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)

Analysis of vaccinia topoisomerase mutants that are impaired in DNA relaxation has allowed the identification of amino acid residues required for the transesterification step of catalysis. Missense mutations of wild-type residues Gly-132----Asp and Arg-223----Gln rendered the protein inert in formation of the covalent enzyme-DNA complex and hence completely inactive in DNA relaxation. Mutations of Thr-147----Ile and Gly-132----Ser caused severe defects in covalent adduct formation that correlated with the extent of inhibition of relaxation. None of these point mutations had an effect on noncovalent DNA binding sufficient to account for the defect in relaxation. Deletion of amino- or carboxyl-terminal portions of the polypeptide abrogated noncovalent DNA binding. Two distinct topoisomerase-DNA complexes were resolved by native gel electrophoresis. One complex, which was unique to those proteins competent in covalent adduct formation, contained topoisomerase bound to the 5'-portion of the incised DNA strand. The 3'-segment of the cleaved strand had dissociated spontaneously. This complex was isolated and shown to catalyze transfer of the covalently bound DNA to a heterologous acceptor oligonucleotide, thereby proving that the covalent adduct between protein and duplex DNA is a true intermediate in strand breakage and reunion. The role of the active site region of eukaryotic topoisomerase in determining sensitivity or resistance to camptothecin was examined by converting the active site region of the resistant vaccinia enzyme (SKRAY274) to that of the drug-sensitive yeast enzyme (SKINY). The SKINY mutation did not alter the resistance of the vaccinia enzyme to the cleavage-enhancing effects of camptothecin.
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PMID:Covalent and noncovalent DNA binding by mutants of vaccinia DNA topoisomerase I. 132 12

Nuclear extracts from teniposide (VM-26)-resistant sublines of the human leukemic cell line CCRF-CEM have decreased levels of DNA topoisomerase II catalytic activity and decreased capacity to form drug-stabilized covalent protein-DNA complexes. The ATP concentration required for equivalent activity in a DNA-unknotting assay is 2- to 8-fold higher in nuclear extracts from drug-resistant cell lines as compared with the parental line. When adenosine 5'-[beta,gamma-imido]triphosphate is substituted for ATP in complex-formation assays, no significant change is seen with drug-sensitive cells, but a 50-65% reduction is seen with VM-26-resistant cells. Collectively, these results indicate that an alteration in ATP binding may be involved in the resistance phenotype. Therefore, we identified regions of the topoisomerase II sequence that conform to previously identified nucleotide-binding sites. Starting with cDNA as the template we determined the sequence of the topoisomerase II mRNA surrounding these sites by sequencing DNA fragments produced by the polymerase chain reaction. In the region corresponding to the consensus B ATP-binding sequence described by Walker et al. [Walker, J. E., Saraste, M., Runswick, M. J. & Gay, N. J. (1982) EMBO J. 1, 945-951], the cDNA from the two VM-26-resistant sublines contained an altered sequence having a G----A base change. This base substitution results in the replacement of the conserved arginine at position 449 with a glutamine. Hybridization with allele-specific oligonucleotides confirmed the presence of both the normal and the altered sequence in the resistant cell lines, whereas only the normal sequence was found in the sensitive CEM cells. A chemical mismatch cleavage procedure for the detection of mispaired bases in DNA duplexes identified no other alterations in the 5' third of the mRNA coding sequence, which contains the complete ATP-binding domain of topoisomerase II. The presence of mRNA encoding topoisomerase II with Gln449 correlates both with the presence of a topoisomerase II protein whose interaction with ATP is altered and with increased resistance to the cytotoxicity of VM-26.
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PMID:Expression of a mutant DNA topoisomerase II in CCRF-CEM human leukemic cells selected for resistance to teniposide. 165 58

The product of the rep gene of ColE2 is required for initiation of ColE2 DNA replication. The rep gene was placed under the control of the promoters, PL and PR, and the heat-labile cl857 repressor of bacteriophage lambda. The Rep protein was identified as a 35 Kd protein by the maxicell method in combination with heat-induced expression. The protein was efficiently expressed from these promoters in unirradiated cells and accumulated up to a few per cent of the total cellular proteins. It was partially purified (about 80% pure) and its properties examined. The amino acid sequence of the amino terminal portion of the partially purified protein agreed well with that predicted from the nucleotide sequence of the rep gene. One of the characteristic features of the rep gene is frequent usage of rare codons, especially those for arginine. The protein specifically stimulated replication of ColE2 DNA but not that of ColE3 DNA in crude cell extracts of Escherichia coli. Specific binding of the protein to plasmid DNA containing the origin region of ColE2 was demonstrated by the filter binding method. Neither endonuclease activity nor topoisomerase activity was detected by using ColE2 DNA.
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PMID:Identification of a plasmid-coded protein required for initiation of ColE2 DNA replication. 182 59

Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.
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PMID:The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase. 184 8

In order to characterize more fully the mechanism by which casein kinase II is regulated in mammalian cells, the effect of epidermal growth factor (EGF) on the activity of the kinase in human A-431 carcinoma cells was examined. Treatment of cells with EGF prior to lysis consistently resulted in a transient 4-fold increase in the activity of cytosolic casein kinase II. Activity rose sharply between 20 and 30 min, peaked at approximately 50 min, and returned to basal levels by approximately 120 min. Similar results were obtained using the casein kinase II specific peptide substrate, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu, or DNA topoisomerase II (which is specifically modified by the kinase in vivo and serves as a high affinity substrate in vitro) as the phosphate acceptor in assays. Identification of casein kinase II as the stimulated activity was confirmed by partial proteolytic mapping and phosphoamino acid analysis of modified topoisomerase II, by inhibition at nanomolar levels of heparin or micromolar levels of nonradioactive GTP, and by the ability to employ radioactive GTP as a direct phosphate donor. The EGF stimulation of casein kinase II was dependent on the availability of intracellular (but not extracellular) calcium. In addition, hormonal action was modulated by calcium/phospholipid-dependent protein kinase (protein kinase C). Casein kinase II stimulation did not require an increase in the concentration of the kinase, protein synthesis, the continual presence of a small effector molecule, or a direct interaction with the EGF receptor/tyrosine kinase. In contrast, hormonal activation of the kinase was dependent on the phosphorylation of casein kinase II or a terminal stimulatory factor.
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PMID:Regulation of casein kinase II activity by epidermal growth factor in human A-431 carcinoma cells. 247 67

Incubation of cultured rat aortic smooth muscle cells (A-10, ATCC CRL 1476) with [8-arginine]vasopressin (AVP) or thrombin increased the amount of DNA strand breakage induced by camptothecin, an inhibitor of topoisomerase I (DNA topoisomerase; EC 5.99.1.2) and transiently stimulated the extractable activity of this enzyme. Both topoisomerase-related responses were prevented by treatment of the cells with AVP or thrombin plus the appropriate receptor antagonist. The increase in strand breakage mediated by AVP and thrombin depended on the concentration of hormone. Neither AVP nor thrombin had any effect on strand breaks obtained with the epipodophyllotoxin VM-26, an inhibitor of topoisomerase II [DNA topoisomerase (ATP-hydrolysing); EC 5.99.1.3]. Pretreatment of the cells with pertussis toxin partially inhibited thrombin-mediated increases in camptothecin-induced strand breakage whereas AVP-mediated increases were unaffected. These results are consistent with the notion that AVP and thrombin induce a transient increase in intracellular topoisomerase I activity via interactions with their respective cell surface receptors and that the effects of the activation of these receptors are mediated by different G-proteins.
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PMID:Stimulation of intracellular topoisomerase I activity by vasopressin and thrombin. Differential regulation by pertussis toxin. 255 99

At concentrations normally used to inhibit eukaryotic type II topoisomerase activity (100-1000 micrograms/ml) novobiocin binds core histones. Approximately 15 moles of novobiocin bind per mole of histone resulting in histone precipitation from solution in either 0.15 M or 2 M NaCl. The interaction between novobiocin and proteins appears to involve arginine residues: histones H3 and H4 (13.5 and 14 mole percent arginine) are precipitated at lower novobiocin concentrations than histones H2A and H2B (9.5 and 6.5 mole percent arginine). Furthermore, polyarginine but not polyornithine competes for novobiocin in histone precipitation. Moreover, histones with arginine residues modified with 1,2-cyclohexanedione are soluble in 1000 micrograms/ml novobiocin. Because novobiocin can remove histones from solution as well as inhibit topoisomerase activity, and because both of these events can alter DNA topology, novobiocin should be used with caution in experiments designed to implicate topoisomerase activity in chromatin dynamics.
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PMID:Novobiocin precipitates histones at concentrations normally used to inhibit eukaryotic type II topoisomerase. 371 93

Mono-conjugation of an anthraquinone nucleus with a range of naturally occurring amino acids chemically modified at their C-terminus has been adopted as a synthetic approach in the rational design of novel topoisomerase (topo) inhibitors. The biochemistry of topo I and II inhibition has been investigated for a series of 16 new compounds (NU/ICRF 500-515) from which structure-activity relationships have been investigated. Only three compounds could be demonstrated to bind to DNA: two serine derivatives (NU/ICRFs 500 and 506) and an arginine derivative (NU/ICRF 510). In decatenation and relaxation assays with purified enzyme, several compounds were shown to be potent catalytic inhibitors of topo II (100% inhibition at 5 micrograms/mL (10-15 microM) or less) without stabilizing cleavable complex formation. These included the three DNA binding species (of which NU/ICRF 506 was the most active) and a dihydroxyphenylalanine analogue (NU/ICRF 513). Both NU/ICRFs 500 and 506 were further shown to antagonize DNA cleavage induced by amsacrine. Only NU/ICRF 506 unequivocally inhibited the catalytic activity of topo I without induction of DNA cleavage, and was the only combined topo I and II catalytic inhibitor. One compound, NU/ICRF 505 (tyrosine conjugate), stabilized topo I cleavable complexes without inhibiting the catalytic activity of topo I and II. Modifications to the structure of NU/ICRF 505 revealed that the presence of an unhindered hydroxyl on the tyrosine ring and a more hydrophobic ethyl ester at the amino acid C-terminal were both essential, suggesting a highly specific interaction between drug, enzyme and DNA in the ternary complex. Molecular modelling studies suggested that the observed differences in topo inhibition are a consequence of major conformational alterations brought about by small changes in the amino acid substituent, and confirmed a rigid structural requirement for the induction of topo I cleavage, in addition to a less rigid structural requirement for topo II inhibition. A strong correlation was observed between topo inhibition and in vitro cytotoxicity against the human ovarian cancer cell line A2780 (IC50 range 3.4-11.6 microM), suggesting a mechanism of cell kill, at least in part, involving topo inhibition.
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PMID:Biochemistry of topoisomerase I and II inhibition by anthracenyl-amino acid conjugates. 759 37

Vaccinia DNA topoisomerase, a member of the eukaryotic type I enzyme family, binds duplex DNA and forms a covalent protein.DNA complex at sites containing a conserved sequence element 5'-CCCTT decreases. The structure of the enzyme in the free and DNA-bound states was probed by limited proteolysis. The free topoisomerase (a 314-amino acid polypeptide) consists of protease-resistant amino- and carboxyl-terminal structural domains flanking a protease-sensitive "hinge." The hinge region, located between residues 135 and 142, is defined by accessibility to three different proteases. The amino-terminal region is punctuated by a trypsin-sensitive "bridge" at Arg-80, suggesting at least a tripartite domain structure overall. A specific subset of residues accessible to proteases in the free enzyme becomes resistant to proteolysis in the DNA-bound state. The trypsin-sensitive site at Arg-80 is protected almost completely in the covalent complex. Within the hinge region, Lys-135, Tyr-136, and Glu-139 are protected from trypsin, chymotrypsin, and V8, respectively. Acquisition of altered protease sensitivity upon DNA binding occurs prior to covalent adduct formation. The 20-kDa carboxyl domain by itself binds noncovalently to duplex DNA, albeit without the sequence specificity characteristic of the full-sized topoisomerase.
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PMID:Proteolytic footprinting of vaccinia topoisomerase bound to DNA. 774 4

The characteristic feature of multidrug resistance (MDR) associated with drugs that interact with DNA topoisomerase II (topo II) is alterations in topo II activity or amount (at-MDR). We have characterized the at-MDR phenotype in human leukemic CEM cells selected for resistance to the topo II inhibitor, VM-26. Compared to drug-sensitive cells, the key findings are that at-MDR cells exhibit (i) decreased topo II activity; (ii) decreased drug sensitivity, activity and amount of nuclear matrix topo II; (iii) increased ATP requirement of topo II; (iv) a single base mutation in topo II resulting in a change of Arg to Gln at position 449, at the start of the motif B/nucleotide binding site; and (v) decreased topo II phosphorylation, suggesting decreased kinase or increased phosphatase activities. Recent results using single-stranded conformational polymorphism analysis reveals the presence of a mutation in the motif B/nucleotide binding site of the topo II alpha gene in CEM at-MDR cells and in another leukemic cell line selected for resistance to m-AMSA. Finally, we have observed marked changes in the nuclear distribution of topo II in cells treated with anti-topo II drugs and have also found these changes to be attenuated in drug-resistant cells. We postulate that traditional inhibitors of topo II alter the equilibrium of the strand-passing reaction such that the number of enzyme-DNA covalent complexes increases. We further suggest that when the enzyme is bound to DNA it is protected from proteolysis, thus allowing more topo II molecules to be detected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Altered DNA topoisomerase II in multidrug resistance. 776 55


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