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)

Those pyrimidine dimers that are repaired in confluent xeroderma pigmentosum Group C cells are clustered together in the genome. Although the average level of repair in this complementation group is of the order of 25% of normal, this percentage represents normal levels of repair in one quarter of the genome and little repair in the remainder. The factors that regulate this clustering process have been investigated using inhibitors of the initial incision step of repair (novobiocin) and of the polymerization step (aphidicolin). Novobiocin at a concentration that permitted 30% of repair to continue reduced the clustering of mended sites only slightly. Aphidicolin, in contrast, at a concentration that permitted 30 to 60% of repair to continue caused the mended sites to be distributed randomly. The clustering of repair sites seen in xeroderma pigmentosum Group C cells, therefore, is produced by an excision repair mechanism in which an aphidicolin-sensitive DNA polymerase, presumably alpha, plays an important regulatory role in determining which damaged sites are mended.
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PMID:Relative importance of incision and polymerase activities in determining the distribution of damaged sites that are mended in xeroderma pigmentosum group C cells. 310 77

The data in this paper show that when the inhibition of growth is measured, xeroderma pigmentosum (XP) complementation groups A, G and D are very sensitive to 4-nitroquinoline-1-oxide (4NQO), whereas only XP groups G and D are very sensitive to 3-methyl-4NQO (3me4NQO). Cells belonging to XP-C group are not particularly sensitive to either agent. Thus there are different epistasis groups for the excision repair of DNA adducts induced by these agents as opposed to the repair of u.v. damage. DNA polymerase alpha is involved in the repair of 4NQO-induced lesions because aphidicolin blocks their repair. XP cells from all the above groups are defective to some extent in this repair. The degree of repair defectiveness follows that seen after u.v., with even the XP-C cell line used having reduced repair (despite the fact that the inhibition of growth by 4NQO in this cell line was not markedly different from normal). Aphidicolin did not induce breaks in the normal or XP cell lines exposed to 3me4NQO, thus the repair of lesions induced by 3me4NQO does not involve DNA polymerase alpha in any of the cell lines. Finally, catalase reduces the alkaline labile lesions induced by 4NQO, but not 3me4NQO, suggesting the latter agent does not induce substantial amounts of DNA damage by the generation of radicals.
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PMID:The response to DNA damage induced by 4-nitroquinoline-1-oxide or its 3-methyl derivative in xeroderma pigmentosum fibroblasts belonging to different complementation groups: evidence for different epistasis groups involved in the repair of large adducts in human DNA. 311 41

DNA polymerases delta and alpha were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase alpha were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases delta and alpha in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase alpha or delta activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase alpha or delta activity. Concentrations of BuPdGTP which totally abolished DNA polymerase alpha activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase delta. Monoclonal antibodies which specifically inhibited polymerase alpha activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase alpha antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-alpha antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase alpha. The remaining replication activity (20-50%) is tentatively attributed to polymerase delta, because it was aphidicolin sensitive and resistant to both anti-polymerase alpha antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase alpha activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of DNA polymerase delta in CV-1 cells: studies implicating both DNA polymerase delta and DNA polymerase alpha in DNA replication. 312 21

A human ovarian cancer cell line, A2780, derived from an untreated ovarian cancer patient and relatively sensitive to cisplatin was treated by stepwise incubation with cisplatin to produce a cisplatin-resistant variant, 2780CP. The relative abilities of these cell lines to repair cisplatin-induced damage to cellular DNA then was examined by measure of [3H]thymidine incorporation into normal density DNA separated from bromodeoxyuridine-substituted DNA on alkaline cesium chloride gradients. These studies revealed that primary cisplatin resistance present in 2780CP was associated with a near twofold-increased ability to repair damage induced by the drug under conditions where 2780CP was approximately 5-fold resistant to cisplatin. Aphidicolin, a specific inhibitor of DNA polymerase alpha, showed a dose-dependent capacity to inhibit DNA repair in this system with maximum inhibition of 63% at 4 micrograms/ml. It was also found that inhibition of DNA repair during and shortly after cisplatin exposure resulted in an approximately threefold increase in the cytotoxicity of cisplatin as monitored by clonogenic cell survival in the resistant but not the sensitive parental cell line.
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PMID:Increased DNA repair as a mechanism of acquired resistance to cis-diamminedichloroplatinum (II) in human ovarian cancer cell lines. 313 81

Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.
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PMID:Thymidylate synthase inhibition in cells with arrested DNA synthesis is not due to an allosteric interaction in the replitase complex. 392 Oct 23

The relationships between replicative DNA synthesis and retinoic acid (RA)-induced differentiation of human promyelocytic leukaemic (HL-60) cells are evaluated with the use of Aphidicolin, a specific and reversible inhibitor of DNA polymerase alpha (alpha). Addition of a sublethal concentration of Aphidicolin (0.4 microM) in culture for 3 days suppresses DNA synthesis to a similar level of the resting stage (day 8) in control cultures. DNA synthesis is reactivated to the level observed in the growing stage of control cultures once Aphidicolin is removed after 3 days in culture. The level of DNA synthesis at the early stage of RA-induction (day 3) is suppressed by only 17% when compared to control cultures. The inhibitory effect of Aphidicolin on DNA synthesis in both control cultures and RA-induced cell cultures is similar. However, no reactivation of DNA synthesis is observed after removal of Aphidicolin on day 3 from RA-induced cell cultures. Flow cytometric analysis of DNA content on day 3 reveals that cells accumulate in G1 and early S phases of the cell cycle after exposure to Aphidicolin with or without RA. Of interest is the fact that, while Aphidicolin alone did not induce cells to differentiate, neither did it interfere with RA-induced cell differentiation (the rate of RA-induced cell differentiation in the presence of Aphidicolin is similar to that of RA-treated cultures in the absence of Aphidicolin). These results suggest that the combined use of Aphidicolin and RA may inhibit leukaemic cell proliferation more effectively without causing severe cytotoxicity and without interfering with RA-induced cell differentiation.
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PMID:Combined effects of aphidicolin and retinoic acid on proliferation and differentiation of human leukaemic (HL-60) cells. 392 6

Aphidicolin, a tetracyclic diterpenoid obtained from the culture filtrates of Cephalosporium aphidicola and other fungi, inhibits the growth of eukaryotic cells and of certain animal viruses (SV40, Herpes and Vaccinia viruses) by selectively inhibiting the cellular replicative DNA polymerase alpha or the viral-induced DNA polymerases. The arrest of cellular or viral growth is thus due to inhibition of cellular or viral replicative DNA synthesis without interference with mitochondrial DNA synthesis, RNA, protein and nucleic acid precursors synthesis or other major metabolic pathways. The inhibition of all sensitive eukaryotic DNA polymerases by aphidicolin is competitive with respect to dCTP. Aphidicolin has thus proved extremely useful in elucidating the functional role of DNA polymerase alpha in nuclear DNA replication, of DNA polymerase gamma in mitochondrial DNA synthesis and both DNA polymerases beta and alpha in DNA repair synthesis. An important laboratory application of aphidicolin is the synchronization of the cell cycle of eukaryotic cells both in culture and in vivo. The properties of aphidicolin have recently aroused considerable interest for its possible exploitation in al practice. The mechanism of action of this drug suggests in fact that it may be useful for controlling excessive cell proliferation in patients with cancer, psoriasis or other dermatitis with little or no adverse effect upon non-multiplying cells. Interestingly, when administered to mice, the highest levels of aphidicolin are found in those tissues most actively proliferating with little or no aphidicolin present in neurons or myocardial cells.
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PMID:Control of cell division by aphidicolin without adverse effects upon resting cells. 393 52

The regulation of trophectoderm differentiation in mouse embryos was studied by inhibiting DNA synthesis with aphidicolin, a specific inhibitor of DNA polymerase alpha. Embryos were exposed to aphidicolin (0.5 micrograms/ml) for 16 h at various preimplantation stages and scored for their ability to form a blastocyst and develop beyond the blastocyst stage. Embryos were most sensitive to aphidicolin at the late 4-cell stage and became progressively less sensitive as they developed. Aphidicolin inhibited blastocyst formation by 70%, 100%, 77%, and 24% after treatment at the 2-cell, 4-cell, noncompacted 8-cell, and compacted 8-cell stages, respectively. Although the inhibitory effect of aphidicolin on blastocyst formation decreased markedly as 8-cell embryos underwent compaction, developmental capacity beyond the blastocyst stage was poor after treatment of either noncompacted or compacted 8-cell embryos. Treatment at the morula and early blastocyst stages was less harmful to embryos than treatment at earlier stages but reduced the number of trophoblast outgrowths by interfering with hatching. Autoradiographic analysis showed that during aphidicolin treatment, incorporation of 3H-thymidine was inhibited over 90% at all stages examined, indicating an inhibition of DNA synthesis. Because inhibition of blastocyst formation by aphidicolin decreased at the compacted 8-cell stage, we suggest that approximately the first half of the fourth DNA replication cycle is critical for subsequent blastocyst formation. Furthermore, the poor further development of blastocysts formed after aphidicolin treatment of compacted 8-cell embryos suggests that the DNA replication requirements for initial trophectoderm differentiation are distinct from requirements for further development of blastocysts in vitro.
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PMID:Inhibition of DNA replication in preimplantation mouse embryos by aphidicolin. 393 86

Aphidicolin is an inhibitor of DNA polymerase alpha and blocks nuclear DNA replication without interfering with mitochondrial DNA synthesis. The efficacy of this mycotoxin as a tool in cell synchronization was evaluated in C3H 10T1/2 clone 8 cells. At concentrations of 1-2 micrograms/mL, aphidicolin quickly reduced the [3H]thymidine uptake to less than 5% of control levels in the first 5 min of incubation. This inhibition was easily reversed by washing and refeeding cells with fresh medium. The synchronization protocol consisted of first blocking cells by confluence arrest, replating them at lower density, and then treating the cells with aphidicolin for 24 h. Once the inhibitor was removed, DNA replication started without any delay. The cell population traversed the S phase in about 8 h and synchronously doubled in cell number. Autoradiography studies revealed a labeling index of 89-93% during the S phase. However, it was also observed that 10T1/2 cells were able to enter S phase in the presence of aphidicolin. The extent of the ensuing replication in the nucleus was dependent on the time that cells remained arrested in early S phase. Analyses of the newly replicated DNA in alkaline sucrose gradients revealed a fairly homogeneous distribution of sizes of nascent DNA in synchronized cells pulse-labeled at the beginning of the S phase. Upon chase in nonradioactive medium, the average molecular weight of the nascent DNA increased linearly with time of DNA synthesis for 2 h. The apparent rate of DNA chain growth determined from pulse and chase experiments was 1.2 micron/min. This rate was strongly inhibited (93%) by aphidicolin at a concentration of 2 micrograms/mL.
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PMID:Kinetics of DNA replication in C3H 10T1/2 cells synchronized by aphidicolin. 393 53

The p-n-butylphenyl- and p-n-butylanilino- substituted analogs of dGTP and dATP, respectively, were tested as inhibitors of purified human placental DNA polymerases alpha and delta. It was observed that DNA polymerase alpha activity was potently inhibited by these analogs with I0.5 values as low as the nanomolar range, whereas DNA polymerase delta activity was poorly inhibited, with I0.5 values of ca. 100 micromolar. These results argue for a distinct identity of these two enzymes, and demonstrate the usefulness of these analogs as probes of DNA polymerase structures. In addition, these analogs provide a rapid method for the discrimination of the two enzyme activities and a means for the selective assay of DNA polymerase delta. Aphidicolin inhibited both DNA polymerases.
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PMID:Differential inhibition of human placental DNA polymerases delta and alpha by BuPdGTP and BuAdATP. 393 20


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