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)

DNA topoisomerases which remove superhelical turns in closed circular DNA have been isolated from cauliflower inflorescences using polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex or CM-cellulose and DNA-cellulose. Two distinct enzymes, topoisomerase-I and ATP-dependent topoisomerase, were separated clearly by CM-Sephadex or CM-cellulose, and partially characterized using agarose gel electrophoresis with plasmid pBR322 DNA. Topoisomerase-I acts like other eucaryotic DNA topoisomerases in the absence of ATP, is stimulated by spermidine and inhibited by EDTA. The ATP-dependent topoisomerase acts like topoisomerase-I only in the presence of ATP in the reaction medium, is inhibited by spermidine and EDTA, and does not introduce supertwists into closed duplex DNA or produce catenate aggregates under the present reaction conditions.
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PMID:Isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescence. 628 25

We have purified a deoxyribonucleic acid topoisomerase to near homogeneity from the nuclei of mature chicken erythrocytes. The enzyme relaxes supercoiled DNA in the absence of ATP or Mg2+. It is unable to resolve topologically knotted circular duplex DNA. These properties resemble those of type I eukaryotic topoisomerases capable of breaking and rejoining one strand of duplex DNA at a time. The sedimentation value of the protein is 4.4 S. The molecular weight of the reduced, denatured protein is 100K. After elution from sodium dodecyl sulfate (NaDodSO4) gels and renaturation, topoisomerase activity is found in the band at 100K and in minor bands at 95K, 78K, and 73K. The minor bands are likely to be proteolytic fragments since the Mr 100K protein is cleaved by trypsin to fragments of similar or even smaller size with retention of activity. At KCl concentrations suboptimal for the 100K form, the trypsin cleaved form is severalfold more active than the 100K form. Single-stranded DNA, but not duplex DNA or RNA, inhibits DNA relaxing activity, presumably by forming a covalent complex at the enzyme active site. Preincubation of the enzyme with single-stranded DNA leads to the depletion, in NaDodSO4-polyacrylamide gels, of protein bands corresponding to the 100K topoisomerase, its putative proteolytic fragments, and its tryptic fragments. The reaction which leads to band depletion requires active topoisomerase and conditions where single-stranded DNA inhibits relaxing activity. The band depletion technique provides a convenient assay for the polynucleotide binding activity of topoisomerases and possibly other proteins. The function of the enzyme in the inactive nuclei of mature chicken erythrocytes is unclear. The estimated content of chicken erythrocyte topoisomerase per unit DNA is comparable to that in nuclei active in replication and transcription.
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PMID:Topoisomerase I from chicken erythrocytes: purification, characterization, and detection by a deoxyribonucleic acid binding assay. 630 1

A type I topoisomerase has been purified from avian erythrocyte nuclei. The most pure fraction contains one major polypeptide of Mr = 105,000 (80% of total) and several minor ones of lower molecular weight. Active forms of the topoisomerase were identified by covalently binding the enzyme to 32P-DNA, digesting with nuclease and detecting 32P labeled peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Topoisomerase activity, as measured by the ability to covalently bind DNA, is associated with the following peptides: Mr = 105, 83, 54 and 30,000. The similar chromatographic properties of the various forms of topoisomerase suggests a common structural identity as previously proposed for the HeLa topoisomerase I (Liu, L.F. and Miller, K.G. (1981) Proc. Natl. Acad. Sci. USA 78, 3487-3491). The avian enzyme is similar to other eucaryotic type I DNA topoisomerases in that it covalently binds double and single stranded DNA forming an enzyme linked to the 3'-phosphoryl end and after binding to single stranded DNA it can transfer the single stranded donor DNA to an acceptor DNA possessing 5'-OH end groups. The binding site size of topoisomerase on DNA has also been determined using micrococcal nuclease to digest unprotected DNA in the native enzyme/DNA complex. The enzyme blocks access to the helix over a span of 25 bp. These findings are discussed in light of the distribution and function of topoisomerase I in chromatin.
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PMID:Biochemical characterization of topoisomerase I purified from avian erythrocytes. 630 57

A type II DNA topoisomerase has been purified from the nuclei of Drosophila melanogaster 6- to 18-h-old embryos. The enzyme, as assayed by its ability to catenate supercoiled DNA, behaved as a single homogeneous species throughout the procedure and the yield was approximately 0.5 mg of protein/100 g of dechorionated embryos. The final product was entirely ATP-dependent and free of topoisomerase I, endonuclease and protease activities. The purified topoisomerase II had a Stokes radius of 69 A and a sedimentation coefficient (S20,w) of 9.2 S, leading to a calculated native molecular weight of approximately 261,000. The protein consists of a single polypeptide of molecular weight 166,000, as determined by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Taken together with the above hydrodynamic studies, the Drosophila enzyme is probably a homodimer, as has been observed for other eukaryotic type II enzymes. Thus, it appears that during the course of evolution the heterologous subunits which comprise bacterial type II topoisomerases have been combined into a single polypeptide chain in eukaryotes.
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PMID:DNA topoisomerase II from Drosophila melanogaster. Purification and physical characterization. 630 10

A type I DNA topoisomerase has been isolated from the nuclei of the flagellate Trypanosoma cruzi, using poly(ethylene glycol) fractionation and chromatography on hydroxyapatite and on phosphocellulose. The relaxation activity was ATP-independent, enhanced by Mg2+ and spermidine. The enzyme removed supercoils from negative and positive superhelical DNAs. Topoisomerase activity was associated with a polypeptide of Mr about 65000 as shown by glycerol gradient centrifugation and by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels.
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PMID:A type I DNA topoisomerase from Trypanosoma cruzi. 630 14

We have previously shown that a DNA topoisomerase I from mouse mammary carcinoma cells is inhibited by heparin. Taking advantage of this enzyme-heparin interaction, we developed a rapid and efficient method of purification of this enzyme to near homogeneity by extraction of chromatin with 0.15 M phosphate buffer followed by two-step column chromatography on heparin-Sepharose and phenyl-Sepharose. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels revealed that the final preparation is composed of two polypeptides with apparent Mr approximately 98,000 (p98) and 102,000 (p102), p98 comprising 70% and p102 30%. Extraction and renaturation of the polypeptides from the gel shows that both p98 and p102 seem to possess topoisomerase activity. Partial proteolytic digestion of p98 and p102 with Staphylococcus aureus V8 and chymotrypsin yielded a series of identical peptides, indicating that the two polypeptides are structurally related. The enzyme sedimented through sucrose density gradient with s20,w of 4.0 S, and thus is monomeric in solution.
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PMID:Rapid purification and characterization of DNA topoisomerase I from cultured mouse mammary carcinoma FM3A cells. 631 74

RecA protein, which is essential for genetic recombination in Escherichia coli, was extensively purified from a strain of E. coli which contained the recA gene cloned in a plasmid (Sancar, A., and Rupp, W. D. (1979) Proc. Natl. Acad. Sci. U. S. A. 76, 3144-3148). Using the DNA-dependent ATPase activity of recA protein as an assay, we obtained about 60 mg of purified recA protein from 100 g of cells. Ten micrograms or 1 microgram of the purified protein exhibited only one detectable band with Mr approximately = 40,000 upon sodium dodecyl sulfate-acrylamide gel electrophoresis. More than 99% of the ATPase activity of purified recA protein was dependent on single-stranded DNA. Purified recA protein had no detectable DNase, topoisomerase, or ligase activities. The enzyme was stable for a least a year when stored at 0-4 degrees C. The half-life of the ATPase activity of 25 microM recA protein was 37 min at 51 degrees C. Purified recA protein binds to single-stranded and double-stranded DNA, unwinds duplex DNA by a mechanism that is stimulated by single-stranded DNA or oligonucleotides, and pairs homologous single strands with duplex DNA.
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PMID:Homologous pairing in genetic recombination. Purification and characterization of Escherichia coli recA protein. 645 91

We have characterized a temperature-sensitive mutant of vaccinia virus, ts16, originally isolated by Condit et al. (Virology 128:429-443, 1983), at the permissive and nonpermissive temperatures. In a previous study by Kane and Shuman (J. Virol 67:2689-2698, 1993), the mutation of ts16 was mapped to the I7 gene, encoding a 47-kDa protein that shows partial homology to the type II topoisomerase of Saccharomyces cerevisiae. The present study extends previous electron microscopy analysis, showing that in BSC40 cells infected with ts16 at the restrictive temperature (40 degrees C), the assembly was arrested at a stage between the spherical immature virus and the intracellular mature virus (IMV). In thawed cryosections, a number of the major proteins normally found in the IMV were subsequently localized to these mutant particles. By using sucrose density gradients, the ts16 particles were purified from cells infected at the permissive and nonpermissive temperatures. These were analyzed by immunogold labelling and negative-staining electron microscopy, and their protein composition was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While the ts16 virus particles made at the permissive temperature appeared to have a protein pattern identical to that of wild-type IMV, in the mutant particles the three core proteins, p4a, p4b, and 28K, were not proteolytically processed. Consistent with previous data the sucrose-purified particles could be labelled with [3H]thymidine. In addition, anti-DNA labelling on thawed cryosections suggested that most of the mutant particles had taken up DNA. On thawed cryosections of cells infected at the permissive temperature, antibodies to I7 labelled the virus factories, the immature viruses, and the IMVs, while under restrictive conditions these structures were labelled much less, if at all. Surprisingly, however, by Western blotting (immunoblotting) the I7 protein was present in similar amounts in the defective particles and in the IMVs isolated at the permissive temperature. Finally, our data suggest that at the nonpermissive temperature the assembly of ts16 is irreversibly arrested in a stage at which the DNA is in the process of entering but before the particle has completely sealed, as monitored by protease experiments.
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PMID:Characterization of ts 16, a temperature-sensitive mutant of vaccinia virus. 747 27

V511 and V513 are Chinese hamster cell lines with acquired resistance to topoisomerase II (topo II) directed agents. These cell lines were obtained by mutagenizing Chinese hamster V79 cells with N-methyl-N'-nitro-N-nitrosoguanidine and subsequently selecting in etoposide (VP-16). We have previously shown that this resistance is not associated with alterations in drug uptake. To elucidate whether any alterations in the functionally important domains of topo II alpha were associated with VP-16 resistance, we used reverse transcriptase-polymerase chain reaction, single-strand conformational polymorphism analysis, and subsequent sequencing of topo II alpha from V79, V511, and V513 to search for mutations in five major functional domains including the regions of the consensus ATP binding sequences (Motif A and Motif B/dinucleotide binding site), the DNA binding domain, and the 5' and 3' flanking regions of the DNA binding position. The V511 cells showed no mutational changes in these regions. However, the topo II alpha gene from V513 showed a point mutation at nucleotide 2552 that resulted in a glycine-to-aspartate mutation at amino acid position 851 in the 3' flanking region of the DNA binding site. This mutation at amino acid position 851 in V513 cells is associated with reduced VP-16-induced cleavable complex formation demonstrated by potassium-sodium dodecyl sulfate assay and band-depletion analysis. Our results suggest that the mutation at amino acid position 851 may play a role in drug resistance, presumably by interfering with enzyme-DNA binding.
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PMID:A novel point mutation in the 3' flanking region of the DNA-binding domain of topoisomerase II alpha associated with acquired resistance to topoisomerase II active agents. 754 41

Several clinically important drugs utilized in cancer chemotherapy inhibit type I (Topotecan) or type II (amsacrine, etoposide) DNA topoisomerases by stabilizing the formation of DNA-topoisomerase complexes (topoisomerase-DNA cross-links). In various cell lines, the magnitude of drug-induced DNA-protein cross-link production correlates with the magnitude of cytotoxicity induced by the drugs. We developed a simple filter-binding assay that can measure drug-induced DNA-protein cross-links in leukemia cells obtained directly from patients because the assays most widely used for assessment of drug-induced DNA-protein cross-links in cells [sodium dodecyl sulfate (SDS)/KCl precipitation and alkaline elution] are not readily applicable for use on patient material. HL-60 human leukemia cells or freshly isolated patients' leukemia cells were incubated with Topotecan, etoposide, or amsacrine; lysed with SDS; and applied to nitrocellulose filters in a low-salt buffer. DNA is retained on the filter only if it is covalently bound to protein. The amount of DNA retained on the filter is quantified by hybridization to the alu sequence of DNA, which is distributed ubiquitously in the human genome. Using radiolabeled cells, we compared the filter-binding assay directly with the SDS/KCl precipitation assay in the detection of etoposide- or amsacrine-induced DNA-protein cross-links in HL-60 cells and amsacrine-resistant HL-60/AMSA cells. Both the SDS/KCl precipitation assay and the filter-binding assay detected etoposide-induced DNA-protein cross-links in HL-60 and HL-60/AMSA cells and detected a greater frequency of amsacrine-induced DNA-protein cross-links in HL-60 cells than in HL-60/AMSA cells. The filter-binding assay detected DNA-protein cross-links in freshly isolated leukemia cells exposed to Topotecan in vitro. The ratios of DNA retention for Topotecan-treated versus untreated cells from leukemia patients ranged from 1.8 to 11.5. The heterogeneity of this detected cross-linking was as might be expected if the assay were predictive of the antileukemic action of Topotecan, which is variable. This new filter-binding technique may be useful for predicting the sensitivity of individual patients' tumors to drugs that inhibit type I or type II DNA topoisomerases.
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PMID:Quantification of topoisomerase-DNA complexes in leukemia cells from patients undergoing therapy with a topoisomerase-directed agent. 800 59


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