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)

Microinjection is shown to be a useful tool for studies of chemical inhibition of DNA synthesis: inhibitor-treated cells were injected with combinations of radioactive precursors and their uptake into DNA was monitored by autoradiography. The results obtained from inhibition by cytosinearabinoside, aphidicolin, trifluorothymidine, and fluorodeoxyuridine agreed well with the common knowledge about these drugs. Short-term (but not long-term) treatments with methotrexate were compensated by injections of thymidine-nucleotides. The effect of hydroxyurea was in part, but not fully, reversed by injection of all four deoxytriphosphates; this implies a second mechanism besides inhibition of ribonucleotide reductase. Regulation of reductase was responsible for the effect of thymidine: the enhanced dTTP caused a depletion of dCTP and dATP. Novobiocin was different from all other drugs tested, DNA polymerase or enzymes of the precursor metabolism are obviously not targets of this drug.
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PMID:Microinjected deoxynucleotides for the study of chemical inhibition of DNA synthesis. 296 41

Novobiocin inhibits DNA topoisomerases. It also inhibits excision repair of DNA photodamage, blocking both repair synthesis and the earlier step of incision at u.v. damage sites (as measured by the accumulation of DNA strand breaks in u.v.-irradiated interphase cells treated with DNA synthesis inhibitors such as hydroxyurea or cytosine arabinoside). It has been supposed, therefore, that novobiocin affects repair by blocking a putative topoisomerase step prior to incision. But we find that novobiocin also has a marked dose- and time-dependent effect on mitochondria: in cells exposed to novobiocin, mitochondria swell and their cristae become disrupted, and the intracellular ATP:ADP ratio is lowered, though the membrane potential is maintained as judged by rhodamine 123 fluorescence. Mitotic cells are more resistant to mitochondrial disruption by novobiocin than are interphase cells. This correlates with a relative resistance of u.v.-irradiated mitotic cells to the inhibition of incision by novobiocin. The chromosomal decondensation that results from the accumulation of DNA breaks due to incision when u.v.-irradiated mitotic cells are treated with hydroxyurea and cytosine arabinoside is largely suppressed by novobiocin. Furthermore, the suppression of induced strand break accumulation is partly due to a suppression by novobiocin of the uptake and phosphorylation of cytosine arabinoside; breaks accumulated in u.v.-irradiated cells in the presence of aphidicolin, an inhibitor of DNA polymerase alpha that does not require phosphorylation, are less novobiocin-sensitive. We conclude that the effects of novobiocin on excision repair are more likely to be due to a non-specific effect on ATP metabolism than to a specific effect on a repair-related topoisomerase.
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PMID:Novobiocin inhibition of DNA excision repair may occur through effects on mitochondrial structure and ATP metabolism, not on repair topoisomerases. 299 34

The influence of ciprofloxacin, nalidixic acid, norfloxacin, novobiocin, and ofloxacin on elements of eucaryotic DNA replication was investigated in vitro. Each of the 4-quinolones, when present in amounts of more than 100 micrograms/ml, reversibly inhibited the DNA synthesis performed by the 95 DNA polymerase alpha primase complex from calf thymus. Novobiocin at 500 micrograms/ml or at higher concentrations irreversibly inactivated DNA polymerase alpha primase complex. The accuracy of in vitro DNA synthesis in the absence of repair mechanisms was determined from amber-revertant assays with phi X174am16(+) DNA as template. The antimicrobial agents did not significantly increase the frequencies of base pairing mismatches during the course of replication, indicating that the basal mutation rate is not affected by novobiocin and the 4-quinolones. The Ki values of 50% inhibition of DNA topoisomerases from calf thymus by ciprofloxacin, norfloxacin, novobiocin, nalidixic acid, and ofloxacin were 300, 400, 1,000 or more, 1,000 or more, and 1,500 or more micrograms/ml, respectively, in the case of topoisomerase I, and the Ki values were 150, 300, 500, 1,000, and 1,300 micrograms/ml, respectively, in the case of topoisomerase II. The procaryotic topoisomerase II is approximately 100-fold more sensitive to inhibition by ciprofloxacin, norfloxacin, and ofloxacin than is its eucaryotic counterpart. Growth curves of lymphoblasts were recorded in the presence of ofloxacin and ciprofloxacin. Neither 1 nor 10 micrograms of ciprofloxacin or of ofloxacin per ml affected cell proliferation. Ofloxacin and ciprofloxacin at 100 micrograms/ml inhibited cell growth; 1,000 micrograms/ml led to cell death. No correlation exists between the antimicrobial and cytotoxic activities of the 4-quinolones.
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PMID:Effect of 4-quinolones and novobiocin on calf thymus DNA polymerase alpha primase complex, topoisomerases I and II, and growth of mammalian lymphoblasts. 301 15

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

In permeable human fibroblasts, novobiocin, coumermycin and nalidixic acid completely inhibit u.v.-induced DNA repair synthesis, with 50% inhibition occurring at 500, 24 and 8800 microM respectively. Novobiocin also inhibits damage-specific incision of DNA in u.v.-irradiated permeable human fibroblasts by at least 75%. It has been suggested that effects of novobiocin on DNA excision repair result from changes in ATP pools; this explanation is not applicable to our data because excision repair in the permeable cell system is entirely dependent on exogenous ATP. It has also been suggested that novobiocin-induced inhibition of repair is mediated by alterations of chromatin structure recognizable by electron microscopy as gross chromatin clumping. There were no ultrastructural alterations, however, in the nuclei of permeable cells that had been incubated with 1 mM novobiocin. We conclude that, in human cells, novobiocin, coumermycin, and nalidixic acid directly inhibit the excision repair of u.v. damage to DNA, and that one locus of inhibition lies at or before the incision step. Because 1 mM novobiocin completely abolishes u.v.-induced repair synthesis in permeable cells, but inhibits damage-specific incision by only 75%, there seems to be a second site of inhibition following the incision step. The similarity between the concentrations of novobiocin, nalidixic acid and coumermycin required to inhibit u.v.-induced excision repair and the concentrations required to inhibit human DNA polymerase alpha suggest that the distal locus of inhibition may be DNA polymerase alpha-mediated repair patch synthesis. The proximal inhibitory site may be a type II DNA topoisomerase.
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PMID:Direct inhibition of u.v.-induced DNA excision repair in human cells by novobiocin, coumermycin and nalidixic acid. 360 79

Bleomycin (BLM) is an antitumor drug which interacts with and damages DNA. We have reported a repair response dependent on DNA polymerase I in toluene-treated Escherichia coli. We report here that DNA polymerase III can also catalyze a repair response in toluene-treated E. coli following exposure to BLM. Polymerase III-mediated synthesis differs because it is ATP-dependent, whereas polymerase I-mediated repair synthesis is not. Polymerase III repair synthesis is independent of replicative synthesis, as demonstrated in a polA-, dnaBts strain, or use of Novobiocin to inhibit replication, and replication persists in the presence of repair synthesis. It appears that ATP-dependent repair synthesis in response to BLM is also present in polA+ strains. Repair synthesis does not require the uvrA gene product.
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PMID:DNA polymerase III-dependent repair synthesis in response to bleomycin in toluene-treated Escherichia coli. 616 Mar 70

Novobiocin inhibits animal DNA polymerase alpha and avian reverse transcriptase activities when these enzymes are assayed in vitro with activated DNA as template. Under the same conditions DNA polymerase beta and gamma are much less inhibited. DNA polymerase alpha and reverse transcriptase are inhibited by different mechanisms: in the case of the retroviral enzyme the effect of novobiocin is not overcome by dilution of the drug, while in the case of polymerase alpha the inhibition disappeared after novobiocin dilution. The inhibition of polymerase alpha by novobiocin is non-competitive with respect to the TTP precursor or activated DNA. The irreversible inactivation of reverse transcriptase by novobiocin leads to the loss of the enzyme affinity for primer tRNATrp. Moreover, novobiocin inhibits the partial unwinding of the 3' end of tRNATrp by reverse transcriptase.
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PMID:The in vitro inhibition of DNA polymerase alpha and avian reverse transcriptase by novobiocin. 620 65

We investigated, in a cloned hamster tracheal epithelial cell line HTE-B, the effects of inhibitors of DNA topoisomerase, novobiocin and nalidixic acid; of DNA polymerase, 1-beta-arabinofuranosylcytosine (ara-C) and 2',3'-dideoxythymidine; of ribonucleotide reductase, hydroxyurea; and of poly(ADP-ribose)synthetase, 3-aminobenzamide, upon the removal of benzo[a]pyrene adducted to DNA [B[a]P--DNA]. A substantial reduction in the rate of removal of the polycyclic hydrocarbon-adducts occurred when nalidixic acid was added to the HTE-B cells that had been previously incubated with B[a]P for 8 h. Novobiocin produced a similar, but less marked, effect. The rate of disappearance of the individual B[a]P--DNA adducts was measured by analysis of the h.p.l.c. profiles. Of the 5 major adducts observed under the h.p.l.c. conditions, 4 were reduced in control cells to 30% of the original levels by 24 h after removal of the B[a]P from the medium; adduct 5 was almost completely removed. In the presence of nalidixic acid, during the 24 h repair period, only the removal of adduct 5 was unimpaired; the removal of the other 4 adducts was significantly retarded. On the other hand, 3-aminobenzamide addition did not affect the rate of removal of B[a]P--DNA adducts from the HTE-B cells. We employed the combinations of ara-C and dideoxythymidine or ara-C and hydroxyurea to allow the accumulation of single strand breaks after incubation of the HTE-B cells with B[a]P. These breaks were assayed by alkaline elution analysis. Inclusion of these inhibitors during the 2 h after removal of the B[a]P from the medium resulted in the accumulation of 4-5 single strand breaks/10(10) daltons of HTE-B DNA. This compares with a minimum estimate of the number of adducts removed during this period of 3 adducts/10(7) daltons. This discrepancy may indicate that the majority of lesions are not repaired by a pathway sensitive to polymerase inhibitors. In the presence of 3-aminobenzamide, we routinely observed a 10% increase in the alkaline elution of the DNA obtained from B[a]P-treated cells (1-2 breaks/10(10) daltons). Our results indicate that an excision repair process may be involved in the removal of at least some of the B[a]P-induced damage to DNA. However, the repair of the multiple adducts is complex and may involve pathways other than classical excision repair.
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PMID:The influence of inhibitors on the repair of benzo[a]pyrene-damaged DNA in hamster tracheal epithelial cells. 632 Oct 50

A DNA-membrane complex was extracted from minicells of an Escherichia coli mutant harboring a "miniplasmid" derivative (11.2 kilobases) of the low-copynumber plasmid RK2 (56 kilobases). The complex contained various species of supercoiled and intermediate forms of plasmid DNA, of which approximately 20% was bound firmly to the membrane after centrifugation in a CsCl density gradient. The plasmid DNA-membrane complex synthesized new plasmid DNA without the addition of exogenous template, enzymes, or other proteins. DNA synthesis appeared to proceed semi-conservatively, was dependent on the four deoxynucleoside triphosphates, partially dependent on ribonucleoside triphosphates, and was sensitive to rifampin, an antibiotic known to inhibit initiation of replication. Novobiocin and nalidixic acid also inhibited synthesis, as did the omission of ATP, N-Ethylmaleimide, an inhibitor of DNA polymerase II and III activity, but not DNA polymerase I activity, also partially inhibited the synthetic reaction, as did chloramphenicol. The plasmid DNA synthetic product was analyzed by alkaline sucrose and dye-CsCl gradient centrifugation, as well as by agarose gel electrophoresis. In each case, the product consisted of parental and intermediate forms of plasmid DNA. Some chromosomal DNA was also synthesized by a contaminating bacterial DNA-membrane complex, but this synthesis was rifampin insensitive and could be separated from plasmid DNA synthesis.
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PMID:Replication of a low-copy-number plasmid by a plasmid DNA-membrane complex extracted from minicells of Escherichia coli. 704 88

We have extended our permeable cell system for measuring DNA excision repair [Roberts, J. D., & Lieberman, M. W. (1979) Biochemistry 18, 4499-4505] so that steps of the repair process, beginning with incision and extending at least through the "rearrangement" of repaired nucleosomes which follows repair synthesis, all take place in permeable cells. In the revised protocol, human fibroblasts are made permeable, damaged with UV or chemicals in suspension, and incubated with a reaction mix containing ATP and the four deoxyribonucleoside triphosphates, one of which is labeled with 32P. By reducing the exogenous dNTP concentration to 3 microM and including 15 mM KCl in the reaction mixture, we have greatly reduced background incorporation in undamaged cells without significantly reducing repair synthesis. This permits us to measure repair synthesis without separating it from replicative synthesis by isopycnic centrifugation. Repair synthesis in this system is very similar to that occurring in intact cells: in response to DNA damage, nucleotides are incorporated into DNA of parental density (when analyzed by the BrdUrd density shift technique), incorporation increases with increasing DNA damage, synthesis is dependent on the presence of all four dNTPs, and the system accurately reflects the genetic UV repair deficiency of xeroderma pigmentosum (XP) cells. Furthermore, as has been observed in intact cells, repair-incorporated nucleotides in these permeable cells are initially overrepresented in staphylococcal nuclease sensitive regions of chromatin and are subsequently redistributed to give a nearly uniform distribution between nuclease-sensitive and -resistant regions. The UV dose curve of permeable cells differs somewhat from that of intact cells; however, the dose differs somewhat from that of intact cells; however, the dose curve for permeable cells treated with N-methyl-N-nitrosourea is very similar to that of intact cells. Repair synthesis in UV-damaged, permeable normal and XP cells is stimulated by addition of Micrococcus luteus UV endonuclease, indicating that the damaged DNA is accessible to exogenous repair enzymes and suggesting that incision, or an obligatory preincision step, is rate limiting for excision repair in these permeable cells. Repair synthesis in this system is inhibited by aphidicolin, but not by high levels of dideoxy-TTP, suggesting involvement of DNA polymerase alpha in excision repair. Novobiocin is also inhibitory alpha and the HeLa cell type II DNA topoisomerase.
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PMID:Characterization of deoxyribonucleic acid repair synthesis in permeable human fibroblasts. 709 2


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