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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An ATP-dependent DNA helicase has been purified to near homogeneity from pea chloroplasts. The enzyme is a homodimer of 68-kDa subunits. The purified enzyme shows DNA-dependent ATPase activity and is devoid of DNA polymerase, DNA topoisomerase, DNA ligase or nuclease activities. The enzyme requires Mg2+ or Mn2+ for its maximum activity. ATP is the most favoured cofactor for this enzyme while other NTP or dNTP are poorly utilized. Pea chloroplast DNA helicase can unwind a 17-bp duplex whether it has unpaired single-stranded tails at both the 5' end and 3' end, at the 5' end or at the 3' end only, or at neither end. However, it fails to act on a blunt-ended 17-bp duplex DNA. The enzyme moves unidirectionally from 3' to 5' along the bound strand. The unwinding activity is inhibited by the intercalating drugs nogalamycin and daunorubicine.
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PMID:Purification and characterization of a DNA helicase from pea chloroplast that translocates in the 3'-to-5' direction. 866 52

We have cloned and sequenced two overlapping DNA fragments (3236 bp) containing a gene encoding the ATPase subunit of a type II DNA topoisomerase from the hyperthermophilic bacterion Thermotoga maritima (Tm Top2B). The deduced protein is composed of 636 aa with a calculated molecular mass of 72415 Da. It shares significant similarities with the ATPase subunits of mesophilic bacterial DNA topoisomerases II, either DNA gyrase (GyrB) or DNA topoisomerase IV (ParE). Although the highest similarity scores are obtained with GyrB proteins (55% identity with Bacillus subtilis DNA gyrase), a detailed phylogenetic analysis of all known DNA topoisomerases II does not allow us to determine if Tm Top2B corresponds to a DNA gyrase or a DNA topoisomerase IV. This hyperthermophilic Top2B protein exhibits a larger amount of charged amino acids than its mesophilic homologues, a feature which could be important for its thermostability. No gyrA-like gene has been found near top2B. A gene coding for a transaminase B-like protein was found in the upstream region of top2B.
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PMID:A gyrB-like gene from the hyperthermophilic bacterion Thermotoga maritima. 886 38

Temperature-sensitive mutations (ts10, ts18, and ts39) of the vaccinia virus RNA helicase nucleoside triphosphate phosphohydrolase II (NPH-II) result in the production of noninfectious progeny virions at the restrictive temperature. The noninfectious mutant particles contain the wild-type complement of virion core and envelope polypeptides, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results of Western blot (immunoblot) analysis indicate that these particles lack NPH-II, whereas other enzymatic components of the virus core are present. These components include the following: DNA-dependent RNA polymerase subunits rpo147, rpo132, rpo94, rpo35, rpo30, rpo22, and rpo18; early transcription initiation factor subunits A8 and D6; mRNA capping enzyme subunits D1 and D12; RNA cap 2'-O-methyltransferase; A18 DNA helicase; DNA-dependent ATPase NPH-I; and DNA topoisomerase. Although RNA polymerase is encapsidated by the mutant viruses, mRNA synthesis in vitro by permeabilized mutant virions is only 5 to 20% that of the wild-type virus, as judged by nucleoside monophosphate incorporation into acid-insoluble material. Moreover, the transcripts synthesized by the mutant particles are longer than normal and remain virion associated. Transcription initiation by mutant virions occurs accurately at an endogenous genomic promoter, albeit at reduced levels (1 to 7%) compared with that of wild-type virions. In contrast, extracts of the mutant virions catalyze the wild-type level of transcription from an exogenous template containing an early promoter. We conclude that NPH-II is required for early mRNA synthesis uniquely in the context of the virus particle. Possible roles in transcription termination and RNA transport are discussed.
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PMID:Vaccinia virions lacking the RNA helicase nucleoside triphosphate phosphohydrolase II are defective in early transcription. 897 Sep 79

The 1429-amino acid residue long yeast DNA topoisomerase II and three of its deletion derivatives, a C-terminal truncation containing residues 1-1202, a 92-kDa fragment spanning residues 410-1202, and an A'-fragment spanning residues 660-1202, were examined by transmission electron microscopy. Analysis of rotary-shadowed images of these molecules shows that the full-length enzyme assumes a tripartite structure, in which a large globular core comprising the carboxyl parts of the dimeric enzyme is connected to a pair of smaller spherical masses comprising the ATPase domains of the enzyme. The linkers bridging the large globular structure and each of the smaller spheres are not visible in most of the images but appear to be sufficiently stiff to keep the relative positions of the connected parts. The angle extended by the pair of spherical masses is variable and falls in a range of 50-100 degrees for the majority of the images. On binding of a nonhydrolyzable ATP analog to the enzyme, this angle is significantly reduced as the two spherical masses swing into contact. These observations, together with results from previous biochemical and x-ray crystallographic studies of the enzyme, provide a sketch of the molecular architecture and conformational states of a catalytically active type II DNA topoisomerase.
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PMID:Study of yeast DNA topoisomerase II and its truncation derivatives by transmission electron microscopy. 911 83

Reverse gyrases are ATP-dependent type I 5'-topoisomerases that positively supercoil DNA. Reverse gyrase from Methanopyrus kandleri is unique as the first heterodimeric type I 5'-topoisomerase described, consisting of a 138-kDa subunit involved in the hydrolysis of ATP (RgyB) and a 43-kDa subunit that forms the covalent complex with DNA during the topoisomerase reaction (RgyA). Here we report the reconstitution of active reverse gyrase from the two recombinant proteins overexpressed in Escherichia coli. Both proteins have been purified by column chromatography to >90% homogeneity. RgyB has a DNA-dependent ATPase activity at high temperature (80 degrees C) and is independent of the presence of RgyA. RgyA alone has no detectable activity. The addition of RgyA to RgyB reconstitutes positive supercoiling activity, but the RgyB and RgyA subunits form a stable heterodimer only after being heated together. This is the first case in which it has been possible to reconstitute an active heterodimeric enzyme of a hyperthermophilic prokaryote from recombinant proteins.
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PMID:Reverse gyrase from Methanopyrus kandleri. Reconstitution of an active extremozyme from its two recombinant subunits. 915 63

Streptococcus pneumoniae is uniquely sensitive to amino alcohol antimalarials in the erythro configuration, such as optochin, quinine, and quinidine. The protein responsible for the optochin (quinine)-sensitive (Opts, Qins) phenotype of pneumococcus is the proteolipid c subunit of the FzeroF1 H(+)-ATPase. OptR/QinR isolates arose by point mutations in the atpC gene and produce different amino acid changes in one of the two transmembrane alpha-helices of the c subunit. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (Opts) and M222 (an OptR strain produced by interspecies recombination between pneumococcus and S. oralis), and S. oralis (OptR) revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin, quinine, and related compounds specifically inhibited the membrane-bound ATPase activity. Equivalent differences between Opts/Qins and OptR/QinR strains, both in growth inhibition and in membrane ATPase resistance, were found. Pneumococci also show a characteristic sensitivity to coumarin drugs, and a relatively high level of resistance to most quinolones. We have cloned and sequenced the gyrB gene, and characterized novobiocin resistant mutants. The same amino acid substitution (Ser-127 to Leu) confers novobiocin resistance on four isolates. This residue position is equivalent to Val-120 of Escherichia coli ryGB, a residue that lies inside the ATP-binding domain but is not involved in novobiocin binding in E. coli, as revealed by crystallographic data. In addition, the genes encoding the ParC and ParE subunits of topoisomerase IV, together with the region encoding amino acids 46 to 172 (residue numbers as in E. coli) of the pneumococcal ryGA subunit, were characterized in respect to fluoroquinolone resistance. The gyrA gene maps to a physical location distant from the gyrB and parEC loci on the chromosome. Ciprofloxacin-resistant (CpR) clinical isolates had mutations affecting amino acid residues of the quinolone resistance-determining region of ParC (low-level CpR), or in both resistance-determining regions of ParC and GyrA (high-level CpR). Mutations were found in residue positions equivalent to Ser-83 and Asp-87 of the E. coli GyrA subunit. Transformation experiments demonstrated that topoisomerase IV is the primary target of ciprofloxacin, DNA gyrase being a secondary one.
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PMID:Molecular bases of three characteristic phenotypes of pneumococcus: optochin-sensitivity, coumarin-sensitivity, and quinolone-resistance. 918 46

Repressive chromatin structures need to be unravelled to allow DNA-binding proteins access to their target sequences. This de-repression constitutes an important point at which transcription and presumably other nuclear processes can be regulated. Energy-consuming enzyme complexes that facilitate the interaction of transcription factors with chromatin by modifying nucleosome structure are involved in this regulation. One such factor, nucleosome-remodelling factor (NURF), has been isolated from Drosophila embryo extracts. We have now identified a chromatin-accessibility complex (CHRAC) which uses energy to increase the general accessibility of DNA in chromatin. However, unlike other known chromatin remodelling factors, CHRAC can also function during chromatin assembly: it uses ATP to convert irregular chromatin into a regular array of nucleosomes with even spacing. CHRAC combines enzymes that modulate nucleosome structure and DNA topology. Using mass spectrometry, we identified two of the five CHRAC subunits as the ATPase ISWI, which is also part of NURF, and topoisomerase II. The presence of ISWI in different contexts suggests that chromatin remodelling machines have a modular nature and that ISWI has a central role in different chromatin remodelling reactions.
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PMID:Chromatin-remodelling factor CHRAC contains the ATPases ISWI and topoisomerase II. 925 92

The nucleotide sequence of a DNA gyrase B subunit gene (gyrB) from Treponema pallidum has been determined. Southern blot analysis of T. pallidum chromosomal DNA indicated that this gene is present as a single copy. The organization of genes flanking the gyrB gene is unique in comparison to that of other bacteria. The gyrB gene encodes a 637 amino acid protein whose deduced sequence has a high degree of homology with type-II topoisomerase ATPase subunits (GyrB and ParE). Five type-II topoisomerase motifs, an ATP-binding site (Walker A), and amino acid residues that putatively interact with ATP, are highly conserved in the T. pallidum GyrB protein.
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PMID:Identification and characterization of the gyrB gene from Treponema pallidum subsp. pallidum. 925 82

The thioether phospholipid ilmofosine (BM 41 440) is a new anti-cancer drug presently undergoing phase II clinical trials. Because resistance to anti-tumour drugs is a major problem in cancer treatment, we investigated the resistance of different cell lines to this compound. Here we report that the multidrug-resistant cell lines MCF7/ADR, CCRFNCR1000, CCRF/ADR500, CEM/VLB100 and HeLa cell lines transfected with a wild-type and mutated (gly/val185) multidrug resistance 1 gene (MDR1) are cross-resistant to ilmofosine compared with the sensitive parental cell lines. In CEMNM-1 cells, in which the resistance is associated with an altered topoisomerase II gene, no cross-resistance to ilmofosine was observed. Ilmofosine is not capable of modulating multidrug resistance and neither does it reduce the labelling of the P-glycoprotein (P-gp) by azidopine nor alter ATPase activity significantly. The resistance to ilmofosine in multidrug-resistant CCRF/VCR1000 cells cannot be reversed by the potent multidrug resistance modifier dexniguldipine-HCI (B8509-035). A tenfold excess of ilmofosine does not prevent the MDR-modulating effect of dexniguldipine-HCl. Treatment of cells with ilmofosine does not alter the levels of MDR1 mRNA. Long-term treatment of an ilmofosine-resistant Meth A subline with the drug does not induce multidrug resistance, indicating that ilmofosine does not increase the level of P-gp. Determination of the MDR2 mRNA levels in the cells revealed that the resistance pattern to ilmofosine is not correlated with the expression of this gene. It is concluded, therefore, that multidrug-resistant cells are cross-resistant to ilmofosine and that the compound is not a substrate of Pgp. No association between the expression of the MDR2-encoded P-gp and resistance to ilmofosine was observed. It is supposed that MDR1-associated alterations in membrane lipids cause resistance to ilmofosine.
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PMID:Resistance to the new anti-cancer phospholipid ilmofosine (BM 41 440). 932 44

Footprinting of yeast DNA topoisomerase II and its NH2- and COOH-terminal truncation derivatives was carried out to map the locations of lysyl side chains that are involved in enzyme-DNA interaction, in the binding of ATP, or in interaction between domains of the same enzyme molecule. Several conclusions were drawn based on these measurements and the crystal structures of a 92-kDa fragment of the yeast enzyme and a 43-kDa fragment of Escherichia coli gyrase B-subunit. First, the footprinting results support the model previously inferred from the 92-kDa fragment crystal structure that the main site of DNA binding is comprised of a pair of semicircular grooves. Second, the binding of a nonhydrolyzable ATP analog to the yeast enzyme appears to affect citraconylation at a minimum of six lysines in the ATPase domain of each polypeptide. Two of these lysines are probably involved in contacting the nucleotide directly, and one probably becomes buried when the two ATPase domains of a dimeric enzyme come into contact upon ATP binding; for the others, changes in lysine reactivity appear to reflect allosteric changes following ATP binding. Third, from a comparison of the footprint of the intact enzyme and those of the truncated polypeptides comprised of either the NH2- or the COOH-terminal half of the intact polypeptide, it appears that there are few contacts between the NH2- and COOH-terminal half of yeast DNA topoisomerase II.
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PMID:Footprinting of yeast DNA topoisomerase II lysyl side chains involved in substrate binding and interdomainal interactions. 938 73


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