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:5.99.1.3 (topoisomerase)
9,911 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.
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PMID:Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins. 630 11

Inhibitors of bacterial DNA gyrase and eukaryotic DNA topoisomerase (novobiocin and nalidixic acid) were investigated with respect to their effect on the activity of RNA-dependent DNA polymerases from murine and avian retroviruses. Purified RNA-dependent DNA polymerase from AKR virus was inhibited more than 90% by 0.3 mg/ml and almost completely by 1 mg/ml of the drugs when poly(A) X oligo(dT)12-18 was used as a template-primer. In contrast to the enzyme from AKR virus, purified enzyme from avian myeloblastosis virus was less sensitive, i.e. nearly 50% activity remained even in the presence of 1 mg/ml of the drugs with the same template-primer. RNA-dependent DNA polymerase activity in AKR virus particles was inhibited, but was resistant to low concentrations of the drugs. The inhibition was not due to specific interaction between drugs and the template-primer or labelled precursor, since RNA-dependent DNA polymerase was inhibited by the drugs with activated calf thymus DNA or poly(C) X oligo(dG)12-18 as the template. Endogenous DNA synthesis by AKR virus particles was inhibited by novobiocin to the same extent.
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PMID:Inhibition of retrovirus RNA-dependent DNA polymerase by novobiocin and nalidixic acid. 631 59

Using the P4 unknotting assay, DNA topoisomerase II has been purified from several mammalian cells. Similar to prokaryotic DNA gyrase, mammalian DNA topoisomerase II can cleave double-stranded DNA and be trapped as a covalent protein-DNA complex. This cleavage reaction requires protein denaturant treatment of the topoisomerase II-DNA complex and is reversible with respect to salt and temperature. The product after reversal of the cleavage reaction remains supertwisted, suggesting that the two ends of the putatively broken DNA are held tightly by the topoisomerase. Alternatively, the enzyme-DNA interaction is noncovalent, and the covalent linking of topoisomerase to DNA is induced by the protein denaturant. Detailed characterization of the cleavage products has revealed that topoisomerase II cuts DNA with a four-base stagger and is covalently linked to the protruding 5'-phosphoryl ends of each broken DNA strand. Calf thymus DNA topoisomerase II cuts SV40 DNA at multiple and specific sites. However, no sequence homology has been found among the cleavage sites as determined by direct nucleotide-sequencing studies.
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PMID:Cleavage of DNA by mammalian DNA topoisomerase II. 631 92

We demonstrate that the activity of the major DNA topoisomerase I from calf thymus is severely inhibited after modification by purified poly(ADP-ribose) synthetase. Polymeric chains of poly(ADP-ribose) are covalently attached to DNA topoisomerase I. These observations with highly purified enzymes suggest that poly(ADP-ribosylation) may be a cellular mechanism for modulating DNA topoisomerase I activity in response to the state of DNA in the nucleus. Although extensive poly(ADP-ribosylation) of the Mr = 100,000 DNA topoisomerase I from calf thymus resulted in greater than 90% enzyme inhibition, exogenous poly(ADP-ribose) does not, by itself, inhibit topoisomerase activity. After modification, the apparent molecular weight of both the topoisomerase enzyme protein and of the topoisomerase enzyme activity was increased. In vitro, the extent of modification of DNA topoisomerase I could be controlled either by changing the ratio of topoisomerase to the synthetase or by varying the reaction time. More than 40 residues of ADP ribose per topoisomerase molecule could be added by the synthetase. Analysis of a poly(ADP-ribosylated) topoisomerase preparation that was about 50% inhibited revealed an average polymer chain length of 7.4, with 1-2 chains per enzyme molecule.
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PMID:Poly(ADP-ribosylation) of DNA topoisomerase I from calf thymus. 632 15

HMG17 protein is shown to greatly facilitate the catention of double-stranded DNA rings catalyzed by DNA topoisomerases. Even at low DNA concentrations such that catenanes are not observable in the absence of HMG17, the addition of the protein promotes the catenation of greater than 95% of the input DNA into networks that do not enter the gel upon electrophoresis. Electron microscopy and restriction enzyme cleavage experiments indicate that these networks are large structures containing many catenated DNA rings. The HMG17-promoted DNA network formation has been observed with calf thymus type II DNA topoisomerase and the type I topoisomerases of Escherichia coli, Micrococcus luteus, and calf thymus.
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PMID:HMG17 protein facilitates the DNA catenation reaction catalyzed by DNA topoisomerases. 632 73

At an early purification stage, DNA polymerase alpha holoenzyme from calf thymus can be separated into four different forms by chromatography on DEAE-cellulose. All four enzyme forms (termed A, B, C, and D) are capable of replicating long single-stranded DNA templates, such as parvoviral DNA or primed M13 DNA. Peak A possesses, in addition to the DNA polymerase alpha, a double-stranded DNA-dependent ATPase, as well as DNA topoisomerase type II, 3'-5' exonuclease, and RNase H activity. Peaks B, C, and D all contain, together with DNA polymerase alpha, activities of primase and DNA topoisomerase type II. Furthermore, peak B is enriched in an RNase H, and peaks C and D are enriched in a 3'-5' exonuclease. DNA methylase (DNA methyltransferase) was preferentially identified in peaks C and D. Velocity sedimentation analyses of the four peaks gave evidence of unexpectedly large forms of DNA polymerase alpha (greater than 11.3 s), indicating that copurification of the above putative replication enzymes is not fortuitous. With moderate and high concentrations of salt, enzyme activities cosedimented with DNA polymerase alpha. Peak C is more resistant to inhibition by salt and spermidine than the other three enzyme forms. These results suggest the existence of a leading strand replicase (peak A) and several lagging strand replicase forms (peaks B, C, and D). Finally, the salt-resistant C form might represent a functional DNA polymerase alpha holoenzyme, possibly fitting in a higher-order structure, such as the replisome or even the chromatin.
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PMID:Mammalian DNA polymerase alpha holoenzymes with possible functions at the leading and lagging strand of the replication fork. 658 75

The interactions of the low cardiotoxic antitumor agents 1,4-dihydroxy-5,8-bis[[2-[(2-hydroxyethyl)amino]ethyl]amino]-9, 10-anthracenedione (mitoxantrone) and 9,10-anthracenedicarboxaldehyde bis[(4,5-dihydro-1H-imidazoyl-2-yl)hydrazone] (bisantrene) with pBR322 and PM2 DNA have been examined by electron microscopy. Direct evidence was obtained for intercalative binding of both drugs, with mitoxantrone causing a 13% average length increase in pBR322 corresponding to approximately 580 drug molecules per circle at saturation and bisantrene causing an 11% increase in length corresponding to approximately 480 drug molecules bound per circle. Considerations of the known GC preference for non-nearest neighbor binding of the drugs and inspection of the known sequence of pBR322 suggest that the available intercalation sites are occupied and that additional external electrostatic binding of the cationic drugs also occurs. An apparent difference in behavior of mitoxantrone as compared with that of bisantrene in causing no net increase in length of supercoiled pBR322 was shown to be attributable to an offsetting compaction due to extensive supercoiling by mitoxantrone molecules. This conclusion was confirmed by independent experiments with PM2 covalently closed-circular DNA--both native, negatively supercoiled and relaxed--with calf thymus topoisomerase, using ethidium for comparison. Ethidium caused a 21.3 +/- 3.6% length increase in nicked, open-circular PM2-DNA, or 2100 molecules bound per 10,300 base pairs. Mitoxantrone caused a 16.6% length increase in nicked PM2-DNA equivalent to approximately 1700 drug molecules per circle. Electron microscopic measurements on relaxed PM2-DNA with progressively increasing proportions of mitoxantrone (from 1.4:1 to 14:1 drug molecules per base pair) revealed the onset of formation of lacelike networks of DNA circles linked together. This phenomenon, which is not produced by bisantrene, is attributed to inter-DNA links by the charged side arms of mitoxantrone and is in accord with previous reports that mitoxantrone causes severe compaction and distortion of chromatin. Electron microscopic examination of the interaction of six additional mitoxantrone derivatives, two of which produced lacelike DNA networks, revealed strict structural requirements for this phenomenon.
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PMID:Interactions of the antitumor agents mitoxantrone and bisantrene with deoxyribonucleic acids studied by electron microscopy. 670 33

In this study we describe a cytometric method to sort apoptotic cell fractions, suitable for biochemical and morphological analyses. Rat thymocytes were used as a model system, as apoptosis naturally occurs in the thymus, where the negative selection of the T cell repertoire takes place. Massive apoptosis was induced in vitro by the topoisomerase-II inhibitor, etoposide. After etoposide treatment, a large fraction of thymocytes showed the morphological and electrophoretic features of apoptotic cells. Unfixed thymocytes were stained for 30 min with propidium iodide (PI: 50 micrograms/ml containing RNase type A and detergent), and analyzed by flow cytometry. Apoptotic thymocytes typically showed sub-G1 DNA contents. Compared to non-apoptotic thymocytes, sub-G1 cells had lower values of low-angle (FSC) scatter and higher values of right-angle (SSC) scatter, so that, in dual parameter cytograms of FSC versus SSC, two distinct cell populations were apparent, and were separated by flow sorting. The purified cell fractions obtained by this procedure had a very well preserved ultrastructural morphology; both early and late apoptotic stages (until the onset of cytoplasm segmentation) were present in the sorted apoptotic fraction.
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PMID:A single-step staining procedure for the detection and sorting of unfixed apoptotic thymocytes. 751 May 45

Several new pyridoacridine alkaloids, dehydrokuanoniamine B (1), shermilamine C (2), and cystodytin J (3), in addition to the known compounds cystodytin A (4), kuanoniamine D (5), shermilamine B (6), and eilatin (7), were isolated from a Fijian Cystodytes sp. ascidian. Their structures were determined by analyses of spectroscopic data. These compounds along with a previously reported pyridoacridine, diplamine (8), showed dose-dependent inhibition of proliferation in human colon tumor (HCT) cells in vitro. All compounds inhibited the topoisomerase (TOPO) II-mediated decatenation of kinetoplast DNA (kDNA) in a dose-dependent manner. The pyridoacridines' ability to inhibit TOPO II-mediated decatenation of kDNA correlated with their cytotoxic potencies and their ability to intercalate into calf thymus DNA. These results suggest that disruption of the function of TOPO II, subsequent to intercalation, is a probable mechanism by which pyridoacridines inhibit the proliferation of HCT cells. Incorporation studies show that pyridoacridines disrupt DNA and RNA synthesis with little effect on protein synthesis. It appears that DNA is the primary cellular target of the pyridoacridine alkaloids. These results are consistent with those for known DNA intercalators.
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PMID:Inhibition of topoisomerase II catalytic activity by pyridoacridine alkaloids from a Cystodytes sp. ascidian: a mechanism for the apparent intercalator-induced inhibition of topoisomerase II. 752 59

A number of DNA minor groove-binding ligands (MGBLs) are known to exhibit antitumor and antimicrobial activities. We show that DNA topoisomerase (Topo) I may be a pharmacological target of MGBLs. In the presence of calf thymus Topo I, MGBLs induced limited but highly specific single-strand DNA breaks. The 3' ends of the broken DNA strands are covalently linked to Topo I polypeptides. Protein-linked DNA breaks are readily reversed by a brief heating to 65 degrees C or the addition of 0.5 M NaCl. These results suggest that MGBLs, like camptothecin, abort Topo I reactions by trapping reversible cleavable complexes. The sites of cleavage induced by MGBLs are distinctly different from those induced by camptothecin. Two of the major cleavage sites have been sequenced and shown to be highly A + T-rich, suggesting the possible involvement of a Topo I-drug-DNA ternary complex at the sites of cleavage. Different MGBLs also exhibit varying efficiency in inducing Topo I-cleavable complexes, and the order of efficiency is as follows: Hoechst 33342 and 33258 >> distamycin A > berenil > netropsin. The lack of correlation between DNA binding and cleavage efficiency suggest that, in addition to binding to the minor grooves of DNA, MGBLs must also interact with Topo I in trapping Topo I-cleavable complexes.
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PMID:DNA minor groove-binding ligands: a different class of mammalian DNA topoisomerase I inhibitors. 769 Jan 43


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