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

The DNA untwisting enzyme has been purified approximately 300-fold from rat liver nuclei. The protein is greater than 90% pure as judged by sodium dodecyl sulfate-acrylamide gel electrophoresis. The native enzyme has a molecular weight between 64 000 and 68 000 and is composed of a single polypeptide chain. Evidence is presented that the protein can act catalytically. A trace amount of endonuclease activity associated with the most pure fraction could be a contaminant or it could be due to the action of the DNA untwisting enzyme itself.
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PMID:Purification and characterization of the DNA untwisting enzyme from rat liver. 18 21

A DNA-relaxing enzyme which catalyzes the conversion of superhelical DNA to a non-superhelical covalently closed form has been purified from Micrococcus luteus to near homogeneity by two chromatographic steps. The enzyme is a single polypeptide chain. As determined by sodium dodecyl sulfate - polyacrylamide gel electrophoresis and gel filtration on Sephadex G 150, the molecular weight is 115,000. The DNA-relaxing activity determined as a function of enzyme concentration follows a sigmoidal curve. The enzyme requires Mg++ for activity. In the presence of 4.5 mM Mg++ addition of 50-250 mM KCl yields incompletely relaxed DNA molecules (intermediates); intermediates are also observed in the absence of KCl, when the reaction is carried out at 0 degree C or at Mg++ concentrations exceeding 10 mM.
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PMID:DNA-relaxing enzyme from Micrococcus luteus. 20 27

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.
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PMID:Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata. 131 98

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

In Escherichia coli, the miniF plasmid CcdB protein is responsible for cell death when its action is not prevented by polypeptide CcdA. We report the isolation, localization, sequencing and properties of a bacterial mutant resistant to the cytotoxic activity of the CcdB protein. This mutation is located in the gene encoding the A subunit of topoisomerase II and produces an Arg462----Cys substitution in the amino acid sequence of the GyrA polypeptide. Hence, the mutation was called gyrA462. We show that in the wild-type strain, the CcdB protein promotes plasmid linearization; in the gyrA462 strain, this double-stranded DNA cleavage is suppressed. This indicates that the CcdB protein is responsible for gyrase-mediated double-stranded DNA breakage. CcdB, in the absence of CcdA, induces the SOS pathway. SOS induction is a biological response to DNA-damaging agents. We show that the gyrA462 mutation suppresses this SOS activation, indicating that SOS induction is a consequence of DNA damages promoted by the CcdB protein on gyrase-DNA complexes. In addition, we observe that the CcdBS sensitive phenotype dominates over the resistant phenotype. This is better explained by the conversion, in gyrA+/gyrA462 merodiploid strains, of the wild-type gyrase into a DNA-damaging agent. These results strongly suggest that the CcdB protein, like quinolone antibiotics and a variety of antitumoral drugs, is a DNA topoisomerase II poison. This is the first proteinic poison-antipoison mechanism that has been found to act via the DNA topoisomerase II.
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PMID:Cell killing by the F plasmid CcdB protein involves poisoning of DNA-topoisomerase II complexes. 132 24

The gene encoding Trypanosoma cruzi type II topoisomerase (TcTOP2) was isolated from a genomic library with a heterologous probe corresponding to part of the Trypanosoma brucei type II topoisomerase (TBrTOP2) gene. Nucleotide sequencing of TcTOP2 showed that the gene consists of an open reading frame of 3696 nucleotides (1232 amino acids), predicting a polypeptide product of 138,413 Da. Comparison of the amino acid sequence with that of type II topoisomerases from T. brucei (TBrTOP2) and Crithidia fasciculata (CfaTOP2), shows a high degree of conservation with estimated identities of 78% and 69%, respectively. TcTOP2 is a single copy gene in the genome of T. cruzi Dm28c and is expressed as a 4.5-kb mRNA. PCR mapping showed two distinct mini-exon addition sites at positions 225 and 203 upstream from the initiator AUG.
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PMID:Cloning and characterization of the gene encoding Trypanosoma cruzi DNA topoisomerase II. 133 85

Topoisomerases catalyse the interconversion of topological isomers of DNA and have key roles in nucleic acid metabolism. Human cells express two distinct type II topoisomerase isozymes, designated topoisomerase II alpha (170 kDa form) and topoisomerase II beta (180 kDa form). We have isolated cDNA clones encoding the beta isozyme from a human B-cell library. The proposed coding region for the topoisomerase II beta protein is 4,863 nucleotides long and would encode a polypeptide with a calculated M(r) of 182,705. The predicted topoisomerase II beta protein sequence shows striking similarity (72% identical residues) to that of the human alpha isozyme, and homology to topoisomerase II proteins from Drosophila, yeast and bacteria. Regions of greatest amino acid sequence divergence lie at the extreme N-terminus and over a C-terminal domain comprising approximately 25% of the total protein. We have quantified the level of topoisomerase II beta mRNA in a panel of human tumour cell lines of different origin using an RNase protection assay, and compared the level to that of topoisomerase II alpha mRNA. Topoisomerase II beta mRNA was expressed in haemopoietic, epithelial and fibroblast cell lines, although to different extents, with U937 cells (promonocytic leukaemia) showing a particularly high level. There was no obvious relationship in terms of level of expression between the topoisomerase II alpha and beta genes. We have localised the gene encoding topoisomerase II beta protein to chromosome 3p24 in the human genome.
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PMID:Isolation of cDNA clones encoding the beta isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. 133 83

The complete nucleotide sequence of a 3484-bp Sau3A fragment, previously shown to carry the replication origin of the Clostridium butyricum NCIB 7423 plasmid pCB101 (6.05 kb), has been determined. Of the four open reading frames (ORF A-D) identified within this fragment, two (B and C) were shown to be encoding by in vitro transcription/translation assays. Evidence was obtained that both polypeptides are required for autonomous replication of the plasmid in Bacillus subtilis. ORF C is immediately preceded by a small ORF (C') that encodes a relatively small polypeptide (50 amino acids) that demonstrates significant homology with RepA of plasmid pLS1. Whereas the ORF C polypeptide (27,100 Da) exhibits no homology to any known protein, that encoded by ORF B (RepB, 43,039 Da) exhibits significant homology with the Rep proteins of the pC194/pUB110 subfamily of single-strand (ss) DNA plasmids, which are widely distributed in gram-positive bacteria. Conserved amino acids include the presumed active site of topoisomerase activity and four cysteine residues in the N-terminus of all Rep proteins compared. The repB gene is preceded by a sequence motif exhibiting substantial homology to the "plus" origins of this family of ss DNA plasmids and was shown to act as a "hot spot" for deletion formation in certain plasmid chimaeras. The compelling suggestion that pCB101 replicates via a rolling circle mechanism was substantiated by the demonstration of ss DNA replication intermediates in B. subtilis cells carrying a pCB101-derived plasmid.
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PMID:Physical characterization of the replication origin of the cryptic plasmid pCB101 isolated from Clostridium butyricum NCIB 7423. 151 9

Monoclonal antibodies directed against four different polypeptide epitopes on the Mr approximately 94,000 steroid-binding subunit of the rat liver cytosolic glucocorticoid receptor (GcR) were used to probe Western blots of epididymal spermatozoa from rats and mice. Two sperm polypeptides with apparent molecular weights of 94,000 (indistinguishable in size from the liver GcR subunit) and 150,000 reacted with these antibodies. Other polypeptides that are present in a wide variety of somatic cells [lamin-A, -B, and -C; topoisomerase-I; poly(ADP-ribose) polymerase; the 62-kilodalton internal nuclear matrix protein; the nucleolar protein B23; and histone H1] could not be detected in these preparations of spermatozoa, thus appearing to rule out contamination by somatic cells. Rat and mouse pachytene spermatocytes and round spermatids contained much lower amounts of the Mr approximately 94,000 and 150,000 polypeptides. These results suggested that the steroid-binding subunit of the GcR might be accumulated late in spermatogenesis. Consistent with this view, a 6-kilobase mRNA (identical in size to a mRNA detected in mouse somatic cell lines) was detected when Northern blots of mouse round spermatid RNA were probed with a cDNA to the steroid-binding GcR subunit. Although the results described above suggest the presence of GcR in rodent sperm, high affinity binding of glucocorticoids to epididymal sperm could not be detected in a whole cell binding assay. Further analysis revealed that the Mr approximately 90,000 heat shock protein (hsp90), a component reportedly required for high affinity ligand binding to the GcR, was present in early germ cells, but absent from rodent epididymal sperm. These results suggest that the Mr approximately 94,000 steroid-binding subunit of the GcR and an immunologically related Mr approximately 150,000 polypeptide are specifically accumulated during the later stages of rodent spermatogenesis, but are not assembled into receptor complexes capable of binding steroid. In addition, these results support the view that hsp90 is required for high affinity binding of glucocorticoids to the Mr approximately 94,000 GcR subunit in intact cells.
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PMID:Evidence that rodent epididymal sperm contain the Mr approximately 94,000 glucocorticoid receptor but lack the Mr approximately 90,000 heat shock protein. 157 14

Western blotting, indirect immunolocalization, flow cytometry, and a functional assay for drug-induced strand breakage were utilized to examine topoisomerase (topo) II levels during granulocytic maturation in HL-60 human progranulocytic leukemia cells and in samples of normal human marrow. Indirect immunofluorescence revealed that the intensity of the signal for topo II in unsynchronized log phase HL-60 cells varied widely. Indirect immunolabeling combined with propidium iodide staining and two-parameter flow cytometry revealed that topo II levels increased an average of 2-fold as cells progressed from G1 to G2/M. When HL-60 cells were induced to mature toward granulocytes, topo II levels progressively decreased and became undetectable by functional assays, by indirect immunoperoxidase staining, and by Western blotting with an antibody which identified Mr 170,000 and Mr 180,000 forms of topo II. Similar changes were detected during normal granulocytic maturation in human marrow in vivo. Western blotting revealed that levels of the Mr 170,000 (proliferation-associated) isoform of topo II were highest in marrow fractions enriched in progranulocytes and myelocytes, intermediate in unfractionated marrow from normal volunteers, and undetectable in mature granulocytes. The Mr 180,000 topo II polypeptide was also diminished or absent from mature granulocytes. In further experiments, marrow samples from normal volunteers were subjected to flow cytometry after labeling of topo II and various cell surface markers. Levels of the Mr 170,000 topo II polypeptide in CD34-positive cells (multipotent and committed progenitors from several hematopoietic lineages) were indistinguishable from levels observed in the HL-60 leukemia cell line. These results suggest that topo II levels in highly proliferative normal human myeloid cells in vivo approach levels found in corresponding neoplastic cell lines in vitro. Conversely, as the same cells mature into granulocytes in vivo or in vitro, levels of both molecular weight forms of topo II diminish. These results provide a framework for the further investigation of topo II levels and drug sensitivity in human leukemia.
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PMID:Topoisomerase II levels during granulocytic maturation in vitro and in vivo. 164 69


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