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 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

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

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K1 values of 10-25 microM for coumermycin and 350 microM for novobiocin. Spectroscopic and fluorescence measurements of drug binding t enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (KD = 27 +/- 5 microM) and two classes of binding sites for novobiocin, one tight site (KD1 = 20 +/- 5 microM) and several weak sites (KD2 = 513 +/- 125 microM; n = 4.9 +/- 0.7). Addition of a stoichiometric amount of DNA to a performed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.
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PMID:Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin. 856 95

Nuclei were treated with VM-26, topoisomerase II inhibitor, or exogenous proteases. Initial stages of multi-step chromatin degradation were analyzed by pulsed-field gel (PFG) electrophoresis. VM-26 induced the total genome breakdown into discrete chromatin fragments containing 0.3 Mbp DNA. Treatment of nuclei with recombinant cathepsin B and chymotrypsin also revealed 0.3-Mbp DNA fragments which were mediated by endonucleolysis. Longer incubation of nuclei with chymotrypsin exhibited the appearance of 0.05-Mbp DNA fragments and their oligomers. Proteases might trigger the detachment of chromatin from nuclear matrix and contribute to the release or activation of the endonucleolytic activity leading to the initial degradation of the DNA into high-molecular weight fragments. The fragments observed correspond in size to the suggested chromatin higher-order structures. The presented data imply that the initial stages of chromatin degradation represents in reality the genome disassembly into two basic units of genome topology--0.3-Mbp DNA chromatin loop-domains which are presumably the bits of biological information, and fundamental 0.05-Mbp DNA loopsize units which might generate the functional loops of different sizes.
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PMID:Disassembly of genome of higher eukaryotes: pulsed-field gel electrophoretic study of initial stages of chromatin and DNA degradation in rat liver and thymus nuclei by VM-26 and selected proteases. 997 90