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At least 19 recordsLinked to original sources
PubMed1987

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides.

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.

Adenosine Kinase↗
PubMed1989

Reinvestigation of deoxyribonucleoside phosphorothioites: synthesis and properties of deoxyribonucleoside-3' dimethyl phosphites.

Further investigation of the synthesis of deoxyribonucleoside-3' (t-butyl) O-(2-cyanoethyl) phosphorothioites as monomer building units for the phosphorothioite approach has led us to conclude that internucleotidic bond formation proceeded via bis(deoxyribonucleoside-3') (2-cyanoethyl) phosphite intermediates, which proved to be activated by iodine, rather than the mechanism previously reported. In connection with this study, deoxyribonucleoside-3' dimethyl phosphites were synthesized and detailed properties of them are also described.

Chemical Phenomena↗
PubMed1982

Incorporation of deoxyribonucleosides into DNA of coryneform bacteria and the relevance of deoxyribonucleoside kinases.

In order to obtain basic knowledge of the salvage pathways for DNA synthesis, the ability of Brevibacterium ammoniagenes ATCC 6872 and Micrococcus luteus ATCC 15932 for incorporation of nucleobases and nucleosides was investigated. Only adenine and uracil are incorporated by B. ammoniagenes, whereas M. luteus additionally can utilize deoxyadenosine and, less efficiently, thymidine. In M. luteus, the demonstration of deoxyadenosine kinase and thymidine kinase explains the incorporation data. Uptake of thymidine is of short duration because of rapid breakdown of exogenously supplied thymidine to thymine. At a 540-fold excess pyrimidine deoxyribonucleosides inhibit 14C incorporation from thymidine nearly totally and purine deoxyribonucleosides cut by half the uptake rate, probably by interfering with transport of thymidine. However, as no cessation of thymidine incorporation occurs at these concentrations of purine deoxyribonucleosides, incorporation is finally enhanced. During the initial period of this reduced uptake considerable protection of thymidine from breakdown to thymine is provided by deoxyguanosine, but not by deoxyadenosine. At a 108-fold excess there is actually no inhibition of thymidine uptake by deoxyguanosine and only an insignificant impairment by deoxyadenosine resulting in an ultimate enhancement of 14C incorporation up to 20% of the exogenously supplied thymidine. As there is no salvage pathway for thymidine in B. ammoniagenes due to the absence of thymidine kinase, labelling with adenine and hydrolyzing of the 'contaminated' RNA fraction with 1 M KOH is recommended for measurements of overall DNA synthesis in this strain.

Brevibacterium↗
PubMed1987

Purine deoxyribonucleosides counteract effects of hydroxyurea on deoxyribonucleoside triphosphate pools and DNA synthesis.

Inhibition of cell growth and DNA synthesis by hydroxyurea is thought to occur via an effect on the enzyme ribonucleotide reductase leading to a block of deoxyribonucleotide synthesis. Earlier attempts to bypass such a block by delivering deoxyribonucleosides to the medium of cultured cells have given equivocal results. Complications arise in such experiments from the specificity of the phosphorylating enzymes since 3 of the 4 deoxyribonucleosides are substrates for the same enzyme, with widely differing Km values, and from allosteric effects exerted by deoxyribonucleotides. We simplify this situation by using a mutant hamster V79 line that lacks the enzyme dCMP deaminase. The cells contain a 20-fold enlarged dCTP pool and require thymidine for optimal growth. Concentrations of hydroxyurea (50 or 100 microM) that in short-term experiments inhibited DNA synthesis depleted the dATP pool without seriously affecting pyrimidine deoxyribonucleotide pools. The dATP pool could be restored by addition of deoxyadenosine but this depleted the dGTP pool. This depletion could be counteracted by the simultaneous addition of deoxyguanosine but then critically depended on the relative concentrations of the two purine deoxyribonucleosides, with optimal results at 1 microM deoxyadenosine + 100 microM deoxyguanosine. Under those conditions the inhibition of DNA synthesis by hydroxyurea was partially reversed.

Animals↗
PubMed1991

Novel route to oligo(deoxyribonucleoside phosphorothioates). Stereocontrolled synthesis of P-chiral oligo(deoxyribonucleoside phosphorothioates).

The synthesis and separation of diastereoisomerically pure 5'-O-DMT-nucleoside 3'-O-(2-thio-1,3,2-oxathiaphospholane) allows their use as synthons in DBU-catalyzed reaction with the 5'-hydroxyl function of solid-support-bound nucleoside moiety. Since this reaction is stereospecific (greater than 99%), this novel method allows preparation of oligo(nucleoside phosphorothioates) with predetermined chirality at each P-chiral internucleotide phosphorothioate centre.

Base Sequence↗
PubMed1986

Interrelations between substrate cycles and de novo synthesis of pyrimidine deoxyribonucleoside triphosphates in 3T6 cells.

Degradation of pyrimidine deoxyribonucleoside triphosphates plays a major role in the regulation of their pool sizes in 3T6 cells. During normal growth, these cells excrete deoxyribonucleosides (mostly deoxyuridine) into the medium. When DNA strand elongation is inhibited, de novo synthesis of dCTP and dTTP continues, followed by degradation of the deoxyribonucleotides. We now demonstrate that inhibition of de novo synthesis with hydroxyurea stops degradation of deoxyribonucleotides. We now demonstrate that inhibition of de novo synthesis with hydroxyurea stops degradation of deoxyribonucleotides and leads to an influx of deoxyuridine from the medium. This effect appears to be caused by a large drop in the size of the intracellular dUMP pool. We propose that substrate cycles, involving phosphorylation of deoxyribonucleosides by kinases and dephosphorylation of deoxyribonucleoside 5'-phosphates by a nucleotidase, participate in the regulation of the size of pyrimidine deoxyribonucleoside triphosphate pools by directing the flow of deoxyribonucleosides across the cell membrane. While kinases are regulated mainly by allosteric effects, the activity of the nucleotidase appears to be regulated by substrate concentration.

Animals↗
PubMed1985

Isolation and purification of deoxyribonucleosides from 90% 13C-enriched DNA of algal cells and their characterization by 1H and 13C NMR.

13C-enriched deoxyribonucleosides have been isolated from the DNA of Algal cells grown in an atmosphere of 90% 13C-labelled carbon dioxide. The 13C enriched DNA was quantitatively hydrolysed with DNase I, snake venom phosphodiesterase I and alkaline phosphatase of intestinal mucosa. The resulting deoxyribonucleosides were separated by preparative reversed-phase high pressure liquid chromatography in 60 minutes with detection by ultraviolet absorption at 254 nm. The final products were obtained in milligram quantities in high purity and in high yield. The 1H resonances of the base and sugar protons of these deoxyribonucleosides appear as well resolved multiplets in the 600 MHz NMR spectrum, due to the extensive 1H-13C couplings. Similarly, the 13C resonances of these deoxyribonucleosides appear as multiplets in the 75.5 MHz 13C NMR spectrum, due to 13C-13C couplings. The 1H-13C and 13C-13C coupling constants were also measured and tabulated. The isotopic enrichment of 13C these deoxyribonucleosides was obtained by integration of the 1H and/or 13C NMR spectra. It was found that the enrichment varied from carbon to carbon and species to species in the range of 70-89%, suggesting differential uptake and assimilation of 90% 13CO2 during metabolism pathways. This protocol provides experimentally useful quantities of 13C-enriched deoxyribonucleosides, which may be incorporated into site-specifically labeled oligonucleotides by chemical synthesis.

Carbon Isotopes↗
PubMed1983

Changes in ribo- and deoxyribonucleoside triphosphate pools within the cell cycle of a synchronized mouse fibroblast cell line.

Intracellular pool levels of ribo- and deoxyribonucleoside triphosphates were monitored throughout the cell cycle of C3H10T1/2 mouse embryo fibroblast cells synchronized by isoleucine deprivation. Absolute pool sizes of ribonucleoside triphosphates were approximately 30 fold greater than those of the corresponding deoxyribonucleoside triphosphates. Of the ribonucleoside triphosphates, pool sizes of ATP exhibited the greatest change, increasing from a low of 32.7 nmol/10(7) cells during G1 to a high of 81.6 nmol/10(7) cells 2 h prior to mid S-phase. Levels of ATP subsequently declined to 40.2 nmol/10(7) cells during late S-phase, followed by a second peak of 65.8 nmol/10(7) with the onset of cell division. No significant changes in the pool sizes of UTP and GTP were found throughout the cell cycle. Of the deoxyribonucleoside triphosphates, pool sizes of pyrimidine deoxyribonucleoside triphosphates were approx. 5-10 fold greater than those of purine deoxyribonucleoside triphosphates. Low levels of deoxyribonucldoside triphosphates during G1 (0.3-1.3 pmol/10(7) cells) increased coordinately with the initiation of DNA synthesis to an initial peak during mid S-phase (0.5-6.4 pmol/10(7) cells). Declining levels of deoxyribonucleoside triphosphates during late S-phase were followed by a subsequent larger second peak (1.7-10.7 pmol/10(7) cells) during G2-M.

Animals↗
PubMed1975

Deoxyribonucleoside triphosphates in human cells: changes in disease and following exposure to drugs.

Deoxyribonucleic acid synthesis requires adequate cellular concentrations of the four deoxyribonucleoside triphosphates. Using a sensitive enzymic assay, we have measured the concentrations (pools) of these compounds in human bone marrow cells and in lymphocytes. The mean concentrations (pmol/10(6) cells) in normal human bone marrow cells were: deoxyadenosine triphosphate (dATP) 1.5; deoxyguanosine triphosphate (dGTP) 0.4; thymidine triphosphate (dTTP) 1.4 and deoxycytidine triphosphate (dCTP) 0.6; and in normal phytohaemagglutinin (PHA)-stimulated lymphocytes (72 h cultures); dATP 3.7; dGTP 1.9; dTTP 9.4 and dCTP 2.9. The deoxyribonucleoside triphosphate concentrations were increased approximately threefold in the nucleated marrow cells from patients with leukaemia and myeloproliferative diseases. PHA-stimulation of lymphocytes caused a marked increase of the deoxyribonucleoside triphosphate concentrations, particularly of dTTP, between 24 and 48 h of culture. In PHA-stimulated lymphocytes, the antifolate drugs methotrexate, pyrimethamine and trimethoprim, all produced a fall in dTTP and a rise in dATP concentrations within 1 h. These effects could be reversed by folinic acid. 5-Fluorouracil caused a fall in dTTP and in dCTP but no consistent changes in dATP; hydroxyurea caused a fall in dATP with a rise in dTTP. BCNU caused a significant fall in dATP and dCTP. Dibutyryl cyclic 3', 5' adenosine monophosphate and theophylline had no consistent effect on the deoxyribonucleoside triphosphate concentrations. 6-Mercaptopurine caused a fall in dATP and dGTP, the fall in dATP being marked after 4 h incubation. It is concluded that measurement of the deoxyribonucleoside triphosphates in human cells provides a new method of studying DNA synthesis in human disease states and of analysing the action of antimetabolite drugs on normal and diseased cells.

Adenosine Triphosphate↗
PubMed1980

Metabolic differences in deoxyribonucleoside triphosphate hydrolysis between rat liver and Yoshida sarcoma cell chromatins.

Enzymatic activities for deoxyribonucleoside triphosphate hydrolysis associated with Yoshida sarcoma and normal rat liver chromatins were comparatively studied. Deoxyribonucleoside triphosphate-hydrolyzing activities were isolated from purified chromatins of Yoshida sarcoma cells and normal rat liver, and gel-filtered on Sephadex G-200 columns. A single peak of dUTP-hydrolyzing activity from the Yoshida sarcoma chromatin and three peaks from the normal liver chromatin were observed. The first peak from the normal chromatin hydrolyzed dATP, dCTP and TTP mainly to the corresponding deoxyribonucleoside diphosphates, and dUTP and dGTP to the corresponding di- and mononucleotide forms. The activity in the first peak from the normal liver chromatin had an apparent Km of 2.5 X 10(-4) M for dUTP, required divalent cations and was inhibited by NaF. The peak from the Yoshida sarcoma chromatin corresponding to the first peak from the normal liver chromatin hydrolyzed all five deoxyribonucleoside triphosphates to deoxyribonucleoside diphosphates with minor co-production of mononucleotides. The enzyme activity from the malignant cells had an apparent Km of 2.5 X 10(-5) M for dUTP, required Mg2+, but was not inhibited by NaF.

Animals↗
PubMed1996

Protection and repair of gamma-radiation-induced lesions in mice with DNA or deoxyribonucleoside treatments.

Mice can survive lethal doses of ionizing radiation if deoxyribonucleosides or 'highly polymerized' salmon sperm DNA (Sigma) are administered 30 min to 24 h post-irradiation. DNA is more effective than deoxyribonucleosides in increasing the survival frequency. At supralethal exposures of gamma-irradiation, Deoxyribonucleosides and DNA are equally effective in reversing radiation damage which otherwise leads to chromosome breakage. The micronucleus frequencies in the polychromatic erythrocytes of bone marrow cells from DNA- or deoxyribonucleoside-treated mice were near the unirradiated control values. This reduction in chromosome breakage was approximately 4-fold when compared with the irradiated, saline-treated control. 'Highly polymerized' DNA protects against mortality if administered 48 and 24 h prior to irradiation. This is somewhat comparable to the effectiveness of the growth factors Interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor-alpha (TNFalpha) administered prior to irradiation. With survival as criterion, the sensitivity of 4 lines of mice to gamma-irradiation is BALB/c > C3H/OuJ > or = C3H/HeJ > C57B1/6.

Animals↗
PubMed1988

Effects of deoxycytidine and thymidine kinase deficiency on substrate cycles between deoxyribonucleosides and their 5'-phosphates.

Substrate cycles constructed from a deoxyribonucleoside kinase and a deoxyribonucleotidase contribute to the metabolism of deoxyribonucleotides in cultured cells. The two enzymes catalyze in opposite directions the irreversible interconversion between a deoxyribonucleoside and its 5'-phosphate. Depending on the balance between the two reactions the net result of the cycle's activity will be synthesis or degradation of the deoxyribonucleotide, and favor import or export of the deoxyribonucleoside. With genetically changed hamster cells (V79 and CHO) deficient in either deoxycytidine or thymidine kinase we now quantify by kinetic isotope flow experiments the contributions of the two kinases to the function of the respective cycles. For each, loss of the relevant kinase was accompanied by an increased degradation of the deoxynucleotide, a slower rate of DNA synthesis, and a longer generation time for the mutant cells. The size of the corresponding deoxyribonucleoside triphosphate pool was apparently not decreased.

Animals↗
PubMed1984

Induction of deoxyribose-5-phosphate aldolase of Bacillus cereus by deoxyribonucleosides.

In Bacillus cereus purine ribonucleosides and deoxyribonucleosides share a common inducible catabolic pathway, leading to the formation of ribose-5-P or deoxyribose-5-P respectively inside the cell, while the purine ring remains in the external medium. Both ribo- and deoxyribonucleosides are inducers of adenosine deaminase, inosine-guanosine phosphorylase and phosphopentomutase, the enzymes of the catabolic pathway. We now show that deoxyribonucleosides, but not ribonucleosides, induce the aldolase specific for deoxyribose-5-P (2-deoxy-D-ribose-5-phosphate acetaldehyde lyase, EC 4.1.2.4), thus allowing the sugar moiety of exogenous deoxyribonucleosides to be utilized as an energy source.

Aldehyde-Lyases↗
PubMed1968

Characteristics of the deo operon: role in thymine utilization and sensitivity to deoxyribonucleosides.

Inability to grow on deoxyribonucleosides as the sole carbon source is characteristic of deo mutants of Escherichia coli. Growth of deoC mutants, which lack deoxyribose 5-phosphate aldolase, is reversibly inhibited by deoxyribonucleosides through inhibition of respiration. By contrast, deoB mutants are not sensitive to deoxyribonucleosides, and deoxyribose 5-phosphate aldolase and thymidine phosphorylase are present at normal levels but are not inducible by thymidine. Organisms with the genotype deoB(-)thy(-) or deoC(-)thy(-) are able to grow on low levels of thymine, whereas deoB(+)thy(-) or deoC(+)thy(-) strains require high levels of thymine for growth. The deoB and deoC mutations are transducible with and map on the counterclockwise side of the threonine marker. They are closely linked to deoA, a gene determining thymidine phosphorylase. Merodiploids heterozygous for either the deoB or deoC genes are resistant to deoxyribonucleosides and, in combination with the thy mutation, require high levels of thymine for growth. Cultures of thy(+)deoC(-) mutants are inhibited by thymidine until this compound has been completely degraded and excreted as deoxyribose and thymine, whereupon growth promptly resumes at a normal rate. The inhibition of respiration in deoC strains and the induction of thymidine phosphorylase and deoxyribose 5-phosphate aldolase in the wild-type organism are considered to result from the accumulation of deoxyribose 5-phosphate.

Bacterial Proteins↗
PubMedDate not supplied

An isocratic high-pressure liquid chromatographic purification method for radioactively labeled deoxyribonucleoside triphosphates.

A method is described for the rapid purification of radioactively labeled deoxyribonucleoside triphosphates from their spontaneously emerging hydrolysis products deoxyribonucleoside diphosphate, deoxyribonucleoside monophosphate, and deoxyribonucleoside. The separations which are finished within 3 min or less are carried out on a 0.1 X 5 cm column filled with LiChrosorb-NH2, using isocratic elution with 0.025 M potassium phosphate, pH 6.8, in a high-pressure liquid chromatograph at room temperature and a flow rate of 30 ml-h-1 (flow velocity 63.7 cm-min-1).

Chromatography, High Pressure Liquid↗
PubMed1979

Evaluation of ribonucleoside and deoxyribonucleoside triphosphate pools in cultured leukemia cells during exposure to methotrexate or methotrexate plus thymidine.

Continuous exposure to inhibitory concentrations of methotrexate produces distinct rates of steady-state growth of murine leukemia L1210 and human leukemia CCRF-CEM cells in culture. Addition of thymidine to the medium produces reversal (6 to 40%) of this steady-state growth rate inhibition. This study utilized combinations of methotrexate and thymidine for an evaluation of the accompanying relationship between steady-state growth rate and changes in the ribo- and deoxyribonucleoside triphosphate pools. In L1210 cells exposed to methotrexate alone, the deoxythymidine 5'-phosphate (dTTP) pools decreased, whereas deoxyadenosine 5'-triphosphate, deoxyguanosine 5'-triphosphate, and deoxycytidine 5'-triphosphate (dCTP) remained relatively constant up to 70% inhibition of growth rate, with dCTP at a constant 112% of controls. The corresponding ribonucleoside triphosphates decreased only slightly. With the combination of methotrexate and thymidine resulting in up to 40% inhibition of growth rate, there was also a decrease in the dTTP pool while the other deoxyribonucleoside triphosphates remained relatively constant, and the corresponding ribonucleoside triphosphates again decreased only slightly. The dCTP pool was reduced to a constant 42% of control comparable to that produced by thymidine alone. With greater than 40% (with thymidine) or 70% (without thymidine) inhibition of growth rate, all pools decreased, but only dTTP was substantially reduced in proportion to the growth rate inhibition caused by methotrexate. The dTTP pool became depleted in spite of the presence of exogenous thymidine. Evaluation of CCRF-CEM cells indicated that inhibition of growth rate and nucleotide pool perturbations by methotrexate were similar to those observed in L1210 cells. However, in the presence of thymidine, inhibition of growth rate appeared related to decreased pools of dCTP, deoxyadenosine 5'-triphosphate, and deoxyguanosine 5'-triphosphate, rather than dTTP as was observed for L1210 cells. Hence, mammalian cells were capable of responding in a differential fashion to pharmacological perturbations, and this capacity may play a role in determining therapeutic selectivity. Since the ribonucleoside triphosphate decreases were slight and relatively uniform during methotrexate-induced perturbations, the deoxyribonucleoside triphosphate pools appear to be more directly related to inhibition of growth rate. The results are consistent with the concept that slight imbalances in the deoxyribonucleoside triphosphate pools dramatically inhibit DNA synthesis, as mediated through their interaction with DNA polymerase.

Animals↗
PubMed1984

Efficient misincorporation by avian myeloblastosis virus reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate.

Misincorporation by avian myeloblastosis virus reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate was studied using 32P-labeled DNA primers annealed to the appropriate template DNA, and polyacrylamide-urea gel electrophoresis to measure the extension of the primer chains. With most primer-template combinations, greater than 50% of the primers were extended by the addition of a single incorrect nucleotide onto the end of the primer chain. Unexpectedly, one primer-template combination was not extended in the presence of dCTP, although misincorporation occurred with the other deoxyribonucleoside triphosphates. In another case, terminal misincorporation of two rather than one dT residue was observed. The primer termini containing unpaired nucleotides were efficiently extended upon addition of the other three deoxyribonucleoside triphosphates, even in the case where the primer terminus contained two unpaired nucleotides. Misincorporation was confirmed by direct sequence analysis. These results indicate that the frequency of mutations following misincorporation by reverse transcriptase in the presence of a single deoxyribonucleoside triphosphate should be sufficiently high to allow detection of mutants by simple screening procedures. An analysis of the sequence of a mutant resulting from misincorporation at the M13mp2 AvaII site revealed that following introduction of the DNA into Escherichia coli cells, mismatch repair preceded replication.

Avian Leukosis Virus↗