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

---

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.
...
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.
...
PMID:DNA-relaxing enzyme from Micrococcus luteus. 20 27

Under some conditions, T4 DNA replication requires the products of the DNA-delay genes, genes 39, 52, 58, and 60. By using an in vitro complementation assay that stimulates DNA replication in T4 39(-)-infected cell extracts, T4 gene 39 protein has been purified. The purified fraction also contains complementing activities for T4 genes 52 and 60. On sodium dodecyl sulfate/polyacrylamide gel analysis the purified preparation exhibits three protein components: a 51,000-dalton protein corresponding to the product of gene 52, a 64,000-dalton protein corresponding to the product of gene 39, and a 110,000-dalton protein. This purified fraction shows a DNA topoisomerase activity that untwists superhelical DNA in an ATP- and Mg2+-dependent reaction. The analogs adenylyl imidodiphosphate and adenyl [beta, gamma-methylene]diphosphonate cannot be used to replace ATP. The topoisomerase activity is not sensitive to the antibiotics oxolinic acid and novobiocin, known antagonists of Escherichia coli DNA gyrase. The possible relationship among the three polypeptides and their biological activities is discussed.
...
PMID:T4 DNA-delay proteins, required for specific DNA replication, form a complex that has ATP-dependent DNA topoisomerase activity. 22 76

Candida albicans is an opportunistic pathogen responsible for life-threatening infections in persons with impaired immune systems. Topoisomerase I is a potential target for novel antifungal agents; however, in order for this enzyme to be a therapeutically useful target, it needs to be demonstrated that the fungal and human topoisomerases differ sufficiently as to allow the fungal topoisomerase to be selectively targeted. To address this question, we isolated the topoisomerase I from C. albicans and compared its biochemical properties with those of the mammalian enzyme. Similar to other eukaryotic type I topoisomerases, the C. albicans type I topoisomerase has an apparent molecular mass of 102 kDa and covalently links to the 3' end of DNA, as shown after the reaction is interrupted by sodium dodecyl sulfate. Topoisomerase poisons such as camptothecin act by stabilizing the cleavage complex formed by the topoisomerase I and DNA. We observed that the C. albicans and mammalian type I topoisomerases differ in that the C. albicans cleavage complex is approximately 10-fold less sensitive to camptothecin than the mammalian cleavage complex is. In addition, we found that the antifungal agent eupolauridine can stabilize the cleavage complex formed by both the C. albicans and human topoisomerases and that the response of the C. albicans topoisomerase I to this drug is greater than that of the human enzyme. Thus, the topoisomerase I from C. albicans is sufficiently distinct from the human enzyme as to allow differential chemical targeting and will therefore make a good target for antifungal drug discovery.
...
PMID:Characterization of DNA topoisomerase I from Candida albicans as a target for drug discovery. 133 88

The object of this study was to devise a purification method for DNA/topoisomerase II complexes, with which to examine the enzyme's cleavage site specificity in cellular differentiation. Retinoic acid-induced differentiation involves topoisomerase II-mediated transient changes in DNA supercoiling, but it is not known whether this occurs at specific sites in the genome. Topoisomerase II forms a covalent DNA enzyme complex as it acts, which can be recovered by the sodium dodecyl sulfate (SDS)/KCl precipitation method, but this method fails to recover significantly more DNA from cells induced to differentiate. This may in part reflect the low numbers of retinoic acid-induced protein-linked breaks in DNA and also the method's relative inefficiency for DNA with few attached topoisomerase molecules. This suggested that an additional purification method would be required to enrich sufficiently for cleavage site DNA to address the issue of site specificity. The principle of our method is to couple poly(ethylene glycol) (PEG) to topoisomerase while it is covalently attached to DNA and then to use phase partitioning in an aqueous two-phase system of PEG and phosphate to separate free DNA from DNA bound to PEG-modified topoisomerases (which have high affinities for the phosphate-rich and PEG-rich phases, respectively). The method can be used in conjunction with DNase protection and, unlike the SDS/KCl method, can fractionate short fragments of DNA to which single protein molecules are attached. Using the SDS/KCl precipitation and new method in series, we have recovered protein-linked DNA from HL60 cells induced to differentiate to the granulocyte lineage (by retinoic acid) or to the monocyte/macrophage lineage (by phorbol myristate acetate) and have demonstrated that specific sequences become protein linked, probably to topoisomerase II, during induced differentiation.
...
PMID:A method for the purification of DNA/protein complexes applied to DNA topoisomerase II cleavage sites. 164 31

To study the mechanism of illegitimate recombination in mammalian cells, we have developed a shuttle vector, pNK1, that contains three bacterial markers, amp (ApR), galK, and neo (KmR). The frequency of deletions occurring in autonomously replicating pNK1 DNA during the growth of monkey COS1 cells was measured by transfecting the plasmid into Escherichia coli cells and counting the number of galK- ApS double mutants among total KmR cells. This method allowed us to test the effects of topoisomerase inhibitors on deletion formation in mammalian cells. The DNA topoisomerase II (TopII) inhibitor, 4'-dimethylepipodophyllotoxin thenylidene-beta-D-glucoside (VM26), stimulated deletions in pNK1 DNA in monkey cells. Since VM26 does not inhibit the strand-break activity of TopII, but rather stabilizes an enzyme-DNA complex in which DNA is cleaved upon treatment with sodium dodecyl sulfate, it is implicated that TopII participates in the deletion process in mammalian cells.
...
PMID:A shuttle vector for analysis of illegitimate recombination in mammalian cells: effects of DNA topoisomerase inhibitors on deletion frequency. 164 63

In the studies reported here we have used topoisomerase II as a model system for analyzing the factors that determine the sites of action for DNA-binding proteins in vivo. To localize topoisomerase II sites in vivo we used an inhibitor of the purified enzyme, the antitumor drug VM-26. This drug stabilizes an intermediate in the catalytic cycle, the cleavable complex, and substantially stimulates DNA cleavage by topoisomerase II. We show that lysis of VM-26 treated tissue culture cells with sodium dodecyl sulfate induces highly specific double-strand breaks in genomic DNA, and we present evidence indicating that these double-strand breaks are generated by topoisomerase II. Using indirect end labeling to map the cleavage products, we have examined the in vivo sites of action of topoisomerase II in the 87A7 heat shock locus, the histone repeat, and a tRNA gene cluster at 90BC. Our analysis reveals that chromatin structure, not sequence specificity, is the primary determinant in topoisomerase II site selection in vivo. We suggest that chromatin organization may provide a general mechanism for generating specificity in a wide range of DNA-protein interactions in vivo.
...
PMID:Chromatin structure, not DNA sequence specificity, is the primary determinant of topoisomerase II sites of action in vivo. 165 19

The binding of purified Drosophila topoisomerase II to the highly bent DNA segments from the SV40 terminus of replication and C. fasciculata kinetoplast minicircle DNA (kDNA) was examined using electron microscopy (EM). The probability of finding topoisomerase II positioned at or near the bent SV40 terminus and Crithidia fasciculata kDNA was two- and threefold higher, respectively, than along the unbent pBR325 DNA into which the elements had been cloned. Closer examination demonstrated that the enzyme bound preferentially to the junction between the bent and non-bent sequences. Using gel electrophoresis, a cluster of strong sodium dodecyl sulfate-induced topoisomerase II cleavage sites was mapped to the SV40 terminus DNA, and two weak cleavage sites to the C. fasciculata kDNA. As determined by EM, Drosophila topoisomerase II foreshortened the apparent length of DNA by only 15 base-pairs when bound, arguing that it does not wrap DNA around itself. When bound to pBR325 containing the C. fasciculata kDNA and the SV40 terminus, topoisomerase II often produced DNA loops. The size distribution was that predicted from the known probability of any two points along linear DNA colliding. In vitro mapping of topoisomerase II on DNA whose ends were blocked by avidin protein revealed that binding is enhanced at sites located near a blocked end as compared to a free end. These observations may contribute towards establishing a framework for understanding topoisomerase II-DNA interactions.
...
PMID:Drosophila topoisomerase II-DNA interactions are affected by DNA structure. 184 28

Type II topoisomerase has been purified from mouse FM3A cells by using P4 phage knotted DNA as a substrate. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 167 and 151 kDa. Partial digestion of the two bands with Staphylococcus aureus V8 protease indicated that the two polypeptides were structurally related. The enzyme required ATP and Mg2+ for activity. dATP could substitute for ATP, and ITP was slightly effective at 5-10 mM. The activity was sensitive to 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA), coumermycin, and ethidium bromide. A protein kinase activity was detected in the partially purified topoisomerase II fraction, and this protein kinase was further purified. The protein kinase phosphorylated the purified topoisomerase II, and the phosphorylation of topoisomerase II by the kinase increased the activity by 8.6-fold over that of the unmodified enzyme. The treatment of the purified topoisomerase II with alkaline phosphatase abolished the enzyme activity almost completely, and the treatment of the dephosphorylated topoisomerase II with the protein kinase restored the enzyme activity. The protein kinase activity was not stimulated by Ca2+ or cyclic nucleotides, and the aminoacyl residue phosphorylated by the kinase was serine. Enzymatic properties of the kinase were very similar to those of the kinase reported to be tightly associated with the Drosophila topoisomerase II [Sander, M., Nolan, J. M., & Hsieh, T.-S. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6938-6942]. The immunoprecipitation of nuclear extracts prepared from 32P-labeled cells with anti-mouse topoisomerase II antiserum indicated that DNA topoisomerase II existed in mouse cells as a phosphoprotein.
...
PMID:Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity. 215 52

Following its cleavage of double-stranded DNA, topoisomerase II is covalently bound to the 5'-termini of both nucleic acid strands. However, in order to isolate this enzyme-cleaved DNA complex in the presence of magnesium (the enzyme's physiological divalent cation), reactions must be terminated by the addition of a strong protein denaturant such as sodium dodecyl sulfate (SDS). Because of the requirement for a protein denaturant, it is unclear whether DNA cleavage in this in vitro system takes place prior to or is induced by the addition of SDS. To distinguish between these two possibilities, experiments were carried out to determine whether topoisomerase II bound DNA contains 3'-OH termini prior to denaturation. This was accomplished by using circular single-stranded phi X174 DNA as a model substrate for the enzyme. As found previously for topoisomerase II mediated cleavage of double-stranded DNA, the enzyme was covalently linked to the 5'-termini of cleaved phi X174 molecules. Moreover, optimal reaction pH as well as optimal salt and magnesium concentrations was similar for the two substrates. In contrast to results with double-stranded molecules, single-stranded DNA cleavage increased with time, was not salt reversible, and did not require the presence of SDS. Furthermore, cleavage products generated in the absence of protein denaturant could be labeled at their 3'-OH DNA termini by incubation with terminal deoxynucleotidyltransferase and [alpha-32P]ddATP. Finally, cleaved phi X174 molecules could be joined to a radioactively labeled double-stranded oligonucleotide by a topoisomerase II mediated intermolecular ligation reaction.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Uncoupling the DNA cleavage and religation activities of topoisomerase II with a single-stranded nucleic acid substrate: evidence for an active enzyme-cleaved DNA intermediate. 217 49


1 2 3 4 5 6 7 Next >>

---