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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)

The virD locus of Agrobacterium tumefaciens Ti plasmid encodes functions necessary for endonucleolytic cleavage of transferred DNA (T-DNA) prior to its transfer to plant cells. For the overproduction of the VIRD proteins in Escherichia coli a tac-virD operon fusion was constructed. A significant increase in the accumulation of VIRD proteins was observed in a lon protease-deficient E. coli host. The presence of an overlapping open reading frame (ORF) upstream of the VIRD1 coding sequence had a negative effect on VIRD1 production. A preparation containing VIRD proteins catalyzes the conversion of supercoiled (form I) DNA to relaxed (form IV) DNA. This activity is similar to that of a DNA topoisomerase. The relaxation activity lacks DNA sequence specificity, requires magnesium ion, and has no requirement for an energy source. Studies with plasmids that had lost defined DNA segments encompassing various virD coding regions showed that VIRD1 is the DNA-relaxing enzyme. In a density gradient centrifugation experiment, the DNA-relaxing activity sedimented as a 21-kDa polypeptide. Earlier studies of Jayaswal et al. [Jayaswal, R., Veluthambi, K., Gelvin, S. & Slightom, J. (1987) (J. Bacteriol. 169, 5035-5045] have shown that in E. coli VIRD2 alone is not sufficient for endonucleolytic cleavage of T-DNA and requires VIRD1 for its activity.
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PMID:The virD operon of Agrobacterium tumefaciens Ti plasmid encodes a DNA-relaxing enzyme. 254 31

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.
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PMID:Studies on DNA polymerases and topoisomerases in archaebacteria. 254 77

We have examined the level of incorporation of 32P into DNA topoisomerase II in vivo in chicken lymphoblastoid cells that were fractionated into the various cell cycle phases by centrifugal elutriation. We find that topoisomerase II is phosphorylated in vivo, with the level of incorporation being approximately 3.5-fold higher in the G2 + M fraction than earlier in the cell cycle. Our antibody studies have revealed that topoisomerase II antigen exists as a number of discrete polypeptide species in these cells. Of these, the 170-kDa intact polypeptide is phosphorylated approximately 4.5-fold more than several antigenic fragments that actually comprise the bulk of the topoisomerase II antigen in these cells at mitosis. Phosphorylation of the 170-kDa form of the enzyme may be involved in activation of the enzyme for its role in the disjunction of sister chromatids at anaphase.
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PMID:In vivo phosphorylation of the 170-kDa form of eukaryotic DNA topoisomerase II. Cell cycle analysis. 254 53

The DNA cleavage reaction of eukaryotic topoisomerase II produces nicked DNA along with linear nucleic acid products. Therefore, relationships between the enzyme's DNA nicking and double-stranded cleavage reactions were determined. This was accomplished by altering the pH at which assays were performed. At pH 5.0 Drosophila melanogaster topoisomerase II generated predominantly (greater than 90%) single-stranded breaks in duplex DNA. With increasing pH, less single-stranded and more double-stranded cleavage was observed, regardless of the buffer or the divalent cation employed. As has been shown for double-stranded DNA cleavage, topoisomerase II was covalently bound to nicked DNA products, and enzyme-mediated single-stranded cleavage was salt reversible. Moreover, sites of single-stranded DNA breaks were identical with those mapped for double-stranded breaks. To further characterize the enzyme's cleavage mechanism, electron microscopy studies were performed. These experiments revealed that separate polypeptide chains were complexed with both ends of linear DNA molecules generated during cleavage reactions. Finally, by use of a novel religation assay [Osheroff, N., & Zechiedrich, E. L. (1987) Biochemistry 26, 4303-4309], it was shown that nicked DNA is an obligatory kinetic intermediate in the topoisomerase II mediated reunion of double-stranded breaks. Under the conditions employed, the apparent first-order rate constant for the religation of the first break was approximately 6-fold faster than that for the religation of the second break. The above results indicate that topoisomerase II carries out double-stranded DNA cleavage/religation by making two sequential single-stranded breaks in the nucleic acid backbone, each of which is mediated by a separate subunit of the homodimeric enzyme.
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PMID:Double-stranded DNA cleavage/religation reaction of eukaryotic topoisomerase II: evidence for a nicked DNA intermediate. 255 67

Escherichia coli contains two type 1 topoisomerases, topoisomerase I and III. Although topoisomerase III can be purified as a potent decatenase, its role in DNA metabolism is unclear. In order to address this issue, the gene encoding topoisomerase III from E. coli has been molecularly cloned and its DNA sequence determined. The cloned fragment of DNA contains an open reading frame that can encode a polypeptide of 73.2 kDa. The first 20 amino acids of this open reading frame are identical to those of topoisomerase III as determined by amino-terminal gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame, using a bacteriophage T7 transient expression system, results in the accumulation of a 74-kDa polypeptide. Soluble extracts prepared from cells overexpressing this gene product show a dramatic increase in topoisomerase activity when compared with control extracts. We propose that this gene be designated topB.
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PMID:Molecular cloning and DNA sequence analysis of Escherichia coli topB, the gene encoding topoisomerase III. 255 98

Immunoscreening of the human placenta cDNA-library in the expression vector lambda gt11 using non-isotope detection based on the avidin-biotin system allowed to identify a number of clones encoding human topoisomerase I. The fusion protein from an extract of Escherichia coli cells infected with the recombinant phage lambda gt11 interacts with the monoclonal antibody raised against topoisomerase I from calf thymus; the dissociation constant being 5.7.10(-8) M. The restricted DNA fragments coding for the topoisomerase polypeptide in the composition of the fusion protein were recloned, and expression in the pEX vector was obtained. The functional analysis of the expression products has enabled localization of the epitope of binding the monoclonal antibody. It was demonstrated that the identified fusion protein can be applied for diagnosis of autoimmune diseases.
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PMID:[Topoisomerase I from human placenta. Functional activity of products of expression of cloned cDNA fragments]. 256 Nov 76

Vaccinia virus encapsidates a type I DNA topoisomerase (EC 5.99.1.2). The enzyme was purified from virus cores to apparent homogeneity, yielding a protein of Mr 32,000. The amino-terminal sequence of the isolated Mr 32,000 polypeptide was determined and used to map the putative structural gene for the vaccinia topoisomerase to the H7r open reading frame of the vaccinia genome. This gene encodes a 314-amino acid polypeptide containing a region homologous to a region of the type I topoisomerase from the yeast Saccharomyces cerevisiae.
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PMID:Identification of a vaccinia virus gene encoding a type I DNA topoisomerase. 282 64

We have recently shown that the aggregation factor (AF) from the sponge Geodia cydonium stimulates DNA synthesis in quiescent, dissociated cells from the same organism; this event was correlated with the release of the two second messengers: inositol trisphosphate and diacylglycerol. Here we describe that after binding of the AF to the plasma membrane-bound aggregation receptor, a rapid and drastic increase in the incorporation of 32Pi into a series of proteins in the pore complex-lamina fraction occurs. Addition of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, to quiescent cells resulted in a similar stimulation of phosphorylation of nuclear proteins. Among them we have selected one protein with a polypeptide Mr of 170,000 (pp170) for detailed studies. By immunoblotting pp170 was identified as DNA topoisomerase II. In vitro studies with nuclei and purified, homogeneous protein kinase C together with the required activators of this enzyme also showed a phosphorylation of pp170. After phosphorylation, DNA topoisomerase II activity was found to be 2.5-fold that of the non-phosphorylated enzyme. From these data we conclude that protein kinase C is involved in AF induced transmembrane signalling, ultimately leading to an initiation of DNA synthesis.
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PMID:Specific phosphorylation of proteins in pore complex-laminae from the sponge Geodia cydonium by the homologous aggregation factor and phorbol ester. Role of protein kinase C in the phosphorylation of DNA topoisomerase II. 283 45

A topoisomerase capable of introducing positive supercoils into closed-circular DNA has been isolated from the extremely thermophilic anaerobic archaebacterium Desulfurococcus amylolyticus. This polypeptide has an Mr of 135,000, as determined by electrophoresis under denaturing conditions. The enzyme is active in the temperature range from 65 degrees C to 100 degrees C and catalyzes positive supercoiling both in negatively supercoiled DNA and in relaxed DNA. These reactions require the presence of ATP. The enzyme's action on a single topoisomer has shown the linking number to increase by an integral number upon the relaxation of negative supercoils and the introduction of positive ones. This means that the reverse gyrase from D. amylolyticus is a type I topoisomerase. The presence of an extended AT sequence within the closed-circular DNA enhances the activity of the Desulfurococcus topoisomerase. Even though the enzyme is isolated from a strictly anaerobic bacterium, it is fully active in the presence of oxygen.
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PMID:Positive supercoiling catalysed in vitro by ATP-dependent topoisomerase from Desulfurococcus amylolyticus. 283 7

We developed monoclonal antibodies against Drosophila topoisomerase II and studied the intracellular forms and the in vivo and in vitro proteolytic degradation of the enzyme. In purified enzyme preparations polyclonal sera and monoclonal antibodies recognized several polypeptides in the 170-132 kD molecular weight range. In vivo, however, the pattern was much simpler. In Drosophila embryos, pupae, fly heads and Schneider S3 tissue culture cells topoisomerase II appeared as a single 166 kD polypeptide. In Drosophila embryos, with two monoclonal antibodies topoisomerase II appeared as a doublet composed of the 166 kD canonical form and a slightly higher molecular weight polypeptide. Topoisomerase II was shown to be present also in fly heads which are composed entirely of nonproliferative tissues.
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PMID:Intracellular forms of Drosophila topoisomerase II detected with monoclonal antibodies. 284 16


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