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)

Experiments were carried out to test for the presence of "channeling" in L1210 cells. L1210 cells were incubated in culture in the presence of labeled cytidine and "cold" deoxycytidine and conversely, in the presence of labeled deoxycytidine and "cold" cytidine. Cytidine did not inhibit the incorporation of [14C]deoxycytidine into DNA while deoxycytidine decreased the incorporation of [14C]cytidine into DNA. Further, in L1210 cells there was not a coordinate inhibition of thymidylate synthetase when either DNA polymerase was inhibited (aphidicolin) or ribonucleotide reductase was inhibited (hydroxyurea). These data indicate that leukemia L1210 cells do not selectively channel ribonucleotides to DNA through a tightly coupled enzyme complex.
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PMID:Studies directed toward testing the "channeling" hypothesis--ribonucleotides----DNA in leukemia L1210 cells. 643 17

Freshly prepared spleen cells from concanavalin A stimulated mice incorporate [3H]thymidine into DNA which can be recovered in detergent-soluble (NP40) and detergent-insoluble forms. The presence of detergent-soluble forms occurs despite the fact that the cells are lysed at 4 degrees C in the presence or absence of 25 mM ethylenediaminetetraacetic acid. After a 2-h pulse with [3H]thymidine, the detergent-soluble fraction contains about 1-3% of the total cellular DNA but 25% of the total labeled high molecular weight material. Since the specific activity of the extensively purified DNA from the detergent-soluble fraction is considerably higher than that of chromosomal DNA, it meets the criteria for being metabolically active. We propose the name "MADS" DNA for metabolically active detergent-soluble DNA. MADS DNA has a density of 1.699 g/mL on cesium chloride gradients and a slightly higher G + C content than chromosomal DNA as determined by high-pressure liquid chromatography. Electrophoresis using native or denaturing agarose gels resolves MADS DNA into discreet sizes between 200 and 4500 base pairs. Nuclease S-1 treatment of native MADS DNA does not alter the size distribution as resolved by means of gel electrophoresis under denaturing conditions. Therefore, MADS DNA is not a collection of single-stranded Okazaki fragments. Southern blot analysis reveals that mitochondrial DNA is a minor component of higher molecular weights above the bulk of the DNA visualized either by staining with ethidium bromide or by incorporation of [3H]thymidine. Inhibitors of ribonucleotide reductase or DNA polymerase alpha inhibit incorporation of [3H]thymidine into MADS DNA, and hence chromosomal DNA synthesis is required for MADS DNA production. Since Southern blot analysis also reveals homology of larger fragments with the 32P-labeled 200 base pair fragment, the presence of repetitive sequences is suggested.
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PMID:Characterization of rapidly labeled detergent-soluble DNA in murine splenocytes. 671 31

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

We investigated the mechanism of cell growth inhibition caused by the deoxyribonucleosides thymidine (dThd), deoxyguanosine (dGuo), deoxyadenosine (dAdo), and deoxycytidine (dCyd). Growth of the cultured human leukemic cells HL-60 and K-562 was measured by cloning in soft agar. Of the deoxyribonucleosides, dGuo was the most potent cell growth inhibitor; however, the potency of added dAdo was probably attenuated by the presence of adenosine deaminase in the tissue culture growth medium. The concentrations of nucleoside causing 50% inhibition of HL-60 cloning were: dCyd, greater than 10,000 microM; dAdo, 500 microM; dThd, 5,000 microM; and dGuo, 80 microM. For K-562 cloning, the concentrations causing 50% inhibition of cloning were dCyd, greater 10,000 microM; dAdo, 1,600 microM; dThd, 880 microM;' and dGuo, 100 microM. Measurement of deoxycytidine 5'-triphosphate (dCTP) pool size in HL-60 cells following incubation with 750 microM deoxyribonucleosides revealed that dGuo caused the greatest reduction of dCTP pools, both in early (passage 10)- and late (passage 71)-passage-derived HL-60 cell cultures (35 and 19% of control, respectively), compared to dThd (61 and 26% of control, respectively) and dAdo (39% of control of HL-60 passage 10). In K-562 cells, reductions in dCTP pool size caused by dAdo, dThd, and dGuo were 68, 46, and 35% of control, respectively. Incorporation of [3H]dCyd into DNA of HL-60 and K-562 cells was enhanced by dThd and dGuo, but the degree of enhancement was greater for dThd than for dGuo. Despite its effect in reducing HL-60 dCTP pool size, dAdo failed to enhance [3H]dCyd incorporation in either HL-60 or K-562 cells. Addition of dCyd to the cultures could only partially rescue the inhibition of HL-60 cloning caused by dThd or dGuo, suggesting that inhibition of cytidine 5'-diphosphate reduction by ribonucleotide reductase is not the only mechanism whereby these nucleosides inhibit leukemic cell cloning. These data suggest that, in addition to inhibiting de novo dCTP production via ribonucleotide reductase, these nucleosides may affect other processes in the salvage pathway such as cellular uptake and phosphorylation or the DNA polymerase reaction itself.
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PMID:Effects of deoxynucleosides on cultured human leukemia cell growth and deoxynucleotide pools. 697 53

Thioredoxin was purified to homogeneity from the Escherichia coli mutant tsnC 7007 that is defective in phage T7 DNA replication and previously shown to contain a missense thioredoxin. Tryptic peptide maps of reduced and carboxymethylated 7007 thioredoxin combined with amino acid sequence analysis revealed one amino acid substitution; Gly-92 in thioredoxin is exchanged to an aspartic acid residue in the 7007 protein. The missense thioredoxin gave no activity with the gene 5 protein of phage T7 in the complementation to active T7 DNA polymerase. It competitively inhibited the complementation of wild type thioredoxin and gene 5 protein and formed a complex with the gene 5 protein that was retained by antithioredoxin Sepharose. The 7007 thioredoxin has reduced catalytic activity with thioredoxin reductase, ribonucleotide reductase, or as a protein disulfide reductase. The apparent Km value of 7007 thioredoxin as a substrate for thioredoxin reductase was increased 3-fold relative to normal thioredoxin, and the Vmax value was decreased 7-fold. The position of GLy-92 in the known three-dimensional structure of thioredoxin-S2 is correlated with the changed functional properties of the substituted mutant protein.
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PMID:A mutant thioredoxin from Escherichia coli tsnC 7007 that is nonfunctional as subunit of phage T7 DNA polymerase. 700 7

9-beta-D-Arabinofuranosyl-2-fluoroadenine (2-F-ara-A), a derivative of 9-beta-D-arabinofuranosyladenine (ara-A) that is resistant to deamination, selectively inhibits DNA synthesis and has activity against mouse leukemia L1210 comparable to that of ara-A plus the adenosine deaminase inhibitor, 2'-deoxycoformycin. To determine if these two nucleosides have similar modes of action, comparisons were made of their effects and those of their triphosphates on enzymes known to be inhibited by ara-A or 9-beta-D-arabinofuranosyladenine 5'-triphosphate. 9-beta-D-Arabinofuranosyl-2-fluoroadenine 5'-triphosphate was more effective than 9-beta-D-arabinofuranosyladenine 5'-triphosphate in inhibiting the reduction of adenosine 5'-diphosphate and cytidine 5'-diphosphate by ribonucleotide reductase from HEp-2 cells or L1210 cells. DNA polymerase alpha from L1210 cells was equally sensitive to 9-beta-D-arabinofuranosyl-2-fluoroadenine 5'-triphosphate and 9-beta-D-arabinofuranosyladenine 5'-triphosphate, and DNA polymerase beta from L1210 cells was much less sensitive to both triphosphates. S-Adenosylhomocysteine hydrolase from L1210 cells was inactivated by 2-F-ara-A and ara-A, but higher concentrations of the fluoro derivative were required. These results are consistent with 2-F-ara-A and ara-A inhibition of DNA synthesis by inhibition of ribonucleotide reductase and DNA polymerase alpha.
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PMID:Comparison of the actions of 9-beta-D-arabinofuranosyl-2-fluoroadenine and 9-beta-D-arabinofuranosyladenine on target enzymes from mouse tumor cells. 704 80

cis-Malonato-diammino platinum(II) significantly inhibited P-388 lymphocytic leukemia cell proliferation at 10 mg/kg/day. Incorporation studies showed that DNA synthesis was inhibited following in vivo drug therapy. The major inhibitory effects appeared to be on thymidine kinase and dihydrofolate reductase activities and on overall purine synthesis, with marginal effects on DNA polymerase and ribonucleotide reductase activities. In addition to the DNA inhibition, a marked increase in cyclic adenosine 3',5'-monophosphate levels was noted, which correlated with a rapid decrease in histone phosphorylation. Other minor effects of the drug included significant reduction of proteolytic activity, suppression of States 4 and 3 respiration, and an increase in adenosine triphosphatase and acid phosphatase activities of P-388 cells.
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PMID:Effects of cis-malonato-diammino platinum (II) on P-388 lymphocytic leukemia cell metabolism. 742 Feb 82

The success of gemcitabine (2',2'-difluorodeoxycytidine; dFdC) resulted in new interest in its purine congeners. Based on the structure-activity relationship studies of catabolism and anabolism, 2',2'-difluorodeoxyguanosine (dFdG) emerged as a lead candidate among the difluoropurine analogs. The cytotoxicity, metabolism, and actions of dFdG on DNA synthesis were studied in the human leukemia lymphoblastoid line CCRF-CEM. The IC50 values of dFdG after a 72-hour continuous incubation were 0.01, 0.03, and 0.28 mumol/L for CCRF-CEM, K562, and HL-60 cells, respectively. A cell line deficient in dCyd kinase was equally sensitive to dFdG, suggesting that, in contrast to dFdC, dFdG may be activated by other deoxynucleoside kinase(s). Consistent with these data, coincubation with dGuo spared the dFdG-mediated toxicity; however, up to 500 mumol/L dCyd failed to reverse the toxicity of dFdG. These observations indicated that dGuo kinase, which phosphorylates arabinosylguanine, also appears to play a major role in activating dFdG. CCRF-CEM cells incubated with varying concentrations of [3H]dFdG accumulated dFdGTP in a dose-dependent manner; a 3-hour incubation with 1 mmol/L dFdG resulted in more than 600 mumol/L intracellular dFdGTP. This is in contrast to the gemcitabine triphosphate accumulation, which is saturated at 10 to 20 mumol/L of exogenous dFdC. dFdG metabolites affected ribonucleotide reductase, resulting in a lowering of the dCTP pool; this is in agreement with the effect of dFdC on dNTP pools in leukemia cell lines. The major effect of dFdG on macromolecular synthesis was inhibition of DNA synthesis. DNA primer extension over a defined template revealed that dFdGTP was a good substrate for DNA polymerase alpha and incorporated opposite C sites of the template. Unlike arabinosyl analogs, but similar to gemcitabine triphosphate, dFdGTP incorporation caused DNA polymerase to pause after one normal deoxynucleotide was incorporated beyond the analog. The unique activation requirements of dFdG, its novel mode of inhibition of DNA synthesis, and its potent toxicity to human leukemia cells make it a promising new antimetabolite.
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PMID:Difluorodeoxyguanosine: cytotoxicity, metabolism, and actions on DNA synthesis in human leukemia cells. 748 47

The emerging clinical success of gemcitabine (2',2'-difluorodeoxycytidine) stimulated interest in the synthesis and evaluation of purine congeners. The cytotoxicity, metabolism, and mechanisms of action of the lead candidate, 2',2'-difluorodeoxyguanosine (dFdGuo), were studied in Chinese hamster ovary cells. Unlike the natural nucleoside deoxyguanosine (dGuo), dFdGuo was not a substrate for purine nucleoside phosphorylase. Wild-type Chinese hamster ovary cells and a mutant line deficient in deoxycytidine (dCyd) kinase were similarly affected by dFdGuo (50% inhibitory concentration, 7.5 and 6.5 microM, respectively), suggesting that unlike gemcitabine, dCyd kinase was not responsible for activation of dFdGuo. This was further confirmed by separation of nucleoside kinases (adenosine kinase, dGuo kinase, and dCyd kinase) of Chinese hamster ovary cells on DEAE-cellulose column chromatography. The kinase activity that phosphorylated dGuo also converted dFdGuo to its monophosphate, suggesting that dGuo kinase activated dFdGuo. Consistent with this result, coincubation with dGuo spared the dFdGuo-mediated toxicity; however, addition of up to 10 mM dCyd did not reverse the toxicity of dFdGuo. Intracellularly, dFdGuo was phosphorylated to its mono-, di-, and triphosphates; dFdGuo triphosphate (dFdGTP) was the major metabolite and accumulated to 45 microM after a 6-h incubation with 30 microM dFdGuo. The elimination of dFdGTP was monophasic with a t1/2 of about 6 h. Deoxynucleotides were decreased in cells incubated with dFdGuo, suggesting that ribonucleotide reductase was inhibited. dATP, which decreased 78% after a 4-h incubation with 30 microM dFdGuo, was most affected. dFdGuo was a potent inhibitor of DNA synthesis. Extension of a DNA primer over a defined template in the presence of dFdGTP revealed that dFdGTP was a good substrate for incorporation opposite C sites of the template by DNA polymerase alpha. dFdGTP incorporation caused DNA polymerase alpha to pause after the polymerization of one additional deoxynucleotide. This pattern of inhibition, which is shared by gemcitabine, distinguishes 2',2'-difluoronucleosides from arabinosylnucleosides which halt primer extension at the incorporation site. dGTP competed effectively with dFdGTP for incorporation by DNA polymerase alpha. The unique activation requirements and patterns of inhibition of DNA synthesis distinguish this promising new antimetabolite from other nucleoside analogues.
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PMID:Cytotoxicity, metabolism, and mechanisms of action of 2',2'-difluorodeoxyguanosine in Chinese hamster ovary cells. 753 64

2-Chloro-9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine (Cl-F-ara-A) is a new deoxyadenosine analogue that is resistant to phosphorolytic cleavage and deamination. Studies with a variety of cell lines demonstrated that Cl-F-ara-A is a potent cytotoxic agent; in cell-free systems, its triphosphate (Cl-F-ara-ATP) inhibited DNA polymerase alpha and ribonucleotide reductase. To further characterize its mechanism of cytotoxicity, the present study investigated the cellular metabolism of Cl-F-ara-A and the actions of its nucleotide metabolites in human T-lymphoblast leukemia CCRF-CEM cells. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, with the monophosphate as its major metabolite. After washing cells into drug-free medium, the elimination of each Cl-F-ara-A nucleotide was nonlinear with a prolonged terminal phase. Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA; a much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. At low Cl-F-ara-ATP:dATP values, incorporation was mainly in phosphodiester linkages at internal sites, whereas at higher Cl-F-ara-ATP:dATP values, Cl-F-ara-AMP was principally detected at terminal sites. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA. These results suggest that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.
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PMID:Metabolism and actions of 2-chloro-9-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)-adenine in human lymphoblastoid cells. 754 Sep 50


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