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)

We have purified a topoisomerase activity from broccoli (Brassica oleracea var. italica) to near homogeneity. The enzyme is an 80 kDa monomer as judged by gel filtration chromatography and SDS gel electrophoresis, though it may represent a proteolytic fragment of a larger protein. The enzyme is capable of removing both negative and positive supercoils in steps of one, does not absolutely require Mg2+, is only very weakly stimulated by NaCl, is inhibited by camptothecin, and cross-reacts with an antibody directed against human DNA topoisomerase I. These properties identify the enzyme as a eukaryotic type I topoisomerase.
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PMID:Purification and properties of DNA topoisomerase I from broccoli. 131 91

Anti-tumor drug VM26 greatly stimulates topoisomerase II mediated DNA cleavage by stabilizing the cleavable complex. Addition of a strong detergent such as SDS to the cleavable complex induces the double stranded DNA cleavage. We demonstrate here that heat treatment can reverse the double stranded DNA cleavage; however, topoisomerase II remains bound to DNA even in the presence of SDS. This reversed complex has been shown to contain single strand DNA breaks with topoisomerase II covalently linked to the nicked DNA. Chelation of Mg++ by EDTA and the addition of salt to a high concentration also reverse the double strand DNA cleavage, and like heat reversion, topoisomerase II remains bound to DNA through single strand DNA break. The reversion complex can be analyzed and isolated by CsCl density gradient centrifugation. We have detected multiple discrete bands from such a gradient, corresponding to protein/DNA complexes with 1, 2, 3, ..... topoisomerase II molecules bound per DNA molecule. Analysis of topoisomerase II/DNA complexes isolated from the CsCl gradient indicates that there are single stranded DNA breaks associated with the CsCl stable complexes. Therefore, topoisomerase II/DNA complex formed in the presence of VM26 cannot be completely reversed to yield free DNA and enzyme. We discuss the possible significance of this finding to the mechanism of action of VM26 in the topoisomerase II reactions.
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PMID:Incomplete reversion of double stranded DNA cleavage mediated by Drosophila topoisomerase II: formation of single stranded DNA cleavage complex in the presence of an anti-tumor drug VM26. 132 36

Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).
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PMID:DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes. 133 86

Relaxation of superhelical DNA by Escherichia coli topoisomerase III (Topo III) was inhibited by the inclusion of tRNA in the reaction mixture. Investigation of the basis of this inhibition revealed that Topo III could bind RNA and establish a cleavage-religation equilibrium. The addition of SDS to these reaction mixtures induced cleavage of the RNA by Topo III. The nucleotide sequences of RNA and DNA cleavage sites were identical, although cleavage site preference differed. Thus, the possibility that Topo III can pass strands of RNA as well as strands of DNA must be considered in accounting for the role of this enzyme in nucleic acid metabolism.
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PMID:Escherichia coli topoisomerase III-catalyzed cleavage of RNA. 138 3

Mitoxantrone-resistant variants of the human HL-60 leukemia cell line are cross-resistant to several natural product and synthetic antineoplastic agents. The resistant cells (HL-60/MX2) retain sensitivity to the Vinca alkaloids vincristine and vinblastine, drugs that are typically associated with the classical multidrug resistance phenotype. Mitoxantrone accumulation and retention are equivalent in the sensitive and resistant cell types, suggesting that mitoxantrone resistance in HL-60/MX2 cells might be associated with an alteration in the type II DNA topoisomerases. We discovered that topoisomerase II catalytic activity in 1.0 M NaCl nuclear extracts from the HL-60/MX2 variant, as measured by the decatenation of Crithidia fasciculata kinetoplast DNA, was reduced 4- to 5-fold compared to that in the parental HL-60 cells. Total cellular topoisomerase II activity in HL-60/MX2 cells was only 50% lower than that in HL-60 cells, however, because the "cytosolic fraction" of the HL-60/MX2 nuclear preparation contained high levels of decatenating activity. Antisera to calf thymus topoisomerase II defined a distinctive immunoreactive pattern of topoisomerase II proteins in crude nuclear extracts from the HL-60/MX2 cells. Both alpha (170 kDa) and beta (180 kDa) forms of topoisomerase II were detected in the HL-60 cell extracts, but only the alpha form was detected in extracts from HL-60/MX2 cells. This finding was associated with the appearance of a new 160-kDa immunoreactive species in nuclear extracts from HL-60/MX2 but not HL-60 cells. Studies were designed to minimize the proteolytic degradation of the topoisomerase II enzymes by extraction of whole cells with hot SDS.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Mitoxantrone resistance in HL-60 leukemia cells: reduced nuclear topoisomerase II catalytic activity and drug-induced DNA cleavage in association with reduced expression of the topoisomerase II beta isoform. 165 25

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1

Previous studies have resulted in conflicting data regarding the recovery of the nuclear enzymes topoisomerase (topo) II and topo I in the nuclear matrix fraction. In the present study we have assessed the effect of systematically altering a single extraction procedure on the distribution of these enzymes during the subfractionation of nuclei from HTC hepatoma tissue culture cells. When nuclear monolayers (prepared by treating attached cells in situ with the neutral detergent Nonidet-P40 at 4 degrees C) were isolated in the presence of the irreversible sulfhydryl blocking reagent iodoacetamide, subsequent treatment with DNase I and RNase A followed by 1.6 M NaCl resulted in structures which were extensively depleted of intranuclear components as assessed by phase contrast microscopy and conventional transmission electron microscopy. These structures contained 12 +/- 4% of the total protein present in the original nuclear monolayers. The lamins and polypeptides with molecular weights comparable to those of actin and vimentin were the predominant polypeptides present on SDS-polyacrylamide gels. Western blotting revealed that less than 5% of the total nuclear topo II molecules were present in these structures. In contrast, when the sulfhydryl cross-linking reagent sodium tetrathionate (NaTT) was substituted for iodoacetamide, the same extraction procedure yielded structures containing components of the nucleolus and an extensive intranuclear network. These structures contained a wide variety of nonlamin, nonhistone nuclear polypeptides including 23 +/- 4% of the total nuclear topo II. SDS-polyacrylamide gel electrophoresis performed under nonreducing conditions revealed that topo II in these nuclear matrices was present as part of a large disulfide cross-linked complex. Treatment of these structures with reducing agents in 1.6 M NaCl released the topo II. In contrast, topo I did not form disulfide cross-linked oligomers and was not detectable in any of these nuclease- and salt-resistant structures prepared at 4 degrees C. To assess the effect of in vitro heat treatment on the distribution of the topoisomerases, nuclear monolayers (isolated in the absence of iodoacetamide and NaTT) were heated to 37 degrees C for 1 h prior to treatment with nucleases and 1.6 M NaCl. The resulting structures (which retained 26 +/- 5% of the total nuclear protein) were morphologically similar to the NaTT-stabilized nuclear matrices and contained 15 +/- 4% of the total nuclear topo II. High-molecular-weight disulfide cross-linked oligomers of topo II were again demonstrated. Attempts to demonstrate these disulfide cross-linked oligomers in intact cells were unsuccessful.
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PMID:Association of topoisomerase II with the hepatoma cell nuclear matrix: the role of intermolecular disulfide bond formation. 184 38

Novobiocin (NB) at the concentration of 2 mmol/l added to the culture medium together with mitoxantrone (MIT) (0.05-0.2 micrograms/ml) reduced the number of MIT-induced single-strand breaks of DNA to approximately one half measured by alkaline DNA unwinding and hydroxyapatite chromatography of DNA and similarly it reduced also the fraction of DNA linked to proteins measured by the K(+) -SDS precipitation method. Neither repair of the induced DNA breaks nor removal of the DNA-protein cross-links were markedly influenced by NB action. The specific inhibitor of topoisomerase II, fostriecin, exerted no effect on the induction of DNA breaks by MIT or their repair. Measurement of intracellular concentration of MIT has revealed that in the presence of NB the uptake of MIT into cells is reduced similarly as the number of induced DNA breaks to approximately one half. The combination of 0.1 mmol araC + 10 mmol HU slightly reduced the number of induced DNA breaks, but did not affect their repair. The present results suggest that (1) MIT induces DNA damage which is not repaired by excision repair, (2) MIT induces protein associated breaks of DNA, (3) topoisomerase II does not probably participate in the formation of DNA breaks induced by MIT, as the specific inhibitor of topoisomerase II, fostriecin exerts no effect on either the induction or repair of these breaks.
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PMID:The influence of DNA-topoisomerase II inhibitors novobiocin and fostriecin on the induction and repair of DNA damage in Chinese hamster ovary (CHO) cells treated with mitoxantrone. 184 36

An ATP-dependent DNA aggregating activity was purified from rat liver by DEAE-cellulose, phosphocellulose, and novobiocin-Sepharose column chromatography. The protein aggregated superhelical, relaxed, single-, or double-stranded DNA in a divalent cation- and ATP-dependent reaction. The DNA aggregating activity was detected by retardation of a DNA-protein complex at the origin on a 1% agarose gel. The protein appeared to exist in solution as a monomer of molecular weight 66,000, and had no DNA polymerase, topoisomerase, recombinase, or ligase activity. The DNA aggregating activity was inhibited by 10 mM nalidixic acid or 1 mM novobiocin but not by 20 mM N-ethylmaleimide or camptothecin. Adenylyl(beta,gamma-methylene)-diphosphonate, adenylyl-imidodiphosphate, or adenosine-5'-O(3-thiotriphosphate) did not substitute for ATP whereas CTP, dTTP, or the ATP analog adenylyl(alpha,beta-methylene)-diphosphonate could replace ATP. The aggregated DNA was only partially dissociated by restriction endonuclease digestion but was completely dissociated by deproteinization with SDS, proteinase K, or chloroform/octanol extraction. On the basis of the molecular weight, thermostability, antigenic property, and amino acid sequence homology in the first 12 positions, we conclude that the rat liver protein is serum albumin and that the ATP-dependent DNA aggregation is a novel function of rat serum albumin.
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PMID:ATP-dependent DNA aggregation is a novel function of rat serum albumin. 189 9

We have previously shown that a cloned 480 bp DNA fragment that spans the 3'-enhancer region of the avian beta-globin gene cluster can become very tightly, perhaps covalently, bound to protein in avian nuclear matrices in vitro [Zenk et al. (1990) Biochemistry 29, 5221-5226]. This binding was not tissue-specific and was probably not mediated by topoisomerase enzymes. In the present study, we have examined avian nuclear matrices (or scaffolds) for the presence of very tight cellular DNA-protein complexes in the region of the beta-globin gene enhancer and of several other avian genes. Nuclear matrices were prepared by both high- and low-salt methods, and protein-DNA complexes were isolated by SDS/K+ precipitation after restriction enzyme digestion. In adult reticulocytes, up to 30% of the intact 3800 bp HindIII-EcoRI fragment that encompasses the beta-globin enhancer element may be very tightly bound to nuclear matrix protein. In adult avian thymus nuclei, the beta-globin enhancer is neither matrix-associated nor tightly bound to protein. In contrast, a 5.0-kb HindIII fragment of the malic enzyme gene is very tightly bound to nuclear matrix-associated protein in thymus cells, but not reticulocytes. The malic enzyme gene is active in thymus cells, and not in reticulocytes. These results suggests that certain regions of cellular DNA are very tightly, perhaps covalently, attached to nuclear matrix-associated proteins. Attachment follows a tissue-specific pattern that is associated with transcriptional activity.
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PMID:Avian nuclear matrix proteins bind very tightly to cellular DNA of the beta-globin gene enhancer in a tissue-specific fashion. 204 26


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