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)

Megaloblastic anaemia is due to a derangement of DNA synthesis caused by insufficient supply of one or other of the four deoxyribonucleoside triphosphate (dNTP) precursors of DNA synthesis or by direct inhibition of one or other DNA polymerase. Reduced supply of the pyrimidine deoxythymidine triphosphate (dTTP) may be caused by folate or vitamin B12 deficiencies or by the action of dihydrofolate reductase inhibitors (e.g. methotrexate, pyrimethamine or trimethoprim), all of which cause reduced supply of the coenzyme 5, 10 methylene tetrahydrofolate (pentaglutamate) needed for thymidylate synthetase. Reduced dTTP supply may also be caused by direct inhibition of thymidylate synthetase by 5-fluorouracil. Reduced supply of both purines, deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP), may be caused by hydroxyurea, 6-mercaptopurine (and probably by another purine antagonist azaserine), whilst reduced supply of both pyrimidine DNA precursors, dTTP and dCTP (deoxycytidine triphosphate) may be due to inherited orotic aciduria or to treatment with azauridine. Cytosine arabinoside directly inhibits DNA polymerase. DNA replication is a discontinuous process and a number of enzymes are concerned with different aspects of the process. The parental strands partly unwind and a large number of initiation points or origins are activated on both strands. A primer RNA is first synthesised using the parental strand of DNA as template. Fragments of new DNA are then synthesised on the parental DNA template, starting at the RNA primer, under the action of one or other DNA polymerase (probably gamma). The RNA primer is then removed and the gap left is filled by further DNA synthesis under the action of a different DNA polymerase (probably alpha). The fragments of new DNA are joined to give newly synthesised stretches of DNA (replicons) which are then liigated together to form bulk DNA of enormous molecular weight. It is suggested here that reduced supply of one or other of the four deoxyribonucleoside triphosphate (dNTP) during the 'S' phase of the cell cycle (due to vitamin B12 or folate deficiency, drug treatment or other congenital or acquired abnormality in synthesis of the dNTP) impairs the cell's ability to elongate newly initiated DNA fragments by preventing gap-filling, the polymerase needed for gap-filling requiring substantially greater concentrations of the deoxyribonucleoside triphosphates than the polymerase involved in chain initiation. Cytosine arabinoside, which also may cause megaloblastosis, may affect principally the synthesis of new DNA fragments. Since active protein synthesis is needed for the cell to enter the S phase and RNA synthesis is needed to prime new DNA synthesis, megaloblastic anaemia may be expected to occur only when DNA synthesis is inhibited but protein and RNA synthesis are relatively unimpaired...
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PMID:Vitamin B12--folate interrelations. 1 Jan 22

A DIRECT APPROACH IS DESCRIBED TO THE QUESTION: Are enzymes of DNA precursor synthesis organized into a supramolecular structure? This approach involved sedimentation analysis of several T4 phage-coded early enzyme activities in crude lysates of infected Escherichia coli. One-third to one-half of several activities tested-dCMP hydroxymethylase, dTMP synthetase, deoxynucleoside 5'-monophosphate kinase, deoxyuridine triphosphatase, and probably dCMP deaminase, but not dihydrofolate reductase or DNA polymerase-sedimented much more rapidly than expected from molecular weight. About 5% of the host cell nucleoside diphosphate kinase, known to participate in T4 DNA precursor synthesis, cosedimented with these activities. To show that this rapidly sedimenting material represents an organized enzyme complex rather than a nonspecific aggregate, we studied the kinetics of formation of dTTP with dUMP as the initial substrate. This three-step reaction sequence reached its maximal rate within a few seconds when catalyzed by enzymes in the aggregate, whereas an equivalent mixture of uncomplexed enzymes required nearly 20 min before dTTP synthesis reached its maximal rate. The effect of aggregation is evidently to decrease the volume into which intermediates are free to diffuse. Because there is reason to believe that intracellular concentration gradients of DNA precursors exist, the properties of this enzyme aggregate in vitro may help to explain how such gradients are maintained.
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PMID:Enzyme associations in T4 phage DNA precursor synthesis. 19 73

The quassinoids bruceantin, brucein D, brucein E, bruceoside A, and brusatol significantly inhibited P-388 lymphocytic leukemic cell RNA and protein synthesis in tissue culture. However, DNA synthesis inhibition seemed to correlate more directly with the anti-neoplastic activity of these compounds in the in vivo P-338 survival system. In vitro, brusatol and bruceoside A marginally inhibited 10-day P-388 lymphocytic leukemia DNA polymerase, RNA polymerase, thymidylate synthetase, dihydrofolate reductase, phosphoribosyl pyrophosphate aminotransferase, and cathepsin protease activities. In vivo studies demonstrated similar inhibition and elevated cyclic AMP levels, correlating positively with the antineoplastic activity of individual compounds. Purine synthesis was inhibited drastically by brusatol in vivo, and one key inhibition site in purine synthesis was at phosphoribosyl pyrophosphate aminotransferase, the regulatory enzyme. Histone phosphorylation and ribonucleotide reductase activity also were inhibited marginally by brusatol.
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PMID:Antitumor agents. XXXIV: Mechanism of action of bruceoside A and brusatol on nucleic acid metabolism of P-388 lymphocytic leukemia cells. 45 10

We have investigated the biochemical basis of the mevalonate dependence of DNA replication. Stimulating quiescent rat hepatoma cells to proliferate in the presence of compactin, an inhibitor of mevalonate synthesis, prevented DNA replication in as many as 80% of these cells. The percentage of cells that failed to replicate DNA increased with the increased duration of quiescence. Aphidicolin-sensitive DNA polymerase and ornithine decarboxylase activities were selectively decreased in compactin-treated cells, whereas RNA and protein synthesis, the level of dihydrofolate reductase and aphidicolin-resistant DNA polymerase activity were unaffected. Adding putrescine, the product of ornithine decarboxylase and the precursor of other polyamines, did not restore DNA replication. Our results demonstrate that the decreased activities of at least two DNA-replication enzymes are among the proximal causes of the failure of mevalonate-deprived cells to synthesize DNA. More importantly, our data indicate that a mevalonate-dependent factor(s) is progressively depleted during quiescence, and that inability to resynthesize this factor(s) may be the ultimate cause of the failure of resting cells to replicate DNA when stimulated to proliferate in the absence of mevalonate.
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PMID:The effect of mevalonic acid deprivation on enzymes of DNA replication in cells emerging from quiescence. 128 82

The sugar boronated thymidine nucleoside, 5' -0-[(triphenylphosphine-boryl) carbonyl]-3'-0-acetyl thymidine 1, and the boron-modified nucleoside phosphotriester, 5'-(diethylphosphite- cyanoborane)-3'-acetylthymidine 2, were successfully synthesized. Both compounds demonstrated differential activity when tested against eight cell lines, with significant cytotoxic activity against the growth of human Tmolt3 leukemia, colon adenocarcinoma, HeLa S3 uterine carcinoma, and osteosarcoma cells. In in vivo studies these agents were found to be active against the growth of Ehrlich ascites carcinoma at 8 mg/kg/day I.P. and to be marginally active against the growth of L1210 and Lewis lung cancers in mice. The mode of action of these thymidine derivatives in Tmolt3 cells was the inhibition of DNA and protein synthesis. Compound 2 was highly effective in inhibiting DNA polymerase alpha and m-RNA, r-RNA and t-RNA polymerase activities. Both compounds inhibited ribonucleoside reductase activity. The de novo purine pathway appeared to be the major site of inhibition of the agents, with IMP dehydrogenase, PRPP amido transferase, and dihydrofolate reductase activities being significantly inhibited. In the pyrimidine pathway, carbamyl phosphate synthetase and aspartate transcarbamylase activities were inhibited by 1. As expected, d[NTP] levels were significantly reduced by treatment with the agents. DNA strand scission was evident after incubating Tmolt3 cells for 24 hr with the agents.
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PMID:Antineoplastic activity of boron-containing thymidine nucleosides in Tmolt3 leukemic cells. 150 1

Examples of collateral sensitivity, even in experimental tumor systems, remain few. Preliminary data from this laboratory indicated that certain tumor cells expressed increased sensitivity to cisplatin after exposure in vitro to x-irradiation. To further clarify whether the type of fractionated radiation procedure used clinically can induce hypersensitivities to certain antitumor drugs we have pre-exposed the human ovarian carcinoma cell line JA-T/P derived from a tumor from an untreated patient to fractionated x-irradiation (total dose 50 Gy) in vitro. The resultant subline JA-T/DXR-10 expressed collateral sensitivity to cisplatin (CDDP), methotrexate (MTX) and fluorouracil (5-FU), but not to acute x-irradiation. Hypersensitivity to CDDP was associated with decreased activity of DNA polymerase beta (3.5-fold, P less than .01), but unaltered glutathione metabolism. Pre-incubation with cyclosporin A or with 3-aminobenzamide significantly enhanced (twofold, P less than .01) CDDP-induced cytotoxicity in JA-T/P cells, but not in the DXR-10 subline. Consistent with MTX hypersensitivity dihydrofolate reductase activity was significantly decreased (2.9-fold, P less than .01). Despite collateral sensitivity to 5-FU, however, thymidylate synthase activity was increased (twofold, P less than .05) suggesting alternative mechanisms for 5-FU-induced cytotoxicity in these JA-T/DXR-10 cells. These data demonstrate that DNA repair and associated reduced folate metabolism can be modified not only by drugs but also by fractionated x-irradiation.
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PMID:Expression of collateral sensitivity to cisplatin, methotrexate, and fluorouracil in a human ovarian carcinoma cell line following exposure to fractionated x-irradiation in vitro. 155 59

We have studied the effect of some specific enzyme inhibitors on DNA repair and replication after UV damage in Chinese hamster ovary cells. The DNA repair was studied at the level of the average, overall genome and also in the active dihydrofolate reductase gene. Replication was measured in the overall genome. We tested inhibitors of DNA polymerase alpha and delta (aphidicolin), of poly(ADPr) polymerase (3-aminobenzamide), of ribonucleotide reductase (hydroxyurea), of topoisomerase I (camptothecin), and of topoisomerase II (merbarone, VP-16). In addition, we tested the effect of the potential topoisomerase I activator, beta-lapachone. All of these compounds inhibited genome replication and all topoisomerase inhibitors affected the overall genome repair; beta-lapachone stimulated it. None of these compounds had any effect on the gene-specific repair.
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PMID:Effect of specific enzyme inhibitors on replication, total genome DNA repair and on gene-specific DNA repair after UV irradiation in CHO cells. 165 49

DNA polymerase beta (beta-pol) and its mRNA are maintained at constitutive levels during the cell cycle and during stages of cell growth in culture. To study biological consequences of variations in the level of this DNA repair enzyme and/or its mRNA, we prepared expression vectors in which cDNA for human beta-pol is inserted under the control of a metallothionein promoter (pMT) in the sense and antisense orientation, respectively, and these vectors then were used for stable transformation of mouse 3T3 cells. Vectors also contained the mouse DHFR gene, such that culture of transformants in medium with increasing concentrations of methotrexate resulted in amplification of inserted DNA. The levels of sense and antisense transcripts are strongly increased by culture of transformants in medium with 65 microM Zn2+, although some expression is detected even without Zn2+ induction. After five days of induction, the beta-pol level was about threefold higher in sense cells and about 10-fold lower in antisense cells than in parallel cultures without induction. The antisense line has a threefold increased cell doubling time in the presence of 65 microM Zn2+ compared with the absence of Zn2+. Zn2+ (65 microM) induction for the sense line results in normal growth for the first three days and, thereafter, a complete cessation of growth. Yet, these blocked cells remain fully viable. The results indicate that sudden deregulation of beta-pol expression alters cell growth in mouse 3T3 cells.
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PMID:Deregulation of DNA polymerase beta by sense and antisense RNA expression in mouse 3T3 cells alters cell growth. 169 88

Dihydrofolate reductase (DHFR; EC 1.5.1.3) is required in folate metabolism for the synthesis of purines, thymidine, and glycine. Although there have been several reports of induction of DHFR enzyme by methotrexate (MTX), a drug that competitively inhibits DHFR, there are no studies reported that examine the effect of MTX on DHFR gene transcription. We have examined the effect of MTX and other inhibitors of DNA synthesis on DHFR transcription using a transient expression assay. MTX stimulates transient expression in a concentration-dependent manner from a hamster DHFR promoter construct containing 150 base pairs 5' to the start of transcription. Addition of either tetrahydrofolate or hypoxanthine plus thymidine prevents the promoter induction in response to MTX, suggesting that stimulation by MTX results from inhibition of these metabolites. Furthermore, two other antimetabolic drugs--fluorodeoxyuridine and hydroxyurea--also stimulate the DHFR promoter in a concentration-dependent manner. In contrast, aphidicolin, which blocks cell growth through inhibition of DNA polymerase alpha, has no effect on the DHFR promoter. The potential relevance of these results to cross-resistance to chemotherapeutic agents and to the process of gene amplification is discussed.
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PMID:Stimulation of dihydrofolate reductase promoter activity by antimetabolic drugs. 183 62

The intracellular distribution and localization of dihydrofolate reductase-thymidylate synthase of wild type suspension carrot cells was analysed using cytochemical and immunocytochemical techniques; in both resting and growing normal cells (E4) the activity appeared to be predominantly cytoplasmic. The pattern of localization of the enzyme was also analysed during the different phases of the cell cycle. To this end carrot cells were synchronized with aphidicolin (an inhibitor of the alpha-like DNA polymerase which blocks cells at the G1/S boundary) and cycle phases checked by labelled-thymidine incorporation. Protoplasts obtained from cells inhibited with aphidicolin or from cells sampled at different times after the removal of the drug (S and G2 phase), failed to show a nuclear localization of DHFR-TS. These results indicate that in carrot the bifunctional enzyme does not change compartment during the cell cycle. Surprisingly Mtx-resistant cells (E2A2, E2A1C6; overproducing DHFR-TS) showed, irrespective of their physiological state (quiescent or growing), also a relevant nuclear or perinuclear immunofluorescence which could not be detected using cytochemical techniques. The reason of this altered localization is not clear and its possible relation with altered cytophysiological parameters is discussed.
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PMID:Cellular localization of dihydrofolate reductase-thymidylate synthase in carrot cells. 186 64


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