans; Methylation; Mice; Nucleotide Deaminases; Thymidine; Thymine Nucleotides
PubMed: 2429505
DOI: 10.1007/978-1-4684-1248-2_24 -
Journal of Genetics and Genomics = Yi... Oct 2014Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study,...
Mutants with abnormal leaf coloration are good genetic materials for understanding the mechanism of chloroplast development and chlorophyll biosynthesis. In this study, a rice mutant st2 (stripe2) with stripe leaves was identified from the γ-ray irradiated mutant pool. The st2 mutant exhibited decreased accumulation of chlorophyll and aberrant chloroplasts. Genetic analysis indicated that the st2 mutant was controlled by a single recessive locus. The ST2 gene was finely confined to a 27-kb region on chromosome 1 by the map-based cloning strategy and a 5-bp deletion in Os01g0765000 was identified by sequence analysis. The deletion happened in the joint of exon 3 and intron 3 and led to new spliced products of mRNA. Genetic complementation confirmed that Os01g0765000 is the ST2 gene. We found that the ST2 gene was expressed ubiquitously. Subcellular localization assay showed that the ST2 protein was located in mitochondria. ST2 belongs to the cytidine deaminase-like family and possibly functions as the dCMP deaminase, which catalyzes the formation of dUMP from dCMP by deamination. Additionally, exogenous application of dUMP could partially rescue the st2 phenotype. Therefore, our study identified a putative dCMP deaminase as a novel regulator in chloroplast development for the first time.
Topics: Chloroplasts; Chromosome Mapping; DCMP Deaminase; Mutation; Oryza; Phenotype; Plant Proteins
PubMed: 25438698
DOI: 10.1016/j.jgg.2014.05.008 -
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