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)

To test whether DNA injury contributes to TNF-induced cytotoxicity, we attempted to enhance DNA injury by inhibiting its repair and then assessing effects on cytotoxicity. DNA repair, assayed as unscheduled DNA synthesis, was first detected in TNF-sensitive targets by 2-3 h of incubation with TNF. Targets resistant to TNF cytotoxicity did not demonstrate significant repair replication. Repair preceded the detection of TNF-induced DNA injury, which was subsequently demonstrated by a double-stranded DNA fragmentation assay, sedimentation of DNA in neutral and alkaline sucrose gradients, and gel electrophoresis of extracted DNA. This suggested that early during exposure to TNF, DNA repair proceeds more rapidly than strand breakage. To inhibit repair, nontoxic concentrations of aphidicolin (inhibitor of DNA polymerase-alpha) and dideoxythymidine (inhibitor of DNA polymerase-beta and gamma) were used. Aphidicolin inhibited repair and consistently sensitized to TNF cytotoxicity, decreasing the ID50 for TNF at least 10- to 50-fold. In contrast, dideoxythymidine had no effect on repair or cytotoxicity. Deoxycytidine, which competitively inhibits binding of aphidicolin to DNA polymerase, blocked the sensitization in a concentration-dependent fashion. In targets sensitized with aphidicolin, TNF-induced strand breakage was accelerated, being detected by 4 h of culture in the sucrose gradient assay. Sensitization to TNF was not due to a heightened activation of poly (ADP-ribose) polymerase. These results indicate that TNF-induced strand breakage participates in TNF-induced cytotoxicity and that the level of DNA repair plays a role in determining relative sensitivity of targets.
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PMID:Inhibition of DNA repair with aphidicolin enhances sensitivity of targets to tumor necrosis factor. 837 4

Calf thymus DNA polymerase epsilon readily uses short, synthetic oligonucleotides as substrates for both polymerase and exonuclease activity. These substrates were used to examine the mechanism of inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol epsilon.DNA complex. Importantly, aphidicolin binds equally well regardless of the identity of the next template base to be replicated (Ki approximately 0.6 microM). Hydrolysis of synthetic templates of defined sequence by the 3'-->5' exonuclease was examined. pol epsilon preferred to hydrolyze single-stranded DNA 3-fold better than double-stranded DNA (Vmax/KM), while under Vmax conditions single-stranded DNA was hydrolyzed 100-fold faster than double-stranded DNA. Aphidicolin did not inhibit exonuclease activity on single-stranded DNA; however, activity on double-stranded DNA was partially inhibited. Formation of an E.[template.primer].aphidicolin ternary complex inhibits exonuclease activity. However, even under conditions where the polymerase site is completely blocked by a template-primer, the exonuclease retains significant activity.
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PMID:DNA polymerase epsilon: aphidicolin inhibition and the relationship between polymerase and exonuclease activity. 839 9

Camptothecin is an S-phase-specific anticancer agent that inhibits the activity of the enzyme DNA topoisomerase-I (topo-I). Irreversible DNA double-strand breaks are produced during DNA synthesis in the presence of camptothecin, suggesting that this agent should not be toxic to nondividing cells, such as neurons. Unexpectedly, camptothecin induced significant, dose-dependent cell death of postmitotic rat cortical neurons in vitro; astrocytes were more resistant. Aphidicolin, an inhibitor of DNA polymerase alpha, did not prevent camptothecin-induced neuronal death, while death was prevented by actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole as well as cycloheximide and anisomycin, inhibitors of RNA and protein synthesis, respectively. Camptothecin-induced neuronal death was apoptotic, as characterized by chromatin condensation, cytoplasmic shrinking, plasma membrane blebbing, and fragmentation of neurites. DNA fragmentation was also confirmed by the use of the in situ DNA end labeling assay. In addition, aurintricarboxylic acid, an inhibitor of the apoptotic endonuclease, partially protected against camptothecin-induced neuronal death. The toxicity of stereoisomers of a camptothecin analogue was stereospecific, demonstrating that toxicity was a result of inhibition of topo-I. The difference in sensitivity to camptothecin between neurons and astrocytes correlated with their transcriptional activity and level of topo-I protein expression. These data indicate important roles for topo-I in postmitotic neurons and suggest that topo-I inhibitors can induce apoptosis independent of DNA synthesis. We suggest a model based on transcriptionally mediated DNA damage, a novel mechanism of action of topo-I poisons.
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PMID:Induction of neuronal apoptosis by camptothecin, an inhibitor of DNA topoisomerase-I: evidence for cell cycle-independent toxicity. 870 53

Aphidicolin, a known inhibitor of eucaryotic deoxyribonucleic acid (DNA) polymerase alpha, efficiently inhibited amplification of ribosomal DNA during oogenesis in Xenopus laevis. DNA polymerase alpha, but not DNA polymerase gamma, as isolated from ovaries, was sensitive to aphidicolin. DNA polymerase beta was not detectable in Xenopus ovary extracts. Therefore, DNA polymerase alpha plays a major role in ribosomal ribonucleic acid gene amplification.
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PMID:Participation of deoxyribonucleic acid polymerase alpha in amplification of ribosomal deoxyribonucleic acid in Xenopus laevis. 927 81

We have used the ciliate Euplotes to study the role of DNA polymerase in telomeric C strand synthesis. Euplotes provides a unique opportunity to study C strand synthesis without the complication of simultaneous DNA replication because millions of new telomeres are made at a stage in the life cycle when no general DNA replication takes place. Previously we showed that the C-strands of newly synthesized telomeres have a precisely controlled length while the G-strands are more heterogeneous. This finding suggested that, although synthesis of the G-strand (by telomerase) is the first step in telomere addition, a major regulatory step occurs during subsequent C strand synthesis. We have now examined whether G- and C strand synthesis might be regulated coordinately rather than by two independent mechanisms. We accomplished this by determining what happens to G- and C strand length if C strand synthesis is partially inhibited by aphidicolin. Aphidicolin treatment caused a general lengthening of the G-strands and a large increase in C strand heterogeneity. This concomitant change in both the G- and C strand length indicates that synthesis of the two strands is coordinated. Since aphidicolin is a very specific inhibitor of DNA pol alpha and pol delta, our results suggest that this coordinate length regulation is mediated by DNA polymerase.
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PMID:Coordinate regulation of G- and C strand length during new telomere synthesis. 936 59

Positive results in the in vitro assay for chromosome aberrations sometimes occur with test chemicals that apparently do not react with DNA, being negative in tests for mutation in bacteria, for DNA strand breaks, and for covalent binding to DNA. These chromosome aberrations typically occur over a narrow concentration range at toxic doses, and with mitotic inhibition. Indirect mechanisms, including oxidative damage, cytotoxicity and inhibition of DNA synthesis induced by chemical exposure, may be involved. Understanding when such mechanisms are operating is important in evaluating potential mutagenic hazards, since the effects may occur only above a certain threshold dose. Here, we used two-parameter flow cytometry to assess DNA synthesis inhibition (uptake of bromodeoxyuridine [BrdUrd]) associated with the induction of aberrations in CHO cells by DNA-reactive and non-reactive chemicals, and to follow cell cycle progression. Aphidicolin (APC), a DNA polymerase inhibitor, induces aberrations without reacting with DNA; 50 microM APC suppressed BrdUrd uptake during a 3-h treatment to <10% of control levels. Several new drug candidates induced aberrations concomitant with marked reductions in cell counts at 20 h (to 50-60% of controls) and suppression of BrdUrd uptake (<15% of control). Several non-mutagenic chemicals and a metabolic poison, which induce DNA double strand breaks and chromosome aberrations at toxic dose levels, also suppressed DNA synthesis. In contrast, the alkylating agents 4-nitroquinoline-1-oxide, mitomycin C, methylnitrosourea, ethylnitrosourea, methylmethane sulfonate and ethylmethane sulfonate, and a topoisomerase II inhibitor, etoposide, produced many aberrations at concentrations that were less toxic (cell counts >/=73% of controls) and gave little inhibition of DNA synthesis during treatment (BrdUrd uptake >/=85% of controls), although cell cycle delay was seen following the 3-h treatment. Thus, inhibition of DNA synthesis at the time of treatment is supporting evidence for an indirect mechanism of aberrations, when there is no direct DNA reactivity.
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PMID:DNA synthesis inhibition as an indirect mechanism of chromosome aberrations: comparison of DNA-reactive and non-DNA-reactive clastogens. 968 28

We have previously demonstrated that DNA polymerase activity of Entamoeba histolytica is inhibited by aphidicolin, which is a specific inhibitor of eukaryotic nuclear replicative DNA polymerases. The present study was aimed to evaluate the effect of aphidicolin on growth and DNA synthesis by this parasite. Aphidicolin blocked the growth of axenic E. histolytica strain HM-1:IMSS. DNA synthesis was also inhibited by aphidicolin when assayed by incorporation of [3H]thymidine into the DNA. The inhibitory effect of aphidicolin on the growth of E. histolytica was abrogated by removal of the drug, and exposure to 3 microg/ml of the drug for at least 48 hr had little effect on the viability. Synchronous growth was observed in the recovery phase after removal of aphidicolin.
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PMID:Effects of aphidicolin on Entamoeba histolytica growth and DNA synthesis. 971 25

We have analyzed the X-ray-sensitive CHO mutant cell line EM9 for sensitivity to the topoisomerase I inhibitor comptothecin. These cells exhibit defective repair of single strand DNA breaks. Recently, EM9 were complemented the DNA ligase III interactive protein, XRCC1. Defective XRCC1 apparently accounts for the low DNA ligase III activity that may explain the single-strand break repair deficiency of EM9 cells. Here, we demonstrate cytotoxic hypersensitivity of EM9 cells following a brief camptothecin treatment. Both the S-phase and non-S-phase populations of EM9 exhibited camptothecin sensitivity relative to the parent cell line AA8. In AA8 cells, only the 55% of the population corresponding to the S-phase subpopulation were sensitive to camptothecin, while the remainder of the population were totally resistant to doses as high as 10 microM. The role of DNA replication in the camptothecin sensitivity was studied using the DNA polymerase inhibitor aphidicolin in co-treatment with camptothecin. Aphidicolin treatment fully protected AA8 cells from camptothecin cytotoxicity. In EM9 cells, aphidicolin protected the S-phase fraction to some degree but all the cells remained sensitive to camptothecin cytotoxicity. These results suggest that EM9 cells are sensitized to camptothecin by a mechanism that is independent of DNA replication and may be a consequence of the XRCC1 mutation or the associated deficiency in DNA ligase III activity. Mechanistic models for the replication-independent cytotoxicity of camptothecin in EM9 cells are discussed.
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PMID:The CHO XRCC1 mutant, EM9, deficient in DNA ligase III activity, exhibits hypersensitivity to camptothecin independent of DNA replication. 973 12

DNA synthesis was effectively inhibited by antisense oligonucleotide A1 complementary to the BamHI-H gene family in Marek's disease virus (MDV)-derived lymphoblastoid MDCC-MSB1 cells. When a cell cycle distribution of a total cell population was analyzed by flow cytometry, the proportion of S-phase cells increased in the cell populations by treatment with oligonucleotide A1. Approximately 60-70% of the cells appeared in the S phase for 24 and 36 hr of incubation in the presence of oligonucleotide A1 (20-30% in the untreated control cells). The inhibition of cell cycle progression by treatment with oligonucleotide A1 was reversible. When the cells were treated with 5 microM aphidicolin for 12 hr, a similar pattern of cell cycle distribution was observed to that obtained after treatment with oligonucleotide A1. Aphidicolin is an inhibitor of cellular DNA polymerase alpha, and it halts progression of the cell cycle at the G1/S border or early S phase. When the cells were treated with aphidicolin for 12 hr and subsequently incubated with oligonucleotide A1, no significant difference was observed in the cycle phase distribution of cells in the presence and absence of oligonucleotide A1. In contrast, when the cells were treated with oligonucleotide A1 for 12 hr and subsequently incubated with aphidicolin, the cell cycle did not progress from the G1/S border or early S phase to the next phase.
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PMID:Antisense oligonucleotide complementary to the BamHI-H gene family of Marek's disease virus induced growth arrest of MDCC-MSB1 cells in the S-phase. 1034 90

DNA polymerase activities in fractionated cell extract of Aeropyrum pernix, a hyperthermophilic crenarchaeote, were investigated. Aphidicolin-sensitive (fraction I) and aphidicolin-resistant (fraction II) activities were detected. The activity in fraction I was more heat stable than that in fraction II. Two different genes (polA and polB) encoding family B DNA polymerases were cloned from the organism by PCR using degenerated primers based on the two conserved motifs (motif A and B). The deduced amino acid sequences from their entire coding regions contained all of the motifs identified in family B DNA polymerases for 3'-->5' exonuclease and polymerase activities. The product of polA gene (Pol I) was aphidicolin resistant and heat stable up to 80 degrees C. In contrast, the product of polB gene (Pol II) was aphidicolin sensitive and stable at 95 degrees C. These properties of Pol I and Pol II are similar to those of fractions II and I, respectively, and moreover, those of Pol I and Pol II of Pyrodictium occultum. The deduced amino acid sequence of A. pernix Pol I exhibited the highest identities to archaeal family B DNA polymerase homologs found only in the crenarchaeotes (group I), while Pol II exhibited identities to homologs found in both euryarchaeotes and crenarchaeotes (group II). These results provide further evidence that the subdomain Crenarchaeota has two family B DNA polymerases. Furthermore, at least two DNA polymerases work in the crenarchaeal cells, as found in euryarchaeotes, which contain one family B DNA polymerase and one heterodimeric DNA polymerase of a novel family.
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PMID:Two family B DNA polymerases from Aeropyrum pernix, an aerobic hyperthermophilic crenarchaeote. 1049 10


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