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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 -
The EMBO Journal Jun 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.
Topics: Decitabine; Humans; DNA (Cytosine-5-)-Methyltransferase 1; Ubiquitin-Protein Ligases; DNA Methylation; Antimetabolites, Antineoplastic; Animals; Sumoylation
PubMed: 38760575
DOI: 10.1038/s44318-024-00108-2 -
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 -
Science Translational Medicine Nov 2019Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the...
Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC.
Topics: Adenocarcinoma; Animals; Biphenyl Compounds; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; DCMP Deaminase; Dihydroorotate Dehydrogenase; Disease Progression; Enzyme Inhibitors; Humans; Lung Neoplasms; Mice; Molecular Targeted Therapy; Oxidoreductases Acting on CH-CH Group Donors; Pancreatic Neoplasms; Pyrimidines; Small Cell Lung Carcinoma; Survival Analysis; Xenograft Model Antitumor Assays
PubMed: 31694929
DOI: 10.1126/scitranslmed.aaw7852 -
Annals of Neurology May 2017Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the...
OBJECTIVE
Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2 ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy.
METHODS
To test these hypotheses, we assessed two therapies in Tk2 mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP.
RESULTS
We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2 animals compared to dCMP+dTMP.
INTERPRETATION
Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.
Topics: Animals; Antimetabolites; DNA, Mitochondrial; Deoxycytidine Monophosphate; Disease Models, Animal; Drug Therapy, Combination; Metabolism, Inborn Errors; Mice; Mice, Transgenic; Mitochondrial Diseases; Tetrahydrouridine; Thymidine; Thymidine Kinase
PubMed: 28318037
DOI: 10.1002/ana.24922 -
Biochimica Et Biophysica Acta. Proteins... Nov 2017The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby deaminases play an essential role in the thymidylate cycle, a crucial step to...
The parasite Schistosoma mansoni possess all pathways for pyrimidine biosynthesis, whereby deaminases play an essential role in the thymidylate cycle, a crucial step to controlling the ratio between cytidine and uridine nucleotides. In this study, we heterologously expressed and purified the deoxycytidylate (dCMP) deaminase from S. mansoni to obtain structural, biochemical and kinetic information. Small-angle X-ray scattering of this enzyme showed that it is organized as a hexamer in solution. Isothermal titration calorimetry was used to determine the kinetic constants for dCMP-dUMP conversion and the role of dCTP and dTTP in enzymatic regulation. We evaluated the metals involved in activating the enzyme and show for the first time the dependence of correct folding on the interaction of two metals. This study provides information that may be useful for understanding the regulatory mechanisms involved in the metabolic pathways of S. mansoni. Thus, improving our understanding of the function of these essential pathways for parasite metabolism and showing for the first time the hitherto unknown deaminase function in this parasite.
Topics: Amino Acid Sequence; Animals; Binding Sites; Cations, Divalent; Crystallography, X-Ray; DCMP Deaminase; Deoxycytosine Nucleotides; Deoxyuracil Nucleotides; Gene Expression; Kinetics; Magnesium; Models, Molecular; Protein Binding; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Protein Multimerization; Protozoan Proteins; Recombinant Proteins; Schistosoma mansoni; Sequence Alignment; Sequence Homology, Amino Acid; Substrate Specificity; Zinc
PubMed: 28807888
DOI: 10.1016/j.bbapap.2017.07.015 -
Scientific Reports Nov 2023Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine...
Deoxycytidine analogues (dCas) are widely used for the treatment of malignant diseases. They are commonly inactivated by cytidine deaminase (CDD), or by deoxycytidine monophosphate deaminase (dCMP deaminase). Additional metabolic pathways, such as phosphorylation, can substantially contribute to their (in)activation. Here, a new technique for the analysis of these pathways in cells is described. It is based on the use of 5-ethynyl 2'-deoxycytidine (EdC) and its conversion to 5-ethynyl 2'-deoxyuridine (EdU). Its use was tested for the estimation of the role of CDD and dCMP deaminase in five cancer and four non-cancer cell lines. The technique provides the possibility to address the aggregated impact of cytidine transporters, CDD, dCMP deaminase, and deoxycytidine kinase on EdC metabolism. Using this technique, we developed a quick and cheap method for the identification of cell lines exhibiting a lack of CDD activity. The data showed that in contrast to the cancer cells, all the non-cancer cells used in the study exhibited low, if any, CDD content and their cytidine deaminase activity can be exclusively attributed to dCMP deaminase. The technique also confirmed the importance of deoxycytidine kinase for dCas metabolism and indicated that dCMP deaminase can be fundamental in dCas deamination as well as CDD. Moreover, the described technique provides the possibility to perform the simultaneous testing of cytotoxicity and DNA replication activity.
Topics: Cytidine; DCMP Deaminase; Deoxycytidine Kinase; Deoxycytidine; Metabolic Networks and Pathways; Cytidine Deaminase
PubMed: 37993628
DOI: 10.1038/s41598-023-47792-4 -
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 -
Gene May 2016Recent studies were mainly focus on the cytidine deaminase family genes, which contained a lot of members that varied on the function of catalytic deamination in RNA or...
Recent studies were mainly focus on the cytidine deaminase family genes, which contained a lot of members that varied on the function of catalytic deamination in RNA or DNA and were involved in the process of growth maintenance, host immunity, retroviral infection, tumorigenesis, and drug resistance with a feature of C-U deamination. In this study, we identified a new member of cytidine deaminase family, NYD-SP15. Previous work showed that the deduced structure of the protein contained two dCMP_cyt_deam domains, which were involved in zinc ion binding. NYD-SP15 was expressed variably in a wide range of tissues, indicating its worthy biological function and creative significances. Sequence analysis, RT-PCR, western blot, flow cytometry, direct-site mutation and GST pull-down assay were performed to analyze the construction and function of NYD-SP15. The results in our studies showed that NYD-SP15 was closely related to deoxycytidylate deaminase and cytidine deaminase, with authentic cytidine deaminase activity in vivo and vitro as well as homo dimerization effects. NYD-SP15 contained nuclear localization sequence (NLS) and nuclear export-signal (NES) and could dynamically shuttle between the nucleus and cytoplasm. Furthermore, NYD-SP15 gene over-expression reduced the cells growth and blocked G1 to S phase, which implied a potential inhibition effect on cell growth.
Topics: Amino Acid Sequence; Base Sequence; Cell Cycle; Cell Nucleus; Cell Proliferation; Cytidine Deaminase; Cytoplasm; DNA-Directed RNA Polymerases; Escherichia coli; Escherichia coli Proteins; Gene Expression Regulation; Humans; MCF-7 Cells; Molecular Sequence Data; Mutation; Nuclear Localization Signals; Phylogeny; Protein Transport; Sequence Homology, Amino Acid; Transcription Factors
PubMed: 26945630
DOI: 10.1016/j.gene.2016.02.048 -
Anti-cancer Agents in Medicinal... 2017Enzymatic activity from tumor and adjacent normal tissue of 200 patients involving deoxycytidine kinase (dCK), uridine/cytidine kinase (U/CK), cytidine deaminase (CD)...
Enzymatic activity from tumor and adjacent normal tissue of 200 patients involving deoxycytidine kinase (dCK), uridine/cytidine kinase (U/CK), cytidine deaminase (CD) and deoxycytidylate deaminase (dCMPD) was quantified. Patients with brain (17), colon (24), and breast (30) tumors, 53, 67, and 73%, respectively, had an elevated T/N value (Specific Activity of tumor/ Specific Activity of normal tissue) involving dCK and dCMPD suggesting chemotherapy with 5-fluorodeoxycytidine (5-FdC) alone or in combination with thymidine plus deoxytetrahydrouridine, or with the radiosensitizer, 5-chlorodeoxycytidine (5-CldC) plus tetrahydrouridine (H4U). Among patients with colon (19) and pancreatic tumors (40), 53 and 68 %, respectively, displayed T/N values >4 for CD suggesting chemotherapy with 5-FdC, 4-N-methylamino-5-FdC, 5-trifluoromethyldeoxycytidine and radiosensitization with 5- CldC, 4-N-methylamino-5-CldC, 5-iododeoxycytidine and 5-bromodeoxycytidine. The percent of patients with tumors with a T/N value >4 for U/CK in lung (72), colon (23) and breast (28) was 47, 61 and 68, respectively, suggesting zebularine (plus thymidine) treatment for tumors involving gene silencing. Evidence is presented that the 4-N-alkylamino-dC substituted nucleosides and those with large 5-substitutions are activated only via CD to thymidine kinase (TK) using end-points of cytotoxicity and/or radiosensitization: H4U, the inhibitor of CD is an antagonist, cells with low CD or no TK are resistant to the analogs, the end points are indifferent to the dCK status of cells, they are poor substrates for dCK and good substrates for CD, whereas 5-FdC and 5-CldC are good substrates for both enzymes. The analogs present opportunities for Collateral Sensitivity for 5-azacytidine and gemcitabine resistant tumors.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cytidine Deaminase; DCMP Deaminase; Deoxycytidine Kinase; Humans; Neoplasms; Pyrimidine Nucleosides; Radiation-Sensitizing Agents; Uridine Kinase
PubMed: 27745548
DOI: 10.2174/1871520616666161013145853