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Ideal conformer Mrv1927 07272110063D starting HoverWatcher_5 Time for openFile(DCP - Ideal conformer Mrv1927 07272110063D 44 45 0 0 0 0 999 V2000 0.3030 0.6270 -5.1690 N 0 0 0 0 0 0 0 0 0 0 0 0 0.7940 -0.6180 -5.0380 C 0 0 0 0 0 0 0 0 0 0 0 0 1.8570 -1.0150 -5.7350 N 0 0 0 0 0 0 0 0 0 0 0 0 2.4600 -0.1890 -6.5790 C 0 0 0 0 0 0 0 0 0 0 0 0 1.9740 1.1240 -6.7360 C 0 0 0 0 0 0 0 0 0 0 0 0 0.8890 1.5110 -6.0250 C 0 0 0 0 0 0 0 0 0 0 0 0 0.2570 -1.3970 -4.2670 O 0 0 0 0 0 0 0 0 0 0 0 0 3.5580 -0.6100 -7.2930 N 0 0 0 0 0 0 0 0 0 0 0 0 -0.8670 1.0340 -4.3890 C 0 0 1 0 0 0 0 0 0 0 0 0 -2.0640 0.1240 -4.7250 C 0 0 1 0 0 0 0 0 0 0 0 0 -2.4580 -0.5010 -3.3650 C 0 0 1 0 0 0 0 0 0 0 0 0 -1.8370 0.4970 -2.3560 C 0 0 2 0 0 0 0 0 0 0 0 0 -0.5950 0.8870 -2.9850 O 0 0 0 0 0 0 0 0 0 0 0 0 -3.8790 -0.5540 -3.2210 O 0 0 0 0 0 0 0 0 0 0 0 0 -1.5730 -0.1870 -1.0130 C 0 0 1 0 0 0 0 0 0 0 0 0 -1.0000 0.7530 -0.1030 O 0 0 0 0 0 0 0 0 0 0 0 0 -0.7500 -0.0300 1.2800 P 0 0 1 0 0 5 0 0 0 0 0 0 -2.0390 -0.5430 1.7960 O 0 0 0 0 0 0 0 0 0 0 0 0 0.2540 -1.2620 1.0250 O 0 0 0 0 0 0 0 0 0 0 0 0 -0.1030 0.9750 2.3580 O 0 0 0 0 0 0 0 0 0 0 0 0 0.1180 0.1290 3.7090 P 0 0 1 0 0 5 0 0 0 0 0 0 -1.1830 -0.3860 4.1880 O 0 0 0 0 0 0 0 0 0 0 0 0 1.1090 -1.1040 3.4130 O 0 0 0 0 0 0 0 0 0 0 0 0 0.7660 1.0790 4.8360 O 0 0 0 0 0 0 0 0 0 0 0 0 0.9600 0.1730 6.1530 P 0 0 1 0 0 5 0 0 0 0 0 0 1.8570 -0.9610 5.8430 O 0 0 0 0 0 0 0 0 0 0 0 0 1.6080 1.0640 7.3270 O 0 0 0 0 0 0 0 0 0 0 0 0 -0.4700 -0.3880 6.6330 O 0 0 0 0 0 0 0 0 0 0 0 0 2.4540 1.8090 -7.4200 H 0 0 0 0 0 0 0 0 0 0 0 0 0.4940 2.5110 -6.1310 H 0 0 0 0 0 0 0 0 0 0 0 0 3.8930 -1.5130 -7.1800 H 0 0 0 0 0 0 0 0 0 0 0 0 3.9950 -0.0050 -7.9130 H 0 0 0 0 0 0 0 0 0 0 0 0 -1.1180 2.0720 -4.6110 H 0 0 0 0 0 0 0 0 0 0 0 0 -2.8890 0.7110 -5.1280 H 0 0 0 0 0 0 0 0 0 0 0 0 -1.7670 -0.6520 -5.4300 H 0 0 0 0 0 0 0 0 0 0 0 0 -2.0200 -1.4930 -3.2510 H 0 0 0 0 0 0 0 0 0 0 0 0 -2.4860 1.3620 -2.2230 H 0 0 0 0 0 0 0 0 0 0 0 0 -4.2110 -1.1180 -3.9330 H 0 0 0 0 0 0 0 0 0 0 0 0 -2.5110 -0.5620 -0.6060 H 0 0 0 0 0 0 0 0 0 0 0 0 -0.8820 -1.0180 -1.1590 H 0 0 0 0 0 0 0 0 0 0 0 0 1.0790 -0.8850 0.6900 H 0 0 0 0 0 0 0 0 0 0 0 0 1.9430 -0.7250 3.1050 H 0 0 0 0 0 0 0 0 0 0 0 0 1.7060 0.4860 8.0950 H 0 0 0 0 0 0 0 0 0 0 0 0 -1.0230 0.3810 6.8240 H 0 0 0 0 0 0 0 0 0 0 0 0 1 2 1 0 0 0 0 1 6 1 0 0 0 0 1 9 1 0 0 0 0 2 3 1 0 0 0 0 2 7 2 0 0 0 0 3 4 2 0 0 0 0 4 5 1 0 0 0 0 4 8 1 0 0 0 0 5 6 2 0 0 0 0 5 29 1 0 0 0 0 6 30 1 0 0 0 0 8 31 1 0 0 0 0 8 32 1 0 0 0 0 9 10 1 0 0 0 0 9 13 1 0 0 0 0 9 33 1 1 0 0 0 10 11 1 0 0 0 0 10 34 1 0 0 0 0 10 35 1 0 0 0 0 11 12 1 0 0 0 0 11 14 1 0 0 0 0 11 36 1 1 0 0 0 12 13 1 0 0 0 0 12 15 1 0 0 0 0 12 37 1 1 0 0 0 14 38 1 0 0 0 0 15 16 1 0 0 0 0 15 39 1 0 0 0 0 15 40 1 0 0 0 0 17 16 1 6 0 0 0 17 18 2 0 0 0 0 17 19 1 0 0 0 0 17 20 1 0 0 0 0 19 41 1 0 0 0 0 21 20 1 6 0 0 0 21 22 2 0 0 0 0 21 23 1 0 0 0 0 21 24 1 0 0 0 0 23 42 1 0 0 0 0 24 25 1 0 0 0 0 25 26 2 0 0 0 0 25 27 1 0 0 0 0 25 28 1 0 0 0 0 27 43 1 0 0 0 0 28 44 1 0 0 0 0 M END): 40 ms reading 44 atoms ModelSet: haveSymmetry:false haveUnitcells:false haveFractionalCoord:false 1 model in this collection. Use getProperty "modelInfo" or getProperty "auxiliaryInfo" to inspect them. Default Van der Waals type for model set to Babel 44 atoms created ModelSet: not autobonding; use forceAutobond=true to force automatic bond creation Script completed Jmol script terminated
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Deoxycytidine triphosphate (dCTP) is a nucleoside triphosphate that contains the pyrimidine base cytosine. The triphosphate group contains high-energy phosphoanhydride bonds, which liberate energy when hydrolized.
DNA polymerase enzymes use this energy to incorporate deoxycytidine into a newly synthesized strand of DNA. A chemical equation can be written that represents the process:
(DNA)n + dCTP ↔ (DNA)n-C + PPi
That is, dCTP has the PPi (pyrophosphate) cleaved off and the dCMP is incorporated into the DNA strand at the 3' end.
Subsequent hydrolysis of the PPi drives the equilibrium of the reaction toward the right side, i.e. incorporation of the nucleotide in the growing DNA chain.
Like other nucleoside triphosphates, manufacturers recommend that dCTP be stored in aqueous solution at −20 °C. |
Read full article at Wikipedia
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InChI=1S/C9H16N3O13P3/c10-7-1-2-12(9(14)11-7)8-3-5(13)6(23-8)4-22-27(18,19)25-28(20,21)24-26(15,16)17/h1-2,5-6,8,13H,3-4H2,(H,18,19)(H,20,21)(H2,10,11,14)(H2,15,16,17)/t5-,6+,8+/m0/s1 |
RGWHQCVHVJXOKC-SHYZEUOFSA-N |
NC1=NC(=O)N(C=C1)[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 |
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Mus musculus
(NCBI:txid10090)
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Source: BioModels - MODEL1507180067
See:
PubMed
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Escherichia coli
(NCBI:txid562)
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See:
PubMed
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Escherichia coli
(NCBI:txid562)
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Found in
whole organism
(UBERON:0000468).
From MetaboLights
See:
MetaboLights Study
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Homo sapiens
(NCBI:txid9606)
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See:
DOI
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Homo sapiens
(NCBI:txid9606)
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Found in
blood
(UBERON:0000178).
See:
PubMed
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Escherichia coli metabolite
Any bacterial metabolite produced during a metabolic reaction in Escherichia coli.
human metabolite
Any mammalian metabolite produced during a metabolic reaction in humans (Homo sapiens).
mouse metabolite
Any mammalian metabolite produced during a metabolic reaction in a mouse (Mus musculus).
(via nucleoside 5'-triphoshate )
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View more via ChEBI Ontology
2'-deoxycytidine 5'-(tetrahydrogen triphosphate)
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2'-deoxy-CTP
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ChEBI
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2'-deoxycytidine 5'-triphosphate
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KEGG COMPOUND
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2'-deoxycytidine triphosphate
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ChEBI
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dCTP
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KEGG COMPOUND
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deoxy-CTP
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HMDB
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deoxycytidine 5'-triphosphate
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KEGG COMPOUND
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deoxycytidine triphosphate
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KEGG COMPOUND
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deoxycytidine-triphosphate
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HMDB
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2056-98-6
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CAS Registry Number
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ChemIDplus
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Kapustina Ž, Jasponė A, Dubovskaja V, Mackevičius G, Lubys A (2021) Enzymatic Synthesis of Chimeric DNA Oligonucleotides by in Vitro Transcription with dTTP, dCTP, dATP, and 2'-Fluoro Modified dGTP. ACS synthetic biology 10, 1625-1632 [PubMed:34110794] [show Abstract] Efficient ways to produce single-stranded DNA are of great interest for diverse applications in molecular biology and nanotechnology. In the present study, we selected T7 RNA polymerase mutants with reduced substrate specificity to employ an in vitro transcription reaction for the synthesis of chimeric DNA oligonucleotides, either individually or in pools. We performed in vitro evolution based on fluorescence-activated droplet sorting and identified mutations V783M, V783L, V689Q, and G555L as novel variants leading to relaxed substrate discrimination. Transcribed chimeric oligonucleotides were tested in PCR, and the quality of amplification products as well as fidelity of oligonucleotide synthesis were assessed by NGS. We concluded that enzymatically produced chimeric DNA transcripts contain significantly fewer deletions and insertions compared to chemically synthesized counterparts and can successfully serve as PCR primers, making the evolved enzymes superior for simple and cheap one-pot synthesis of multiple chimeric DNA oligonucleotides in parallel using a plethora of premixed templates. | Li K, Luo Y, Huang K, Yang Z, Wan Y, Xu W (2020) Single universal primer recombinase polymerase amplification-based lateral flow biosensor (SUP-RPA-LFB) for multiplex detection of genetically modified maize. Analytica chimica acta 1127, 217-224 [PubMed:32800127] [show Abstract] In this study, an isothermal paper biosensor, combining single universal primer recombinase polymerase amplification (SUP-RPA) and the lateral flow technique was developed for the multiplex detection of genetically modified maize (GMM). In pre-amplification stage, the event-specific primers contain a universal sequence at the 5' end, with a biotin-labeled deoxycytidine triphosphate (dCTP) deoxynucleotide providing additional amplification, which improves their amplification ability and ensures consistent multiplex amplification efficiency. In the signal recognition strategy, the SUP-RPA products are identified visually using the lateral flow biosensor (LFB) through dual hybridization. The accumulation of gold nanoparticles (AuNPs) produces a characteristic red band. Through this biosensor, a limit of detection of at least 50 copies was achieved, which is sensitive enough to detect MON810, MON863 and MON89034 simultaneously. The entire process of analysis was completed within 30 min and without any large-scale instrumentation. This biosensor, therefore, provides a novel rapid and portable multiple detection method for point-of-care applications, especially genetically modified organism (GMO) event-specific detection. | Martínez-Arribas B, Requena CE, Pérez-Moreno G, Ruíz-Pérez LM, Vidal AE, González-Pacanowska D (2020) DCTPP1 prevents a mutator phenotype through the modulation of dCTP, dTTP and dUTP pools. Cellular and molecular life sciences : CMLS 77, 1645-1660 [PubMed:31377845] [show Abstract] To maintain dNTP pool homeostasis and preserve genetic integrity of nuclear and mitochondrial genomes, the synthesis and degradation of DNA precursors must be precisely regulated. Human all-alpha dCTP pyrophosphatase 1 (DCTPP1) is a dNTP pyrophosphatase with high affinity for dCTP and 5'-modified dCTP derivatives, but its contribution to overall nucleotide metabolism is controversial. Here, we identify a central role for DCTPP1 in the homeostasis of dCTP, dTTP and dUTP. Nucleotide pools and the dUTP/dTTP ratio are severely altered in DCTPP1-deficient cells, which exhibit an accumulation of uracil in genomic DNA, the activation of the DNA damage response and both a mitochondrial and nuclear hypermutator phenotype. Notably, DNA damage can be reverted by incubation with thymidine, dUTPase overexpression or uracil-DNA glycosylase suppression. Moreover, DCTPP1-deficient cells are highly sensitive to down-regulation of nucleoside salvage. Our data indicate that DCTPP1 is crucially involved in the provision of dCMP for thymidylate biosynthesis, introducing a new player in the regulation of pyrimidine dNTP levels and the maintenance of genomic integrity. | Kandeel M, Al-Taher A (2020) Metabolic drug targets of the cytosine metabolism pathways in the dromedary camel (Camelus dromedarius) and blood parasite Trypanosoma evansi. Tropical animal health and production 52, 3337-3358 [PubMed:32926292] [show Abstract] Trypanosomiasis is a major illness affecting camels in tropical and subtropical regions. Comparisons of camel and Trypanosoma evansi genomes can lead to the discovery of new drug targets for treating Trypanosoma infections. The synthesis pathways of cytosine, cytidine, cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP) deoxycytidine, deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP) were compared in the dromedary camel (Camelus dromedarius) and T. evansi. None of the enzymes involved in cytosine pathway were detected in camels and T. evansi. Notably, cytidine kinase (CK) and 5'-nucleotidase, which interconverts cytidine to CMP, were not detected in T. evansi but were present in camels. UMP/CMP kinase was not predicted in T. evansi. Therefore, the presence of enzymes involved in the CTP synthesis cascade was not predicted in T. evansi. CMP synthesis might also be encoded by other enzymes, e.g., purine nucleotides kinases. Both camel and T. evansi share an efficient enzyme system for converting CDP to CTP. In conclusion, CTP synthase is important for homeostasis of cytosine nucleotides in T. evansi and could be a potential drug target against the parasite. In addition, the inhibition of UMP synthesis might contribute to parasite death as it is a shared source for CTP synthesis. | Fesenko DO, Guseinov TO, Lapa SA, Kuznetsova VE, Shershov VE, Spitsyn MA, Nasedkina TV, Zasedatelev AS, Chudinov AV (2018) [Substrate Properties of New Fluorescently Labeled Deoxycytidine Triphosphates in Enzymatic Synthesis of DNA with Polymerases of Families A and B]. Molekuliarnaia biologiia 52, 533-542 [PubMed:29989586] [show Abstract] The efficiency of the incorporation of fluorescently labeled derivatives of 2'-deoxycytidine in DNA synthesized de novo has been studied using PCR with Taq and Tth polymerases of family A and Vent (exo-) and Deep Vent (exo-) polymerases of family B. Four derivatives of 5'-triphosphate-2'-deoxycytidine (dCTP) have different chemical structures of the indodicarbocyanine dye and Cy5 analogue attached to position 5 of cytosine. The kinetics of the accumulation of the PCR products and the intensity of the fluorescent signals in the hybridization analysis with immobilized DNA probes depend on the modification of the fluorescently labeled dCTP counterpart, its concentration, and the type of DNA polymerase. All labeled triphosphates showed some inhibitory effects on PCR. The best balance between the efficiency of incorporating labeled cytidine derivatives and the negative effect on the PCR kinetics has been shown in the case of Hot Taq polymerase in combination with the Cy5-dCTP analogue, which contains containing electrically neutral chro-mophore, the axis of which is a continuation of the linker between the chromophore and the pyrimidine base. | Huang SW, Tzeng HF (2012) Simultaneous determination of deoxycytidine diphosphate and deoxycytidine triphosphate by capillary electrophoresis with transient isotachophoretic stacking: a sensitive monitoring method for ribonucleotide reductase activity. Electrophoresis 33, 536-542 [PubMed:22212996] [show Abstract] A simple and rapid capillary electrophoretic method was developed for simultaneous determination of sub-micromolar 2'-deoxycytidine 5'-diphosphate (dCDP) and 2'-deoxycytidine 5'-triphosphate (dCTP) levels in enzyme assays without using radioactively labeled substrates. The separation was performed at 25°C using MES in the BGE as the terminating ion, the chloride ions in the sample buffer as the leading ion, and PEG 4000 in the BGE as the EOF suppressor for sample stacking by transient isotachophoresis (tITP). Several parameters affecting the separation were investigated, including the pH of the BGE, the concentration of sodium chloride in the sample buffer, and the concentrations of MES and PEG 4000 in the running buffer. Good separation with high separation efficiency was achieved within 6 min under optimal conditions. In comparison with the simple CZE method, the present tITP-CZE method enabled a 150-fold increase in the injection time without any decrease in resolution and the sensitivity was enhanced up to two orders of magnitude with the new method. The linear range of the method was 0.1-10 μM for dCDP and dCTP. The limits of detection of dCDP and dCTP were 85 and 73 nM, respectively. The proposed method was successfully applied for the activity assay of ribonucleotide reductase from Hep G2 and Sf9 cells. | Crauste C, Lefebvre I, Hovaneissian M, Puy JY, Roy B, Peyrottes S, Cohen S, Guitton J, Dumontet C, Perigaud C (2009) Development of a sensitive and selective LC/MS/MS method for the simultaneous determination of intracellular 1-beta-D-arabinofuranosylcytosine triphosphate (araCTP), cytidine triphosphate (CTP) and deoxycytidine triphosphate (dCTP) in a human follicular lymphoma cell line. Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 877, 1417-1425 [PubMed:19328748] [show Abstract] A method was developed for the quantification of araCTP, CTP and dCTP in a human follicular lymphoma cell line. This method involves solid phase extraction (SPE) using a weak anion-exchanger (WAX) cartridge, a porous graphitic carbon high-performance liquid chromatography (HPLC) column separation, and tandem mass spectrometry (MS/MS) detection. By using a triple quadrupole mass spectrometer operating in negative ion multiple reaction monitoring (MRM) mode, the method was able to achieve a lower limit of quantification (LLOQ) of 0.1 microg mL(-1) for araCTP and of 0.01 microg mL(-1) for both CTP and dCTP. The method was validated and used to determine the amount of araCTP, CTP and dCTP formed after incubation of araC and an araCMP prodrug in the human follicular lymphoma cell line RL. | Yamauchi T, Ueda T (2005) A sensitive new method for clinically monitoring cytarabine concentrations at the DNA level in leukemic cells. Biochemical pharmacology 69, 1795-1803 [PubMed:15935150] [show Abstract] Cytarabine (ara-C), a major antileukemic agent, is phosphorylated in the cell to cytarabine triphosphate (ara-CTP), which is then partly incorporated into DNA. The drug incorporation into DNA poisons the extending primer against further incorporation of deoxyribonucleotides including dCTP, ultimately inhibiting DNA synthesis. While intracellular ara-CTP concentration has been found to predict clinical outcome, cytotoxicity in vitro is determined primarily by the extent of drug incorporation into DNA. However, clinically appropriate quantitation methods for ara-C at the DNA level have not been available. We developed a sensitive new method for monitoring ara-C incorporated into DNA in vivo. After DNA from leukemic cells was fractionated using the Schmidt-Thannhauser-Schneider method, it was degraded to constituent nucleosides to release ara-C, which was isolated from the nucleosides using HPLC and then measured by radioimmunoassay. Recovery for DNA fractionation, ara-C release by degradation, and ara-C isolation were 92.0+/-6.4%, 90.7+/-9.4%, and 98.5+/-1.4%, respectively. The method was found to determine ara-C incorporation into DNA of ara-C-treated HL 60 cells in vitro with minimal interassay variation. The values determined were compatible with those determined by scintillation counting in parallel experiments using tritiated ara-C. Our method could be used to monitor DNA-incorporated ara-C concentrations during ara-C therapy, together with plasma ara-C and intracellular ara-CTP concentrations. ara-C incorporation into DNA appeared to be associated with intracellular retention of ara-CTP or persistence of plasma ara-C. Thus, the present method is sensitive, accurate, precise, and may permit therapeutic drug monitoring at the DNA level for better individualization of antileukemic regimens. | Choi JY, Guengerich FP (2005) Adduct size limits efficient and error-free bypass across bulky N2-guanine DNA lesions by human DNA polymerase eta. Journal of molecular biology 352, 72-90 [PubMed:16061253] [show Abstract] The N2 position of guanine (G) is one of the major sites for DNA modification by various carcinogens. Eight oligonucleotides with varying adduct bulk at guanine N2 were analyzed for catalytic efficiency and fidelity with human DNA polymerase (pol) eta, which is involved in translesion synthesis (TLS). Pol eta effectively bypassed N2-methyl(Me)G, N2-ethyl(Et)G, N2-isobutyl(Ib)G, N2-benzyl(Bz)G, and N2-CH2(2-naphthyl)G but was severely blocked at N2-CH2(9-anthracenyl)G (N2-AnthG) and N2-CH2(6-benzo[a]pyrenyl)G (N2-BPG). Steady-state kinetic analysis showed proportional decreases of kcat/Km in dCTP insertion opposite N2-AnthG and N2-BPG (73 and 320-fold) and also kcat/Km in next-base extension from a C paired with each adduct (15 and 51-fold relative to G). Frequencies of dATP misinsertion and extension beyond mispairs were also proportionally increased (70 and 450-fold; 12 and 44-fold) with N2-AnthG and N2-BPG, indicating the effect of adduct bulk on blocking and misincorporation in TLS by pol eta. N2-AnthG and N2-BPG also greatly decreased the pre-steady-state kinetic burst rate (25 and 125-fold) compared to unmodified G. N2-AnthG decreased dCTP binding affinity (2.6-fold) and increased DNA substrate binding affinity. These results and the small kinetic thio effects (S(p)-dCTPalphaS) suggest that the early steps, possibly conformational change, are interfered with by the bulky adducts. In contrast, human pol delta bypassed adducts effectively up to N2-EtG but was strongly blocked by N2-IbG and larger adducts. We conclude that TLS DNA polymerases may be required for the efficient bypass of pol delta-blocking N2-G adducts bulkier than N2-EtG in human cells, and the bulk size can be a major factor for efficient and error-free bypass at these adducts by TLS DNA polymerases. | van 't Wout AB (2005) Gene expression profiling of HIV-1 infection using cDNA microarrays. Methods in molecular biology (Clifton, N.J.) 304, 455-459 [PubMed:16061997] [show Abstract] To illustrate the methods employed in gene expression profiling using cDNA microarrays, infection of CD4+ T cell lines with HIV-1LAI is used to identify expression changes relevant to in vitro HIV-1 infection. Cell lines are infected at a high multiplicity of infection to ensure a population of near-synchronously infected cells to be compared to uninfected cells. Infection status is verified using flow cytometry to determine the intracellular expression of the viral gag p24 protein before samples are harvested for total RNA extraction. Total RNA is extracted and amplified using commercially available kits, and RNA quality is verified using Bioanalyzer technology. To obtain fluorescently labeled cDNA probes, the amplified RNA is reverse-transcribed to yield cDNA, using random nonamers in the presence of dye-labeled dCTP. After first-strand cDNA synthesis, RNA is degraded and the probes are purified. For each infection condition (LAI and mock), two slides are hybridized with identical probes generated from the same RNAs, but with fluorescent labels reversed on one of the slides to control for dye-specific effects. Troubleshooting strategies and issues to consider prior to starting the experiment are discussed in detail in the notes section. | Moriarty TJ, Marie-Egyptienne DT, Autexier C (2005) Regulation of 5' template usage and incorporation of noncognate nucleotides by human telomerase. RNA (New York, N.Y.) 11, 1448-1460 [PubMed:16120835] [show Abstract] Telomerase accurately synthesizes telomeric DNA by reverse transcription of a tightly defined template region in the telomerase RNA (TR). Reverse transcription past the 5' boundary of the template can cause the incorporation of noncognate nucleotides into telomeric DNA, which can result in disruption of normal telomere function. The products synthesized by human telomerase do not contain the nucleotide cytosine, which is encoded by an hTR residue 2 nucleotides (nt) 5' of the template boundary. We examined dCTP incorporation by a series of telomerases reconstituted with N- and C-terminally mutated human telomerase reverse transcriptases (hTERTs). We found that altering sequences in the N-terminal RNA interaction domain 1 (RID1) and C terminus caused dCTP-dependent catalytic phenotypes suggestive of reverse transcription of sequences 5' of the template boundary. A RID1 mutant that exhibited a dCTP-dependent phenotype interacted less efficiently with a human telomerase RNA (hTR) variant in which the 5' template boundary-defining P1b element was disrupted, whereas C-terminal mutations did not alter hTR interactions in a P1b-dependent fashion. Disruption of P1b or template linker sequences between P1b and the 5' template boundary also impaired 5' template usage in RID1 and C-terminal hTERT mutants. These observations identify overlapping roles for hTR sequences and structures 5' of the template in regulating both 5' template boundary definition and 5' template usage, and implicate hTERT N- and C-terminal regions in 5' template usage and suppression of noncognate nucleotide incorporation. | Brzeziańska E, Zdzieszyńska M, Goś R, Lewiński A (2004) [Genetic analysis of rhodopsin and peripherin genes in patients with autosomal dominant retinitis pigmentosa (adRP) in Polish families]. Klinika oczna 106, 743-748 [PubMed:15787173] [show Abstract]
PurposeThe aim of that study was to identify the mutations in rhodopsin and peripherin genes in Polish families with autosomal dominant form of retinitis pigmentosa and determine the population polymorphism in both genes in adRP families.Material and methodsWe performed ERG, visual acuity, Goldman visual fields, intraocular pressure measurements and fundoscopy in all the patients included in the study. On the basis of disease history, the families pedigree was made and the mode of inheritance was analyzed. The molecular analysis of DNA for each family with adRP was conducted. Genomic DNA was obtained from leucocytes by phenol-chloroform procedure according to Maniatis protocol. DNA was amplified by the PCR reaction in a volume of 50 microl containing 100 ng/microl of genomic DNA, water, Cetus buffer pH 8.4 (1 n Tris, 1 n MgCl, 1 n KCl, 2% gelatin), 0.25 microM of each primer, 200 microM of each of dATP, dTTP, dCTP, and dGTP and 2.5 U Taq polymerase (Promega). For amplification of rhodopsin gene 30 cycles of PCR were carried out. Each cycle consisting of denaturation at 95 degrees C for 5 min, annealing: at 58 degrees C (exon 1), 63 degrees C (exon 2 and 3), 68 degrees C (exon 4) and 2 min extension at 72 degrees C min. For amplification of peripherin gene 30 cycles of PCR were carried out with annealing at 60 degrees C. The entire PCR product was in electrophoresis on 8% PAA. The PCR-RFLP PCR-HD PCR-SSCP and analysis of polymorphism (CA)n dinucleotide repetition was performed.ResultsMolecular study demonstrated, that mutations in rhodopsin gene were cause of retinitis pigmentosa in case of two families. In any study families mutations in peripherin gene were not identified. Two kinds of bases polymorphism were identified: restriction fragments length polymorphism (RFLP) in rhodopsin gene in exon 1 and 3 and single strand conformation polymorphism (SSCP) in exon 1 and 3 in rhodopsin gene and in exon 3 in peripherin gene. The confirmed mutations in rhodopsin gene, cosegregation with adRP, whereas two kinds of population polymorphism did not correlate with clinical symptoms. Natural polymorphism appeared to be a frequent feature in rhodopsin gene while a less frequent feature in peripherin gene.ConclusionsGenetic investigations in patients with adRP allow to confirm the diagnosis and evaluate the prognosis. The mutation in rhodopsin gene should be confirmed in directly sequencing reaction in next study. | Hinz M, Gottschling D, Eritja R, Seliger H (2000) Synthesis and properties of 2'-deoxycytidine triphosphate carrying c-myc tag sequence. Nucleosides, nucleotides & nucleic acids 19, 1543-1552 [PubMed:11200258] [show Abstract] The synthesis of 2'-deoxycytidine triphosphate carrying mercaptoethyl groups at position 4 of cytosine is described. This nucleoside triphosphate was reacted with a maleimido-peptide carrying the c-myc tag-sequence to yield a peptide-nucleoside triphosphate chimera. Primer extension studies showed that the nucleoside triphosphate modified with the peptide sequence is incorporated by DNA polymerases opposite guanine. | Traut TW (1994) Physiological concentrations of purines and pyrimidines. Molecular and cellular biochemistry 140, 1-22 [PubMed:7877593] [show Abstract] The concentrations of bases, nucleosides, and nucleosides mono-, di- and tri-phosphate are compared for about 600 published values. The data are predominantly from mammalian cells and fluids. For the most important ribonucleotides, average concentrations +/- SD (microM) are: ATP, 3,152 +/- 1,698; GTP, 468 +/- 224; UTP, 567 +/- 460 and CTP, 278 +/- 242. For deoxynucleosides-triphosphate (dNTP), the concentrations in dividing cells are: dATP, 24 +/- 22; dGTP, 5.2 +/- 4.5; dCTP, 29 +/- 19 and dTTP 37 +/- 30. By comparison, dUTP is usually about 0.2 microM. For the 4 dNTPs, tumor cells have concentrations of 6-11 fold over normal cells, and for the 4 NTPs, tumor cells also have concentrations 1.2-5 fold over the normal cells. By comparison, the concentrations of NTPs are significantly lower in various types of blood cells. The average concentration of bases and nucleosides in plasma and other extracellular fluids is generally in the range of 0.4-6 microM; these values are usually lower than corresponding intracellular concentrations. For phosphate compounds, average cellular concentrations are: Pi, 4400; ribose-1-P, 55; ribose-5-P, 70 and P-ribose-PP, 9.0. The metal ion magnesium, important for coordinating phosphates in nucleotides, has values (mM) of: free Mg2+, 1.1; complexed-Mg, 8.0. Consideration of experiments on the intracellular compartmentation of nucleotides shows support for this process between the cytoplasm and mitochondria, but not between the cytoplasm and the nucleus. | Flickinger RA, Richman R (1983) Incorporation of deoxycytidine triphosphate and thymidine triphosphate into nuclear DNA in vitro during early and late S phase in frog embryos. Cell differentiation 12, 233-237 [PubMed:6601514] | Harris AW, Reynolds EC, Finch LR (1979) Effect of thymidine on the sensitivity of cultured mouse tumor cells to 1-beta-D-arabinofuranosylcytosine. Cancer research 39, 538-541 [PubMed:761229] [show Abstract] Assays of thirteen cell lines, derived from mouse lymphomas, myelomas, myeloid tumors, and a mastocytoma, for sensitivity to growth inhibition by 1-beta-D-arabinofuranosylcytosine (ara-C) revealed a spectrum between the most and least sensitive which differed 100-fold from each other. An inverse correlation between sensitivity and cellular deoxycytidine 5'-triphosphate (dCTP) content was found, and this suggested that sensitivity of cells might be increased if the dCTP content was lowered during cell exposure to ara-C. Previous work has shown that thymidine treatment of cells lowers their dCTP content, and the effect of thymidine on the sensitivity of six of the cell lines to ara-C was therefore measured. Concentrations of thymidine below those inhibitory to cell growth by themselves caused an increase in ara-C sensitivity by up to 3-fold in 4 cell lines in which thymidine causes a depression in dCTP content but not in 2 myeloid lines in which the dCTP content was found not to be depressed by the same thymidine treatment. The results confirm an important role for dCTP in determining cellular sensitivity to ara-C. The finding that the sensitivity of 2 lymphoma cell lines to ara-C could be increased by concentrations of thymidine in the region of 10 micrometer, which are attainable clinically in humans, suggests that a combination of ara-C with thymidine might be useful in the treatment of some human tumors. |
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