Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Analysis of ribonucleotide reductase and DNA polymerase activities in extracts of Pseudomonas stutzeri by centrifugation in discontinuous sucrose gradients indicated that these two enzymes are associated with two different high molecular weight cellular components. In addition, 95% of the ribonucleotide reductase activity was pelleted by centrifugation of extracts for 4 hr at 200,000 X G. The reductase activity remained particulate (sedimentable) following sonication whereas some 90% of the DNA polymerase activity was rendered soluble (non-sedimentable) by this technique. This data indicate that the P. stutzeri ribonucleotide reductase is not a cytosolic enzyme, but is associated with a macromolecular component in the cell.
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PMID:Sedimentation analysis of ribonucleotide reductase activity in extracts of Pseudomonas stutzeri. 388 16

Pool sizes of deoxyribonucleoside triphosphates (dNTPs) in cultured cells are tightly regulated by i.al., the allosteric control of ribonucleotide reductase. We now determine the in situ activity of this enzyme from the turnover of the deoxycytidine triphosphate (dCTP) pool in rapidly growing 3T6 mouse fibroblasts, as well as in cells whose DNA replication was inhibited by aphidicolin or amethopterin, by following under steady state conditions the path of isotope from [5-3H]cytidine into nucleotides, DNA, and deoxynucleosides excreted into the medium. In normal cells as much as 28% of the dCDP synthesized was excreted as deoxynucleoside (mostly deoxyuridine), leading to an accumulation of deoxyuridine in the medium. Inhibition with amethopterin slightly increased ribonucleotide reductase activity, while aphidicolin halved the activity of this enzyme (and thymidylate synthase). In both instances all dCDP synthesized was degraded and excreted as nucleosides. This continued synthesis and turnover in the absence of DNA synthesis is in contrast to the earlier found inhibition of dCTP (and dTTP) turnover when hydroxyurea, an inhibitor of ribonucleotide reductase, was used to block DNA synthesis. To explain our results, we propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, participate in the regulation of pool sizes. Within the cycles, a block of the reductase activates net phosphorylation, while inhibition of DNA polymerase stimulates degradation.
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PMID:Evidence for the involvement of substrate cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 cells. 392 65

Microbial siderophores represent a class of iron chelators characterized by their high affinity (i.e., formation constants, greater than 10(40) M) for ferric iron. Previously, we demonstrated that the bacterial siderophores, N-[3-(2,3-dihydroxybenzamido)propyl]-N-[4-(2, 3-dihydroxybenzamino)butryl]-2-(2-hydroxyphenyl) trans-5-methyloxazoline-4-carboxamide (Parabactin) and N1,N8-bis(2,3-dihydroxybenzoyl)spermidine (Compound II), inhibit the growth of L1210 cells and the replication of DNA (but not RNA) viruses at low micromolar concentrations (Biochem. Biophys. Res. Commun., 121: 848-854, 1984). The basis for this antiproliferative effect on L1210 cells has now been investigated further. Onset of growth inhibition induced by 5 microM Parabactin occurs much earlier than with an equimolar concentration of Compound II but, once established by either chelator, inhibition appears to be irreversible. Growth inhibition was fully preventable with exogenous FeCl3 when given at the same time as the chelators. Flow cytometric analysis revealed a G1-S cycle block following treatment for 4 h with either 5 microM Parabactin or 30 microM Compound II. The block was readily reversed with exogenous FeCl3, allowing cells to progress to mid-S phase by 3 h and to G1 again by 9 h. Thereafter, cells accumulated at a second block located at S phase. The treatment conditions required for the initial cell cycle block (at 4 h) were adapted for subsequent studies. Clonogenicity of L1210 cells in soft agar following a 4-h exposure was reduced to 22% of control by 5 microM Parabactin and to 16% by 30 microM Compound II. Neither growth inhibition in suspension culture nor decreased clonogenicity in soft agar could be reversed with exogenous iron, following treatment with the chelators. Both chelators caused an early and significant decrease in [14C]thymidine incorporation over the 4-h period (50% inhibitory concentration at 4 h, 0.4 microM for Parabactin and 6.0 microM for Compound II). [3H]Uridine incorporation was inhibited later than [14C]thymidine and to a much lesser extent, while [3H]leucine incorporation was not significantly affected. Treatment of cells with 5 microM Parabactin or Compound II for 4 h decreased deoxy-adenosine triphosphate pools by 38 and 70%, respectively, and increased deoxythymidine triphosphate pools by 67 and 36%, respectively, suggesting interference with ribonucleotide reductase. Indeed, extracts of cells treated for 4 h with either 5 microM Parabactin or 30 microM Compound II exhibit a 97 to 98% decrease in cytidine-5'-diphosphate reductase activity compared to control, whereas DNA polymerase was elevated slightly.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Characterization of L1210 cell growth inhibition by the bacterial iron chelators parabactin and compound II. 402 62

Ribonucleotide reductase is an essential enzyme in mammalian DNA replication. In quiescent BHK-21/C13 cells exhibiting a low level of ribonucleotide reductase activity, infection with herpes simplex virus (HSV) resulted in the early induction of an altered ribonucleotide reductase. The extent of the induction was dependent upon the m.o.i. and could be diminished or prevented by u.v. treatment of the viral stock, or by inhibitors of mRNA synthesis or protein synthesis. The induction followed the same course of synthesis as viral thymidine kinase and DNA polymerase, and could thus be classified with them as a beta polypeptide. These results suggested that the new activity was produced as a consequence of the virus genome expression. Comparisons of the properties of ribonucleotide reductase extracted from exponentially growing BHK-21/C13 cells showed that the HSV-induced enzyme differed from the cellular isozyme by its insensitivity to inhibition by dTTP, dATP or araATP and its resistance to high salt concentrations. On the other hand, the virus-induced enzyme and the cellular isozyme exhibited a similar sensitivity to hydroxyurea. Therefore, the reported inhibition of HSV DNA replication by hydroxyurea could be the result of inhibition of both HSV-induced and cellular reductase activities.
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PMID:Characterization of ribonucleotide reductase induction in BHK-21/C13 Syrian hamster cell line upon infection by herpes simplex virus (HSV). 617 49

The metabolism of pyrimidine nucleotides in various tissues and tumor cells of rodents was investigated. Ribonucleoside diphosphate reductase, thymidine monophosphate kinase and DNA polymerase (alpha, beta) were specifically localized in tumor cells, i.e., the activities of these enzymes in tumor cells were at least three times higher than those in normal tissues, including rapidly growing tissues, such as bone marrow, thymus, and spleen. The activities of deoxycytidine monophosphate deaminase and all the nucleoside kinase were high not only in tumor cells, but also in rapidly growing normal tissues, so that these enzymes are unsuitable as targets for cancer chemotherapy. The tissue distribution of other enzymes, including orotate phosphoribosyltransferases, cytidine triphosphate synthetase, thymidine monophosphate synthase, nucleoside phosphorylases and cytidine deaminase had no relation with the cell growth rate. AH130 tumor cells and the thymus showed specific increases in the activities of enzymes involved in de novo DNA synthesis. In contrast, Yoshida sarcoma and bone marrow showed high activities of enzymes in the salvage pathway of DNA synthesis, which suggested that the two tumors have different patterns of nucleotide metabolism.
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PMID:Metabolism of pyrimidine nucleotides in various tissues and tumor cells from rodents. 627 49

The activities of 5-methyltetrahydrofolate (5-CH3-THF)-related enzymes [5-CH3-THF homocysteine methyltransferase and 5,10-methylenetetrahydrofolate (5,10-CH2-THF) reductase] and DNA polymerase alpha were measured in normal and malignant hematopoietic cells. The 5-CH3-THF homocysteine methyltransferase activity was significantly correlated with 5,10-CH2-THF reductase activity, indicating that the hematopoietic cells with active biosynthesis of tetrahydrofolate from 5-CH3-THF also actively synthesize 5-CH3-THF from 5,10-CH2-THF. The activities of 5-CH3-THF-related enzymes had a tendency to be high in lymphoid cells and low in myeloid cells, and were not correlated with the percentage of blasts and immature cells in the samples examined. Fairly good correlations were observed among these three enzymes in non-malignant bone marrow cells. However, the activities of two of the enzymes correlated only weakly overall with DNA polymerase alpha activity in normal and malignant hematopoietic cells. Generally speaking, DNA polymerase alpha activity correlated well with the percentage of blasts and immature cells in the samples examined.
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PMID:5-methyltetrahydrofolate-related enzymes and DNA polymerase alpha in normal and malignant hematopoietic cells. 635 15

Infection of monkey kidney (BSC-40) cells with vaccinia virus strain WR resulted in a marked increase in ribonucleoside diphosphate reductase (EC 1.17.4.1) activity as measured by CDP reduction in cell-free extracts. After a synchronous infection, increased activity was detected at 2 h, peaked at 4 to 5 h, and then declined between 6 and 8 h to the endogenous cellular level. The induction, detectable at 0.5 PFU/cell, correlated strongly with multiplicity of infection to 10 PFU/cell and continued to increase to 50 PFU/cell. It paralleled the previously described induction of viral DNA polymerase and thymidine kinase, suggesting that the reductase may also be a product of early transcription of the viral genome. The inhibition of DNA synthesis throughout infection resulted in prolonged accumulation of reductase activity and delayed and incomplete down-regulation at 8 h, suggesting that repression involves late functions. Rescue of fluorodeoxyuridine-inhibited DNA synthesis with exogenous thymidine restored the normal pattern. Preferential association of the induced reductase with the cytoplasmic sites of vaccinia virus DNA replication (virosomes) was not detected. The induced enzyme is similar in several respects to other eucaryotic ribonucleotide reductases, but is distinct from host cell reductase in response to certain modulators of reductase activity (M. B. Slabaugh and Christopher K. Mathews, J. Virol. 52:501-506, 1984). Full activity required an activator, exogenous reducing equivalents, and iron. Hydroxyurea, EDTA, dATP, and dTTP inhibited CDP reduction, setting this reductase apart from T4 reductase, which is not inhibited by dATP, and from herpesvirus reductase, which requires no activation and is insensitive to deoxyribonucleoside triphosphate inhibition.
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PMID:Vaccinia virus induces ribonucleotide reductase in primate cells. 638 75

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.
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PMID:Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells. 675 37

N-Substituted indan-1.3-diones have proven to be potent cytotoxic agents effective against the growth of single cell leukemia tumors and cell lines derived from solid tumors. A number of the derivatives were active against growth of solid tumors e.g. colon, lung bronchogenic and osteosarcoma for which few effective agents are available to inhibit their growth. These agents inhibited DNA and RNA synthesis of L1210 cells. The de novo purine synthetic pathway was inhibited at PRPP amido transferase and IMP dehydrogenase. The pyrimidine synthetic pathway was inhibited at aspartate transcarbamylase. Other sites which demonstrate minor inhibition were DNA polymerase alpha, r- and t-RNA polymerase, ribonucleoside reductase, dihydrofolate reductase, nucleoside kinases and thymidylate synthetase. In addition d(NTP) pool levels were reduced by the drugs. L1210 DNA strand scission was evident after exposure to drugs for 24 hr. at 100 microM.
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PMID:Cytotoxicity and mode of action of substituted indan-1, 3-diones in murine and human tissue cultured cells. 784 49

N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c,e]azepines demonstrated significant cytotoxic activity against the growth of murine and human cells. These derivatives were active against leukemias, carcinomas and sarcomas. Different derivatives with N-substitutions showed specific activity against the growth of several tumor types. These agents inhibited L1210 leukemia IMP dehydrogenase and PRPP amido transferase activities; this was reflected in the inhibition of purine and DNA synthesis. Other sites inhibited to a minor degree by these agents included DNA polymerase alpha, r- and tRNA polymerases, ribonucleoside reductase, dihydrofolate reductase, pyrimidine synthesis, and nucleoside kinase. d(NTP) pool levels were reduced after 24 h incubation with these derivatives. L1210 DNA strand scission was evident after drug treatment.
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PMID:The cytotoxicity of N-substituted diphenimides and 6,7-dihydro-5H-dibenz[c,e]azepines. 829 66


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