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 present a novel assay for the study of protein-protein interactions involving DNA topoisomerase II. Under various conditions of incubation we observe that topoisomerase II forms complexes at least tetrameric in size, which can be sedimented by centrifugation through glycerol. The multimers are enzymatically active and can be visualized by electron microscopy. Dephosphorylation of topoisomerase II inhibits its multimerization, which can be restored at least partially by rephosphorylation of multiple sites within its 200 C-terminal amino acids by casein kinase II. Truncation of topoisomerase II just upstream of the major phosphoacceptor sites reduces its aggregation, rendering the truncated enzyme insensitive to either kinase treatments or phosphatase treatments. This is consistent with a model in which interactions involving the phosphorylated C-terminal domain of topoisomerase II aid either in chromosome segregation or in chromosome condensation.
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PMID:Topoisomerase II forms multimers in vitro: effects of metals, beta-glycerophosphate, and phosphorylation of its C-terminal domain. 793 13

Evidence for multiprotein complexes playing a role in DNA replication has been growing over the years. We have previously reported on a replication-competent multiprotein form of DNA polymerase isolated from human (HeLa) cell extracts. The proteins that were found at that time to co-purify with the human cell multiprotein form of DNA polymerase included: DNA polymerase alpha, DNA primase, topoisomerase I, RNase H, PCNA, and a DNA-dependent ATPase. The multiprotein form of the human cell DNA polymerase was further purified by Q-Sepharose chromatography followed by glycerol gradient sedimentation and was shown to be fully competent to support origin-specific and large T-antigen dependent simian virus 40 (SV40) DNA replication in vitro [Malkas et al. (1990b): Biochemistry 29:6362-6374]. In this report we describe the further characterization of the human cell replication-competent multiprotein form of DNA polymerase designated MRC. Several additional DNA replication proteins that co-purify with the MRC have been identified. These proteins include: DNA polymerase delta, RF-C, topoisomerase II, DNA ligase I, DNA helicase, and RP-A. The replication requirements, replication initiation kinetics, and the ability of the MRC to utilize minichromosome structures for DNA synthesis have been determined. We also report on the results of experiments to determine whether nucleotide metabolism enzymes co-purify with the human cell MRC. We recently proposed a model to represent the MRC that was isolated from murine cells [Wu et al. (1994): J Cell Biochem 54:32-46]. We can now extend this model to include the human cell MRC based on the fractionation, chromatographic and sedimentation behavior of the human cell DNA replication proteins. A full description of the model is discussed. Our experimental results provide further evidence to suggest that DNA synthesis is mediated by a multiprotein complex in mammalian cells.
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PMID:Further characterization of the human cell multiprotein DNA replication complex. 853 May 40

Vaccinia topoisomerase forms a covalent protein-DNA intermediate at sites containing the sequence 5'-CCCTT. The T nucleotide is linked via a 3'-phosphodiester bond to Tyr-274 of the enzyme. Here, we report that the enzyme catalyzes hydrolysis of the covalent intermediate, resulting in formation of a 3'-phosphate-terminated DNA cleavage product. The hydrolysis reaction is pH-dependent (optimum pH = 9.5) and is slower, by a factor of 10(-5), than the rate of topoisomerase-catalyzed strand transfer to a 5'-OH terminated DNA acceptor strand. Mutants of vaccinia topoisomerase containing serine or threonine in lieu of the active site Tyr-274 form no detectable covalent intermediate and catalyze no detectable DNA hydrolysis. This suggests that hydrolysis occurs subsequent to formation of the covalent protein-DNA adduct and not via direct attack by water on DNA. Vaccinia topoisomerase also catalyzes glycerololysis of the covalent intermediate. The rate of glycerololysis is proportional to glycerol concentration and is optimal at pH 9.5.
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PMID:DNA strand transfer reactions catalyzed by vaccinia topoisomerase: hydrolysis and glycerololysis of the covalent protein-DNA intermediate. 915 7

Melanoplus sanguinipes entomopoxvirus (MsEPV) encodes a 328 amino acid polypeptide related to the type I topoisomerases of six other genera of vertebrate and insect poxviruses. The gene encoding MsEPV topoisomerase was expressed in bacteria, and the recombinant protein was purified by ion-exchange chromatography and glycerol gradient sedimentation. MsEPV topoisomerase, a monomeric protein, catalyzed the relaxation of supercoiled plasmid DNA at approximately 0.6 supercoils/s. Like other poxvirus topoisomerases, the MsEPV enzyme formed a covalent adduct with duplex DNA at the target sequence CCCTT downward arrow. The kinetic and equilibrium parameters of the DNA transesterification reaction of MsEPV topoisomerase were k(cl) = 0.3 s(-1) and K(cl) = 0.25. The introduction of a 5'-bridging phosphorothiolate at the scissile phosphate increased the cleavage equilibrium constant from 0.25 to >/=30. Similar phosphorothiolate effects were observed with vaccinia topoisomerase. Kinetic analysis of single-turnover cleavage and religation reactions established that the altered equilibrium was the result of a approximately 10(-4) decrement in the rate of topoisomerase-catalyzed attack of 5'-SH DNA on the DNA-(3'-phosphotyrosyl)-enzyme intermediate. 5'-bridging phosphorothiolates at the scissile phosphate and other positions within the CCCTT element had no significant effect on k(cl).
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PMID:Melanoplus sanguinipes entomopoxvirus DNA topoisomerase: site-specific DNA transesterification and effects of 5'-bridging phosphorothiolates. 1056 6

Vaccinia topoisomerase forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at sites containing the sequence 5'-CCCTT downward arrow. The covalently bound topoisomerase can religate the CCCTT strand to a 5'-OH-terminated polynucleotide or else transfer the strand to a non-DNA nucleophile such a water or glycerol. Here, we report that vaccinia topoisomerase also catalyzes strand transfer to hydrogen peroxide. The observed alkaline pH-dependence of peroxidolysis is consistent with enzyme-mediated attack by peroxide anion on the covalent intermediate. The reaction displays apparent first-order kinetics. From a double-reciprocal plot of k(obs) versus [H(2)O(2)] at pH 10, we determined a rate constant for peroxidolysis of 6.3 x 10(-)(3) s(-)(1). This rate is slower by a factor of 200 than the rate of topoisomerase-catalyzed strand transfer to a perfectly aligned 5'-OH DNA strand but is comparable to the rate of DNA strand transfer across a 1-nucleotide gap. Strand transfer to 2% hydrogen peroxide is 300 times faster than strand transfer to 20% glycerol and approximately 2000 times faster than topoisomerase-catalyzed hydrolysis of the covalent intermediate. Hydroxylamine is also an effective nucleophile in topoisomerase-mediated strand transfer (k(obs) = 6.4 x 10(-)(4) s(-)(1)). The rates of the peroxidolysis, hydroxylaminolysis, glycerololysis, and hydrolysis reactions catalyzed by the mutant enzyme H265A were reduced by factors of 100-700, in accordance with the 100- to 400-fold rate decrements in DNA cleavage and religation by H265A. We surmise that vaccinia topoisomerase catalyzes strand transfer to DNA and non-DNA nucleophiles via a common reaction pathway in which His-265 stabilizes the scissile phosphate in the transition state rather than acting as a general acid or base.
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PMID:DNA strand transfer catalyzed by vaccinia topoisomerase: peroxidolysis and hydroxylaminolysis of the covalent protein-DNA intermediate. 1082 56

Bioassay-directed isolation and purification of the hexane extract of Apium graveolens L. seeds led to the characterization of three compounds: beta-selinene (1), 3-n-butyl-4,5-dihydrophthalide (2) and 5-allyl-2-methoxyphenol (3). The structures of these compounds were established by using (1)H and (13)C NMR spectral methods. Compounds, 1-3 demonstrated 100% mortality on fourth-instar Aedes aegyptii larvae at 50, 25, and 200 microg mL(-)(1), respectively, in 24 h. Also, 2 inhibited the growth of Candida albicans and Candida kruseii at 100 microg mL(-)(1). It inhibited both topoisomerase-I and -II enzyme activities at 100 microg mL(-)(1). Compound 2 displayed 100% mortality at 12.5 and 50 microg mL(-)(1), respectively, when tested on nematodes, Panagrellus redivivus and Caenorhabditis elegans. The triglyceride, 1,3-di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9, 12-octadecadienoyl]glycerol (4) and 3 were isolated for the first time from A. graveolens seeds, although 4 was not biologically active.
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PMID:Bioactive compounds and 1,3-Di[(cis)-9-octadecenoyl]-2-[(cis,cis)-9, 12-octadecadienoyl]glycerol from Apium graveolens L. seeds. 1099 71

Tyrosyl-DNA phosphodiesterase (Tdp1) is a DNA repair enzyme that catalyzes the hydrolysis of a phosphodiester bond between a tyrosine residue and a DNA 3'-phosphate. The only known example of such a linkage in eukaryotic cells occurs normally as a transient link between a type IB topoisomerase and DNA. Thus human Tdp1 is thought to be responsible for repairing lesions that occur when topoisomerase I becomes stalled on the DNA in the cell. Tdp1 has also been shown to remove glycolate from single-stranded DNA containing a 3'-phosphoglycolate, suggesting a role for Tdp1 in repair of free-radical mediated DNA double-strand breaks. We report the three-dimensional structures of human Tdp1 bound to the phosphate transition state analogs vanadate and tungstate. Each structure shows the inhibitor covalently bound to His263, confirming that this residue is the nucleophile in the first step of the catalytic reaction. Vanadate in the Tdp1-vanadate structure has a trigonal bipyramidal geometry that mimics the transition state for hydrolysis of a phosphodiester bond, while Tdp1-tungstate displays unusual octahedral coordination. The presence of low-occupancy tungstate molecules along the narrow groove of the substrate binding cleft is suggestive evidence that this groove binds ssDNA. In both cases, glycerol from the cryoprotectant solution became liganded to the vanadate or tungstate inhibitor molecules in a bidentate 1,2-diol fashion. These structural models allow predictions to be made regarding the specific binding mode of the substrate and the mechanism of catalysis.
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PMID:Insights into substrate binding and catalytic mechanism of human tyrosyl-DNA phosphodiesterase (Tdp1) from vanadate and tungstate-inhibited structures. 1247 Sep 49

DE-310 is a novel macromolecular prodrug of the topoisomerase-I inhibitor DX-8951. DX-8951 is covalently linked to carboxymethyl dextran polyalcohol (CM-Dex-PA) via a Gly-Gly-Phe-Gly (GGFG) tetrapeptide spacer. The present study was conducted to identify the portions of DX-8951 linked to DE-310, as well as to quantify the number of DX-8951 molecules associated with DE-310. Two different structures terminated with either glycolaldehyde (CM-GA-GGFG-DX-8951) or glycerol (CM-Glr-GGFG-DX-8951) are obtained when the polymer backbone is fragmented with 1 M HCl. The two products, i.e., CM-GA-GGFG-DX-8951 and CM-Glr-GGFG-DX-8951, indicate linkage of GGFG-DX-8951 with carboxymethyl (CM) group at C-2 and C-4 position of the glucose units, respectively. In the present study, CM-GA-GGFG-DX-8951 was reduced to CM-ethyleneglycol (EG)-GGFG-DX-8951 in order to improve stability prior to HPLC analysis. Hydrolysis results revealed that the amount of CM-GA-GGFG-DX-8951 liberated was 84.7 nmol/mg DE-310 and the amount of CM-Glr-GGFG-DX-8951 was 71.8 nmol/mg DE-310. Considering the ratio of generation between CM-GA-GGFG-DX8951 and CM-Glr-GGFG-DX8951, it suggested that slightly larger amount of GGFG-DX-8951 was linked to carboxymethyl groups at the C-2 position of glucose units in DE-310. The sum of the amounts of CM-GA-GGFG-DX-8951 and CM-Glr-GGFG-DX-8951 agrees well with the amount of G-DX-8951 produced from DE-310 by alpha-chymotrypsin treatment (157.5 nmol/mg DE-310). The data indicate that the established hydrolysis give a quantitative evaluation of the DX-8951 linked to DE-310.
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PMID:Quantitative acid hydrolysis of DE-310, a macromolecular carrier system for the camptothecin analog DX-8951f. 1712 25

In order to study DNA replication and expression in wheat mitochondria our laboratory has been seeking to develop a system that supports DNA synthesis and transcription, either in isolated mitochondria from wheat embryos or in a mitochondrial lysate from the same source deprived of endogenous DNA in vitro. We have characterized some of the enzymes involved in the DNA synthesis and transcription process. In this study we describe a DNA topoisomerase activity.Broken mitochondria from wheat embryos can actively relax negatively supercoiled DNA (pBR322, pAT153, etc...). The enzyme is intramitochondrial: the activity is detected only when intact organelles are broken by non-ionic detergent. Most of the topoisomerase activity found in the broken mitochondria is recovered in the mitochondrial lysate. It is stimulated by Mg(2+) and has an optimum salt concentration, KCl or NaCl, between 50 mM and 100 mM. ATP has no effect on this activity. Ethidium bromide, berenil, novobiocine and nalidixic acid, compounds currently used to characterize DNA topoisomerases, do not effect the relaxation of supercoiled DNA by the wheat mitochondrial activity. On the other hand N-ethylmaleimide has a strong inhibitory effect indicating that sulfhydryl groups are essential for enzyme activity. The molecular weight of the enzyme as determined by glycerol gradient sedimentation, is about 110 kd. Another important feature of the mitochondrial lysate DNA topoisomerase is the ability to relax positively supercoiled DNA, a property of eukaryotic topoisomerases I.
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PMID:A DNA topoisomerase type I from wheat embryo mitochondria. 2430 19


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