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

---

Query: EC:5.99.1.2 (topoisomerase)
9,166 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.
...
PMID:The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae. 608

In order to study the double-strand DNA passage reaction of eukaryotic type II topoisomerases, a quantitative assay to monitor the enzymic conversion of supercoiled circular DNA to relaxed circular DNA was developed. Under conditions of maximal activity, relaxation catalyzed by the Drosophila melanogaster topoisomerase II was processive and the energy of activation was 14.3 kcal . mol-1. Removal of supercoils was accompanied by the hydrolysis of either ATP or dATP to inorganic phosphate and the corresponding nucleoside diphosphate. Apparent Km values were 200 microM for pBR322 plasmid DNA, 140 microM for SV40 viral DNA, 280 microM for ATP, and 630 microM for dATP. The turnover number for the Drosophila enzyme was at least 200 supercoils of DNA relaxed/min/molecule of topoisomerase II. The enzyme interacts preferentially with negatively supercoiled DNA over relaxed molecules, is capable of removing positive superhelical twists, and was found to be strongly inhibited by single-stranded DNA. Kinetic and inhibition studies indicated that the beta and gamma phosphate groups, the 2'-OH of the ribose sugar, and the C6-NH2 of the adenine ring are important for the interaction of ATP with the enzyme. While the binding of ATP to Drosophila topoisomerase II was sufficient to induce a DNA strand passage event, hydrolysis was required for enzyme turnover. The ATPase activity of the topoisomerase was stimulated 17-fold by the presence of negatively supercoiled DNA and approximately 4 molecules of ATP were hydrolyzed/supercoil removed. Finally, a kinetic model describing the switch from a processive to a distributive relaxation reaction is presented.
...
PMID:DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA. 630 11

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [3H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a 'swivelase' role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, D-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.
...
PMID:The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria. 630 36

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.
...
PMID:Effects of ATP and inhibitory factors on the activity of vaccinia virus type I topoisomerase. 631 84

We have recently demonstrated by electron microscopic cytochemical methods that unfixed human fibroblasts exhibit intense MG2+ dependent adenosine triphosphatase (nATPase) activity in circumscribed areas of the cell nucleoli. The nATPase was specific for ATP and dATP and was inhibited by other ribonucleoside triphosphates. Its intranucleolar localization relative to nucleolar chromatin, and segregation into nucleolar zones after actinomycin treatment of the cells, suggested that the reaction took place in fibrillar centers. This ATPase has now been further characterized by electron microscopic cytochemistry. It was determined that short fixation permitted retention of most of the ATPase activity, and that the enzyme was active at high ionic strength (up to 400 mM KCl), but that the enzyme activity was very sensitive to elevated temperatures. DNA dependence of the enzyme was shown by inhibition of the reaction by DNase pretreatment in parallel with the removal of DNA from the cell, while pretreatment with RNase had no significant effect. The nATPase activity was also selectively inhibited by treatment of the cells with antagonists of the B subunit of DNA gyrase, novobiocin, and coumermycin, but not by nalidixic or oxolinic acids, which interfere with the A subunit of gyrase. Inhibitors of RNA synthesis, actinomycin D and aminonucleoside of puromycin, potentiate rather than inhibit nATPase reaction. The results suggest that nATPase functions to alter the degree of supercoiling or catenation of nucleolar organizer DNA, and is in reality a DNA topoisomerase that hydrolyzes ATP during its action.
...
PMID:DNA dependence and inhibition by novobiocin and coumermycin of the nucleolar adenosine triphosphatase (ATPase) of human fibroblasts. 646 Aug 2

A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.
...
PMID:Purification and properties of human DNA helicase VI. 754 99

The rate of relaxation of supercoiled DNA by purified vaccinia topoisomerase I is stimulated 20-fold by 5 mM ATP. A similar effect is elicited by GTP, CTP, UTP, dATP, and adenosine 5'-(beta, gamma-imido)triphosphate. ATP-mediated rate enhancement requires salt as a coactivator. ADP and inorganic pyrophosphate also stimulate relaxation 10-20-fold, whereas AMP and inorganic phosphate have little effect. A model for allosteric activation of topoisomerase by nucleotides is suggested.
...
PMID:Stimulation of vaccinia topoisomerase I by nucleoside triphosphates. 796 68

DNA sequence analysis of Escherichia coli parC and parE, encoding the subunits of topoisomerase IV (Topo IV) (Kato, J.-I., Suzuki, H., and Ikeda, H. (1992) J. Biol. Chem. 267, 25676-25684), showed that ParC was 22 amino acids longer on the N terminus and ParE was 29 amino acids longer on the C terminus than reported previously. E. coli strains bearing bacteriophage T7 RNA polymerase-based expression plasmids carrying both intact and truncated parC and parE were used to overproduce the ParC and ParE proteins. Full-length ParC and ParE were required to reconstitute Topo IV activity, whereas the truncated ParC and ParE were inactive. Topo IV activity was supported only by ATP or dATP. The [ATP]1/2 for DNA relaxation was 0.45 mM, almost 25-fold higher than the [ATP]1/2 for decatenation of kinetoplast DNA. Topo IV activity was inhibited by the quinolone and coumarin antibiotics, although the concentrations required for 50% inhibition of activity were 3-30-fold higher than those required to inhibit DNA gyrase. The norfloxacin-induced DNA cleavage patterns of Topo IV and DNA gyrase were distinct but overlapping. The native forms of ParC and ParE were a dimer and a monomer, respectively; whereas the active form of Topo IV was a heterotetramer, ParC2ParE2. The inactivity of the truncated forms of ParC and ParE could be attributed to their failure to form the heterotetramer.
...
PMID:Escherichia coli topoisomerase IV. Purification, characterization, subunit structure, and subunit interactions. 822

Using a strand-displacement assay with 32P labeled oligonucleotide annealed to M13 ssDNA we have purified to apparent homogeneity and characterized a novel DNA unwinding enzyme from HeLa cell nuclei, human DNA helicase V (HDH V). This is present in extremely low abundance in the cells and has the highest turnover rate among other human helicases. From 300 grams of cultured cells only 0.012 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The enzyme also shows ATPase activity dependent on single-stranded DNA and has an apparent molecular weight of 92 kDa by SDS-polyacrylamide gel electrophoresis. Only ATP or dATP hydrolysis supports the unwinding activity. The helicase requires a divalent cation (Mg2+ > Mn2+) at an optimum concentration of 1.0 mM for activity; it unwinds DNA duplexes less than 25 bp long and having a ssDNA stretch as short as 49 nucleotides. A replication fork-like structure is not required to perform DNA unwinding. HDH V cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids; it unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand, a polarity similar to the previously described human DNA helicases I and III (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acid Res. 20, 5329-5337, 1992) and opposite to that of human DNA helicase IV (Tuteja et al. Nucleic Acid Res. 19, 3613-3618, 1991).
...
PMID:Human DNA helicase V, a novel DNA unwinding enzyme from HeLa cells. 838 37

Certain drugs used in the treatment of lung cancer and other human malignancies are cytotoxic because of their ability to interact with the two isoforms of topoisomerase II (topo II), topo IIalpha and topo IIbeta. As part of an effort to evaluate the contribution of topo II alterations to drug sensitivity and resistance in lung cancer, we have developed a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assay to measure levels of topo II alpha and beta mRNAs simultaneously using a single pair of primers with sequences common to both isoforms. The PCR products derived from the topo II alpha and beta mRNAs are both 446 bp but have different electrophoretic mobilities in a nondenaturing polyacrylamide gel, allowing sensitive, rapid quantitation when the products are radiolabeled with [35S]-dATP. Using this RT-PCR method, poly(A+) RNA from 13 non-small cell lung cancer (NSCLC) cell lines was analyzed. The results obtained indicated that the cell lines express a wide range of topo II alpha mRNA levels (12-fold) and topo IIbeta mRNA levels (5.5-fold). Tumor and normal lung tissues from 25 patients with NSCLC were also examined. In the tumor samples, the levels of the topo II alpha and beta mRNAs were similar. However, mean topo IIalpha mRNA levels in the tumors were approximately 7-fold higher than those of the paired normal lung tissues. In contrast, topo IIbeta mRNA levels were similar in both tumor and normal lung. Topo II alpha and beta mRNA levels were both significantly lower in the squamous cell tumors than in the adenocarcinoma samples. Topo IIbeta mRNA levels in the squamous cell tumors were also significantly lower than those in paired normal lung tissue. The RT-PCR method described is reliable and convenient, and for the first time, makes the rapid simultaneous direct comparison of topo IIalpha and topo IIbeta mRNA levels feasible in large numbers of clinical samples.
...
PMID:Simultaneous quantitation of topoisomerase II alpha and beta isoform mRNAs in lung tumor cells and normal and malignant lung tissue. 1087 30


<< Previous 1 2 3 Next >>

---