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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Fifteen specific inhibitors of DNA topoisomerases I and II were used to elucidate whether these enzymes participate in the excision repair of UV-induced DNA damage, monitoring DNA repair synthesis in confluent saponin-permeabilized human fibroblasts. To achieve a sufficient degree of accuracy dose--response experiments were performed, analysed by linear regression, and the concentrations at which repair activity was reduced to 50% were calculated and designated K50. Camptothecin, a specific inhibitor of topoisomerase I did not markedly diminish DNA repair synthesis. Similarly, when combined with topoisomerase II inhibitors [nalidixic acid, oxolinic acid, 4'-demethylepipodophyllotoxin-9-(4,6-O-ethylidene-beta-D-glucop yra noside) (etoposide), 4'-demethylepipodophyllotoxin-thenylidene-beta-D-glucoside (teniposide), 1,4-dihydroxy-5,8-bis ((2-[(2-hydroxyethyl)amino]ethyl)amino)-9,10-anthracenedione (mitoxantrone), 5-(N-phenyl-carboxamido)-2-thiobarbituric acid (merbarone) or 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA)], it did not lower K50 values determined for topoisomerase II-specific drugs in separate experiments. The effects observed can be classified according to the mechanism of action the inhibitors exhibit. (i) Novobiocin and coumermycin, inhibitors of the ATPase subunit of topoisomerase II, completely reduced DNA repair synthesis. (ii) Inhibition of repair was also found for ethidium bromide, quinacrine and distamycin, drugs known to modify the DNA substrate by intercalation or binding to the DNA minor groove. (iii) Inhibitors acting through intercalation and, simultaneously, binding to the cleavable DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) also suppressed reparative DNA synthesis. (iv) Only small effects were observed for etoposide, nalidixic acid and oxolinic acid, whereas teniposide caused marked inhibition of DNA repair synthesis. (v) Merbarone, a novel type of topoisomerase II inhibitor, blocked UV-induced DNA repair drastically. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kDa form of topoisomerase II is the main target enzyme for inhibitors which suppressed DNA excision repair and that this isozyme is involved in steps preceding repair-specific DNA incision.
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PMID:The function of DNA topoisomerases in UV-induced DNA excision repair: studies with specific inhibitors in permeabilized human fibroblasts. 133 77

A sedimentable complex of enzymes for DNA synthesis was partially purified from the combined low-salt nuclear extract-postmicrosomal supernatant solution of HeLa cell homogenates by poly(ethylene glycol) precipitation in the presence of 2 M KCl, discontinuous gradient centrifugation, Q-Sepharose chromatography, and velocity gradient centrifugation. In addition to the previously described 640-kDa multiprotein DNA polymerase alpha-primase complex [Vishwanatha et al. (1986) J. Biol. Chem. 261, 6619-6628], the enzyme complex also has associated topoisomerase I, DNA-dependent ATPase, RNase H, DNA ligase, a simian virus 40 origin recognition, dA/dT sequence binding protein [Malkas & Baril (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 70-74], and proliferating cell nuclear antigen. Essentially all of the T antigen dependent simian virus 40 in vitro replication activity in the combined nuclear extract-postmicrosomal supernatant solution resides with the sedimentable complex of enzymes for DNA synthesis. Sedimentation analysis on a 10-35% glycerol gradient in the presence of 0.5 M KCl indicates that the enzyme complex is 21S. The associated enzymes for DNA synthesis and in vitro simian virus 40 replication activity cofractionate throughout the purification of the 21S complex. The DNA polymerase and in vitro simian virus 40 replication activities are both inhibited by monoclonal antibody (SJK 132-20) to human DNA polymerase alpha and by 5-10 microM butylphenyl-dGTP, indicating that the association of DNA polymerase alpha with the 21S enzyme complex is essential for the initiation of SV40 DNA replication in vitro.
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PMID:A 21S enzyme complex from HeLa cells that functions in simian virus 40 DNA replication in vitro. 216 68

TNF is a pleiotropic cytokine that mediates diverse cellular responses, including cytotoxicity, cytostasis, proliferation, differentiation, and the expression of specific genes. Many of these processes require the activity of DNA topoisomerases I and II. We have investigated the interactions of TNF with inhibitors of both topoisomerases in 16-h assays using the murine L929 and human ME-180 cell lines, which undergo a cytotoxic TNF response. Camptothecin, a specific inhibitor of topoisomerase I, enhanced TNF cytotoxicity 150-fold against both cell lines. The topoisomerase II inhibitors VM-26 and VP-16, which stabilize covalent DNA-topoisomerase intermediates, greatly enhance TNF cytotoxicity against both cell lines. The most effective, VM-26, can lower the TNF LD50 to femtomolar levels. In contrast, the topoisomerase II inhibitors novobiocin and coumermycin, which bind to the enzyme ATPase site, protect L929 cells from TNF cytotoxicity but enhance TNF cytotoxicity in ME-180 cells. The large changes in TNF sensitivity induced by drug concentrations that by themselves show no effect, and the opposing synergistic effects of inhibitors with different inhibitory mechanisms (in L929 cells), suggest the active involvement of topoisomerases in TNF-mediated cytotoxicity. The correlation of cytotoxic synergy with the stabilization of DNA strand breaks indicates that DNA damage may play a significant role in TNF-mediated cytotoxicity.
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PMID:Synergistic interactions between tumor necrosis factor and inhibitors of DNA topoisomerase I and II. 217 May 26

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).
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PMID:In vitro replication of DNA containing either the SV40 or the polyoma origin. 289 81

Vaccinia virus cores contain a type I topoisomerase which promotes the relaxation of superhelical DNA of either handedness (Bauer et al., Proc. Natl. Acad. Sci. U.S.A. 74:1841-1845, 1977). The activity of partially purified vaccinia virus topoisomerase (VV-Topo I) was determined in the presence of ATP, dATP, GTP, ADP, and ATP analogs in which hydrolysis of the alpha, beta or beta, gamma phosphate bond is restricted. Topoisomerase activity was stimulated 2.5-fold by the addition of 2 to 4 mM ATP or dATP to standard assay mixtures; 2 mM GTP produced no significant effect on enzyme activity. The addition of 2 mM beta, gamma-imido ATP or 2 mM gamma-thiophosphate ATP reduced VV-Topo I activity by 80 and 65%, respectively. In contrast, 4 mM alpha, beta-methylene ATP produced no significant change in topoisomerase activity compared to ATP itself. Assays performed in the presence of 4 mM ADP exhibited an 80% reduction in enzyme activity. The preparations of VV-Topo I used for these studies showed, however, no detectable DNA-dependent or -independent ATPase activity. The activity of VV-Topo I was similarly measured in the presence of the antibiotics novobiocin and coumermycin A1, which inhibited enzyme activity by 50% at concentrations of 180 and 40 microM, respectively. Comparable inhibition of VV-Topo I activity was observed in the presence of 1 mM beta, gamma-imido ATP. We determined that novobiocin inhibits vaccinia core transcription at the same concentrations which inhibit vaccinia core topoisomerase I activity. These results suggest that the vaccinia DNA topoisomerase may play a role in the ATP-dependent transcription of viral genes from intact core particles.
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PMID:Effects of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase. 631 84

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.
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PMID:Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme. 632 14

Reverse gyrase, an ATP-dependent topoisomerase that positively supercoils DNA, has been purified to near-homogeneity from the hyperthermophile Methanopyrus kandleri. It migrates on SDS-polyacrylamide gel electrophoresis as two principal bands with apparent molecular masses of 150 and 50 kDa. Both proteins remain associated throughout all chromatographic steps. Transfer of a radioactive phosphate from DNA to the 50-kDa protein and gel retardation experiments indicate that this protein forms the covalent complex with DNA. A blot overlay assay identifies the 150-kDa protein as the potential ATPase. This is the first evidence that a reverse gyrase can be a topoisomerase consisting of two protomers. In analogy with the DNA gyrase A subunit (DNA breakage and reunion activity) and the B subunit (ATPase), the 50- and 150-kDa components of Mka reverse gyrase have been designated the A and B subunits, respectively. Methanopyrus reverse gyrase changes DNA linking number in steps of one and its A subunit covalently binds to the 5'-DNA phosphoryl group. It nicks DNA at sites that predominantly have a cytosine at the -4-position. The same rule was derived previously for monomeric reverse gyrase from sulfur-metabolizing hyperthermophiles and for topoisomerase I from mesophilic bacteria. Based on these results, Mka reverse gyrase is classified as belonging to group A of type I topoisomerases. The structural diversity of type I group A topoisomerases parallels the diversity of type II enzymes and suggests the evolution of an essential function by gene fusion.
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PMID:A reverse gyrase with an unusual structure. A type I DNA topoisomerase from the hyperthermophile Methanopyrus kandleri is a two-subunit protein. 815 33

A function for topoisomerases I and II in DNA excision repair can be postulated from the organization of the mammalian chromosome, involving nucleosomal structures and matrix-attached DNA loops. To analyse this function we determined UV-induced DNA incision in confluent human fibroblasts in the presence of 16 inhibitors of topoisomerases I and II which belonged to at least five different drug categories, based on their mechanism of action. Dose-response experiments were performed, analysed by linear regression and the concentrations at which DNA-incising activity was reduced to 50% were calculated (K50 values). The majority of these values represent concentrations for which interfering cell toxicity could be excluded. K50 concentrations, which were determined by extrapolating dose-response data, may hit the toxicity range, nevertheless, we deem our K50 scale useful for making biochemical comparisons. With respect to topoisomerase I, camptothecin and topotecan diminished repair-specific DNA incision to a small extent, whereas distamycin, which binds to the minor groove of DNA, caused a stronger effect. With respect to topoisomerase II the results were as follows. (i) The DNA intercalator ethidium bromide decreased DNA-incising activity at rather low concentrations, which indicates marked inhibitory potency. Quinacrine was less effective. (ii) Inhibitors intercalating and binding to the 'cleavable' DNA-topoisomerase complex (m-AMSA, mitoxantrone, doxorubicin and daunorubicin) strongly suppressed reparative DNA incision. (iii) Only small effects were observed using several drugs which act by trapping the 'cleavable' DNA-enzyme complex, namely nalidixic acid and oxolinic acid. In contrast, etoposide and teniposide inhibited post-UV DNA cleavage sizeably. (iv) Merbarone had to be applied at very high concentrations to reduce UV-induced DNA incision. (v) Novobiocin, an inhibitor of the ATPase subunit of topoisomerase II, markedly diminished repair-specific DNA cleavage. A comparison of the K50 values for DNA incision with those for DNA repair synthesis (1) shows that the majority of the investigated drugs inhibited both repair parameters. There were, however, differences in the concentrations required to achieve the 50% inhibition level. The results are best explained by assuming that in UV-irradiated human fibroblasts the 180 kd form of topoisomerase II is a target enzyme for inhibitors which suppressed repair and that this isozyme is involved in steps preceding repair-specific DNA incision.
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PMID:Various inhibitors of DNA topoisomerases diminish repair-specific DNA incision in UV-irradiated human fibroblasts. 824 65

The epipodophyllotoxins, etoposide (VP-16) and teniposide (VM-26), inhibit topoisomerase II activity by stabilization of the cleavable complex between the enzyme and DNA and formation of protein-bound double-stranded DNA breaks. While it is thought that these agents are cytotoxic by preventing cells from completing the S phase or undergoing mitosis, recent evidence suggests that these agents are also potent inducers of programmed cell death or apoptosis in both normal and malignant cells. We have examined the intracellular pathway leading to epipodophyllotoxin-induced apoptosis in normal mouse thymocytes. Epipodophyllotoxin-induced apoptosis may proceed via a mechanism that is independent of inhibition of topoisomerase activity per se because novobiocin and coumermycin, which inhibit the ATPase subunit of topoisomerase II, were relatively inefficient inducers of apoptosis in these cells, under conditions where strong apoptosis by the epipodophyllotoxins and dexamethasone could be observed. In addition, camptothecin, which inhibits topoisomerase I by stabilization of the cleavable complex between that enzyme and DNA, was also a poor inducer of apoptosis in these cells. Our data suggest that epipodophyllotoxin-induced mouse thymocyte apoptosis, like that induced by dexamethasone, proceeds via a mechanism that involves protein kinase C (PKC) or a similar enzyme. Apoptosis induced by VM-26 or by dexamethasone was inhibited by 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine dihydrochloride (H7), an inhibitor of both PKC and cAMP-dependent protein kinases, but was relatively unaffected by N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA1004), a more specific inhibitor of cAMP-dependent protein kinases. A more specific inhibitor of PKC, sangivamycin, also inhibited both VM-26-induced and dexamethasone-induced apoptosis. Both VM-26- and dexamethasone-induced apoptosis were unaffected by EGTA, a calcium (Ca2+) chelator, under conditions that inhibited apoptosis induced by the Ca2+ ionophore A23187. Moreover, while strong increases in intracellular Ca2+ were observed in thymocytes treated with A23187, we failed to detect increases in intracellular Ca2+ in cells induced to apoptose with either VM-26 or dexamethasone within the first 2 hr of culture. These results suggest that in mouse thymocytes there are at least two intracellular pathways leading to apoptosis: one, utilized by glucocorticoid and the epipodophyllotoxins, that proceeds in the absence of detectable increases in intracellular Ca2+ and possibly requires a novel Ca(2+)-independent PKC-like enzyme and another, utilized by Ca2+ ionophores, that is at least partially dependent on increased intracellular Ca2+.
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PMID:The mechanism of epipodophyllotoxin-induced thymocyte apoptosis: possible role of a novel Ca(2+)-independent protein kinase. 840 39

The Escherichia coli UvrA protein possesses a stretch of amino acids, 494 to 513, that matches the consensus sequence of the helix-turn-helix motif of many sequence-specific DNA binding proteins. It also has two zinc finger motif regions and two ATP binding sites. To study the potential roles of both helix-turn-helix and zinc finger motifs in the functioning of UvrA protein, random mutations were created in these motif regions by degenerate oligonucleotide-directed mutagenesis. Using this method, 12 single substitution mutants (eight in the helix-turn-helix motif region, one in the N-terminal zinc finger region, and three in the C-terminal zinc finger region) were isolated that failed to confer UV resistance in the E. coli strain deleted of the uvrA gene. One "hyper" UV-resistant mutant, G275A, was identified that conferred significantly more UV resistance than the wild type in the MH1-delta A strain. To further investigate the mechanism of failure of these mutant UvrA proteins to support nucleotide excision repair, two mutant UvrA proteins, G502D and V508D, were selected for purification and characterization, since they carry mutations at the positions offered as the putative constellation for the helix-turn-helix motif. The binding affinity of these two mutants for nonirradiated plasmid DNA was unaffected by the mutations. Both mutant proteins exhibited substantial ATPase activity, and together with the UvrB protein, they were capable of generating positively supercoiled plasmid DNA from the relaxed form in the presence of ATP and bacterial topoisomerase I. However, both mutant proteins failed to respond to UV damage in the filter binding assay and were incapable of forming 2 x SSC-resistant nucleoprotein complexes with UvrB protein on UV-irradiated plasmid DNA. Taking these properties together, it appears that the mutations in the helix-turn-helix motif region impaired the UvrA protein's ability to recognize UV damage, while its other activities were largely unaffected. Interestingly, ERCC-3, a human DNA repair protein, also has a similar helix-turn-helix motif. Given the highly conserved nature of repair proteins in general, this observation raises the possibility that both procaryotes and eucaryotes might use similar mechanisms to recognize damaged sites in their genomes.
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PMID:Mutations in the helix-turn-helix motif of the Escherichia coli UvrA protein eliminate its specificity for UV-damaged DNA. 844 6


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