Purification and characterization of a type II DNA topoisomerase from bovine calf thymus

We report here the large scale purification of DNA topoisomerase II from calf thymus glands, using the unknotting of naturally knotted P4 phage DNA as an assay for enzymatic activity. Topoisomerase II was purified more than 1300-fold as compared to the whole cell homogenate, with 22% yield. Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two bands of apparent molecular masses of 125 and 140 kDa. Tryptic maps of the two bands indicated that they derive from the same protein. Using these fragments, specific polyclonal antisera to topoisomerase II were raised in rabbits. Immunoblotting of whole cell lysates from various species indicated that topoisomerase II is well conserved among mammals and has a native subunit molecular mass of 180 kDa. Analytical sedimentation and gel filtration were used to determine a sedimentation coefficient of 9.8 S and a Stokes radius of 68 A. The calculated solution molecular mass of 277 kDa implies a dimer structure in solution. The purified topoisomerase II unknots P4 DNA in an ATP-dependent manner and is highly stimulated in its relaxation activity by ATP. A DNA-stimulated ATPase activity, as has been found with other type II topoisomerases, is associated with the purified enzyme. Approximate kinetic parameters for the ATPase reaction were determined to be: a Vmax of 0.06 nmol of ATP/(micrograms of protein) (min) and Km of 0.2 mM in the absence of DNA, and a Vmax of 0.2 nmol of ATP/(micrograms of protein) (min) and Km of 0.4 mM ATP in the presence of supercoiled plasmid DNA.     

Purification and characterization of type II DNA topoisomerase from mouse FM3A cells: phosphorylation of topoisomerase II and modification of its activity

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.     

Vaccinia virus encapsidates a novel topoisomerase with the properties of a eucaryotic type I enzyme

A DNA topoisomerase has been purified from vaccinia virus cores. The native enzyme is composed of a single subunit of 32,000 daltons. The enzyme relaxes both positively and negatively supercoiled DNA in the absence of an energy cofactor. Enzymatic activity is stimulated by magnesium ions and inhibited by ATP, and during relaxation the topoisomerase changes the linking number of the DNA substrate by steps of one. Trapping of the covalent DNA-enzyme intermediate has been achieved, and analysis of the cleavage of duplex DNA by the enzyme reveals that it makes a single-strand break and forms a covalent bond through the 3'-end of the broken strand. Enzymatic activity and formation of the trapped intermediate are inhibited by the drugs novobiocin, coumermycin A1, and berenil. The virally encapsidated enzyme has novel properties but most closely resembles a eucaryotic type I topoisomerase.     

Activity of DNA topoisomerase I and quiescence of embryo cells in seeds of Pisum sativum L.

DNA topoisomerase activity is present at a very early stageof germination in nuclear extract of pea root meristems. Theactivity of this enzyme changes before the onset of replicativeDNA synthesis, thus suggesting the existence of a correlationbetween DNA topoisomerase I and events taking place during therelease of cells from a quiescent state. An antibody preparedagainst a human topoisomerase I is able to immuno-precipitatepart of the DNA topoisomerase activity present in pea nuclearextracts, and recognizes a protein with a molecular weight of45 kDa. We suggest that the 45 kDa protein is a DNA topoisomeraseI; its presence during embryogenesis and its storage in dryseeds would explain the presence of DNA topoisomerase I activityduring early stages of germination. Key words: Pisum sativum L, DNA topoisomerase, nuclei, quiescence, proliferation     

Purification and characterization of a type-I topoisomerase from cultured tobacco cells

Cultured tobacco (Nicotiana tabacum, var Xanthi) cells contain a topoisomerase that removes positive and negative supercoils from DNA. The enzyme has an estimated molecular mass of 30,000 daltons under denaturing conditions, but may exist as a multimeric protein in the native state. Activity is enhanced significantly by either MgCl₂ or CaCl₂, but other divalent cations are much less effective in stimulating DNA relaxation. The purified enzyme acts by altering the linking number in topological steps of one and is inhibited by berenil or camptothecin, not novobiocin. Taken together, these data identify this enzyme as a type I topoisomerase.     

Purification and properties of DNA topoisomerase I from broccoli

We have purified a topoisomerase activity from broccoli (Brassica oleracea var. italica) to near homogeneity. The enzyme is an 80 kDa monomer as judged by gel filtration chromatography and SDS gel electrophoresis, though it may represent a proteolytic fragment of a larger protein. The enzyme is capable of removing both negative and positive supercoils in steps of one, does not absolutely require Mg²⁺, is only very weakly stimulated by NaCl, is inhibited by camptothecin, and cross-reacts with an antibody directed against human DNA topoisomerase I. These properties identify the enzyme as a eukaryotic type I topoisomerase.     

The purification and characterization of DNA topoisomerases I and II of the yeast Saccharomyces cerevisiae

The ATP-independent type I and the ATP-dependent type II DNA topoisomerase of the yeast Saccharomyces cerevisiae have been purified to near homogeneity, and the purification procedures are reported. Both purified topoisomerases are single subunit enzymes with monomer weights of Mr = 90,000 and 150,000 for the type I and type II enzyme, respectively. Sedimentation and gel filtration data suggest that the type I enzyme is monomeric and the type II enzyme is dimeric. Similar to other purified eukaryotic topoisomerases, the yeast type I enzyme does not require a divalent cation for activity, but is stimulated 10-20-fold in the presence of 7-10 mM Mg(II) or Ca(II). Mn(II) is about 25% as efficient as Mg(II) in this stimulation but Co(II) is inhibitory. The yeast type II topoisomerase has an absolute requirement for a divalent cation: Mg(II) is the most effective, whereas Mn(II), Ca(II), or Co(II) supports the reaction to a lesser extent. The type II enzyme also requires ATP or dATP; the nonhydrolyzable ATP analogues adenylyl imidodiphosphate and adenylyl (beta,gamma-methylene)diphosphonate are potent inhibitors. Both yeast topoisomerases are completely inhibited by N-ethylmaleimide at 0.5 mM. In addition, the type II enzyme, but not the type I enzyme, is inhibited to various extents by coumermycin, ethidium, and berenil. Both topoisomerases are nuclear enzymes; no topoisomerase specific to mitochondria has been detected.     

Regulation of Xenopus laevis DNA topoisomerase I activity by phosphorylation in vitro

DNA topoisomerase I has been purified to electrophoretic homogeneity from ovaries of the frog Xenopus laevis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most purified fraction revealed a single major band at 110 kDa and less abundant minor bands centered at 62 kDa. Incubation of the most purified fraction with immobilized calf intestinal alkaline phosphatase abolished all DNA topoisomerase enzymatic activity in a time-dependent reaction. Treatment of the dephosphorylated X. laevis DNA topoisomerase I with a X. laevis casein kinase type II activity and ATP restored DNA topoisomerase activity to a level higher than that observed in the most purified fraction. In vitro labeling experiments which employed the most purified DNA topoisomerase I fraction, [gamma-32P]ATP, and the casein kinase type II enzyme showed that both the 110- and 62-kDa bands became phosphorylated in approximately molar proportions. Phosphoamino acid analysis showed that only serine residues became phosphorylated. Phosphorylation was accompanied by an increase in DNA topoisomerase activity in vitro. Dephosphorylation of DNA topoisomerase I appears to block formation of the initial enzyme-substrate complex on the basis of the failure of the dephosphorylated enzyme to nick DNA in the presence of camptothecin. We conclude that X. laevis DNA topoisomerase I is partially phosphorylated as isolated and that this phosphorylation is essential for expression of enzymatic activity in vitro. On the basis of the ability of the casein kinase type II activity to reactivate dephosphorylated DNA topoisomerase I, we speculate that this kinase may contribute to the physiological regulation of DNA topoisomerase I activity.     

Purification of GidA protein,a novel topoisomerase II inhibitor produced by Streptomyces flavoviridis

The presence of topoisomerase II inhibition activities in the intracellular extract of Streptomyces flavoviridis was investigated. One active compound inhibiting relaxation activity of topoisomerase II was determined to be a protein. This active principle was purified to homogeneity by gel filtration followed by ion exchange chromatography. The apparent molecular mass was 42 kDa as determined by SDS-PAGE. MALDI TOF peptide mass fingerprinting analysis confirmed this topoisomerase II inhibitor, as glucose-inhibited division protein A (GidA) by MOWSE score of 72. The effects of purified GidA protein on DNA relaxation and decatenation by topoisomerase II were investigated. It inhibited topoisomerase II activity and acted as a topoisomerase poison that significantly stabilized the covalent DNA-topoisomerase II reaction intermediate “cleavable complex”, as observed with etoposide. Collectively, these findings indicate that GidA is a potent inhibitor of topoisomerase II enzyme, which can be exploited for rational drug design in human carcinomas.     

Characterization of VP-16-induced DNA damage in isolated nuclei from L1210 cells

Based on the observation that VP-16-induced DNA damage can be demonstrated in isolated nuclei but not in purified DNA, and that this effect is temperature-dependent, it is postulated that the mechanism of action of VP-16 involves an essential intranuclear event, perhaps enzyme-mediated, which is a prerequisite for the cleavage of DNA. Using alkaline elution to assay single-strand breaks in isolated L1210 nuclei, we have further characterized conditions influencing this putative intranuclear reaction. We have found drug activity to be dependent on magnesium and pH and to be stimulated by low concentrations of ATP (0.05–1 mM), an effect which was not observed with a nonhydrolyzable analog of ATP. Heat-labile activity in a nuclear non-histone protein extract was critical to VP-16-mediated DNA damage. This new evidence lends further credence to the hypothesis that activity of an intranuclear enzyme, possessing characteristics consistent with a type II DNA topoisomerase, is a prerequisite for the cleavage of DNA by VP-16.     

Isolation of intercalator-dependent protein-linked DNA strand cleavage activity from cell nuclei and identification as topoisomerase II

DNA intercalating agents such as 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) have previously been found to induce in mammalian cells the formation of protein-associated DNA single- and double-strand breaks. In the current work, an activity characterized by the production of DNA-protein links associated with DNA strand breaks and by stimulation by m-AMSA was isolated from L1210 cell nuclei and was shown to be due to topoisomerase II. Nuclei were extracted with 0.35 M NaCl, and the extract was fractionated by gel filtration, DNA-cellulose chromatography, and glycerol gradient centrifugation. A rapid filter binding assay was devised to monitor the fractionation procedure on the basis of DNA-protein linking activity. The active DNA-cellulose fraction contained both topoisomerase I and topoisomerase II whereas the glycerol gradient purified material contained only topoisomerase II activity. The properties of the active material were studied at both stages of purification. m-AMSA enhanced the formation of complexes between purified topoisomerase II and SV40 DNA in which the DNA sustained a single- or double-strand cut and the enzyme was covalently linked to the 5' terminus of the DNA. This action was further enhanced by ATP, as well as by nonhydrolyzable ATP analogues. m-AMSA inhibited the topoisomerization and catenation reactions of topoisomerase II, probably because of trapping of the enzyme-DNA complexes. The activity showed a dependence on the type of DNA intercalators used, analogous to what was previously observed in intact cells. m-AMSA had no effect on topoisomerase I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorylation of mammalian DNA topoisomerase I and activation by protein kinase C

The influence of mammalian DNA topoisomerase I phosphorylation on enzyme activity has been investigated. Dephosphorylation by calf intestine alkaline phosphatase abolished the DNA relaxing activity of DNA topoisomerase I and the sensitivity of the enzyme to its specific inhibitor, camptothecin. DNA topoisomerase I could be reactivated by incubation with purified protein kinase C. DNA topoisomerase I was then able to relax supercoiled DNA processively, like the native enzyme, and to cleave 32P-end-labeled SV40 DNA fragments at the same sequences as the native enzyme in the presence of camptothecin. These results show that active DNA topoisomerase I is a phosphoprotein and suggest a possible regulatory role of protein kinase on topoisomerase I activity and on its sensitivity to camptothecin.     

A homogeneous type II DNA topoisomerase from HeLa cell nuclei

Using kinetoplast DNA networks as a substrate in a decatenation assay, we have purified to apparent homogeneity a type II DNA topoisomerase from HeLa cell nuclei. The most pure preparations contain a single polypeptide of 172,000 daltons as determined by sodium dodecyl sulfate-gel electrophoresis. The molecular weight of the native protein, based on sedimentation and gel filtration analyses, is estimated to be 309,000. These results suggest that the enzyme is a dimer of 172,0090-dalton subunits. The enzyme is a type II topoisomerase as demonstrated by its ability to change the linking number of DNA circles in steps of two and to decatenate or unknot covalently closed DNA circles. No gyrase activity is detectable. ATP is required for the relaxation, decatenation, and unknotting of DNA, and a DNA-dependent ATPase activity is present in the most pure fractions. ATP is hydrolyzed to ADP in this properties to T4 DNA topoisomerase (Liu, L. F., Liu, C. C., and Alberts, B. M. (1979) Nature 281, 456-461).     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.     

Effects of the DNA intercalators 4'-(9-acridinylamino)methanesulfon-m-anisidide and 2-methyl-9-hydroxyellipticinium on topoisomerase II mediated DNA strand cleavage and strand passage

DNA topoisomerase II is believed to be the enzyme that produces the protein-associated DNA strand breaks observed in mammalian cell nuclei treated with various intercalating agents. Two intercalators--4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA, amsacrine) and 2-methyl-9-hydroxyellipticinium (2-Me-9-OH-E+)--differ in their effects on protein-associated double-strand breaks in isolated nuclei. m-AMSA stimulates their production at all concentrations, whereas 2-Me-9-OH-E+ stimulates at low concentrations and inhibits at high concentrations. We have reproduced these differential effects in experiments carried out in vitro with purified L1210 DNA topoisomerase II, and we have found that concentrations of 2-Me-9-OH-E+ above 5 microM prevent the trapping of DNA-topoisomerase II cleavable complexes irrespective of the presence of m-AMSA. It also stimulated topoisomerase II mediated DNA strand passage, again with or without inhibitory amounts of m-AMSA (this result suggests that extensive intercalation by 2-Me-9-OH-E+ destabilized the cleavable complexes). From these data, it is concluded that intercalator-induced protein-associated DNA strand breaks observed in intact eukaryotic cells and isolated nuclei are generated by DNA topoisomerase II and that intercalators can affect mammalian DNA topoisomerase II in more than one way. They can trap cleavable complexes and inhibit DNA topoisomerase II mediated DNA relaxation (m-AMSA and low concentrations of 2-Me-9-OH-E+) or destabilize cleavable complexes and stimulate DNA relaxation (high concentrations of 2-Me-9-OH-E+).     

Poly(ADP ribose) polymerase-defective mutant cell clone of mouse L1210 cells.

By a sequential mutation and selection utilizing N-methyl-N'-nitro-N-nitrosoguanidine as a mutagen, we succeeded in separating a poly(ADP ribose) polymerase-defective mutant clone (Cl-3527) from a mouse L1210 cell clone (Cl-3). The enzyme activity per cell in Cl-3527 cells was only 8% of that in wild type L1210 (CCL 219) cells. Immunoblot analysis of the enzyme protein in crude extracts of the mutant and wild type cells revealed that the enzyme defect was manifested as the loss of a 113-kDa wild type enzyme band in Cl-3527. Further analysis of partially purified enzyme from Cl-3527 by immunoblotting revealed that the molecular size of the enzyme in Cl-3527 was 108 kDa and that the amount of the mutant enzyme protein was markedly decreased in Cl-3527. The mutant enzyme was much more heat-labile than the wild type enzyme but the Km for NAD+, requirements for Mg2+ and nicked DNA, and the inhibition by 3-aminobenzamide, a potent inhibitor of the enzyme, however, were not so different from those of wild type enzyme. The mutant cells showed prolonged doubling time, increased temperature-sensitivity, increased percentage of active enzyme on a treatment of cells at high temperature, and increased expression of plasma membrane NADase, compared to wild type cells. Introduction of wild type ADPR pol gene into Cl-3527 cells partially restored the ADPR pol activity and the heat-resistance.     

A type-II DNA topoisomerase and a catenating protein from the transplantable VX2 carcinoma

It has recently been suggested that topoisomerases could be important targets for several DNA intercalating drugs used in cancer therapy. This prompted us to purify and characterize a type II topoisomerase in a highly tumorigenic transplantable rabbit tumor isolated from a skin carcinoma associated with cottontail rabbit papillomavirus. We have found that the decatenating activity present in tumor cells was 40-100 times higher than that in the rabbit liver, while no activity could be found in skin extracts. The type II topoisomerases purified from tumor and liver cells consist of two subunits with molecular masses of about 160 kDa. The conditions of the reactions of relaxation, unknotting and decatenation catalyzed by these topoisomerases II were found to be similar to those observed with enzymes of other eukaryotic cells. In the course of the purification of the VX2 enzyme, we isolated and characterized a protein of about 30 kDa in whose presence the topoisomerase II was able to catenate very efficiently supercoiled DNA molecules. This protein has the same electrophoretic mobility as an H1-2 histone, and cross-reacts with an anti-H1 antiserum. The VX2 topoisomerase II as well as the VX2 tumor should constitute useful models for assays of antitumoral drugs.     

In vitro and in vivo studies of Trypanosoma cruzi DNA polymerase

One major DNA polymerase has been purified and characterized from Trypanosoma cruzi. The enzyme has a sedimentation coefficient of 6.8 S corresponding to an approximate molecular weight of 180,000 assuming a globular shape. The enzyme recognizes activated DNA very efficiently, as well as synthetic polydeoxynucleotides, whereas poly rA-dT12 is very poorly utilized. Trypanosoma cruzi DNA polymerase is not inhibited at all by aphidicolin, while araCTP inhibits the enzyme very slightly. The purified enzyme is strongly inhibited by N-ethyl maleimide, dideoxyTTP, ethidium bromide and berenil. All our attempts to find a DNA polymerase sensitive to aphidicolin in vitro have failed, nor have we been able to find a low molecular weight DNA polymerase in this organism. However, when DNA synthesis was studied in whole trypanosomes, aphidicolin was shown to inhibit DNA synthesis more efficiently than ethidium bromide and berenil.     

In vitro analysis of a type I DNA topoisomerase activity from cultured tobacco cells

The role of DNA topoisomerases in plant cell metabolism is currently under investigation in our laboratory. Using a purified type I topoisomerase from cultured tobacco, we have carried out a biochemical characterization of enzymatic behavior. The enzyme relaxes negatively supercoiled DNA in the presence of MgCl₂, and to a lesser extent in the presence of KCl. Phosphorylation of the topoisomerase does not influence its activity and it is not stimulated by the presence of histones H1 or H5. The enzyme may act in either a processive or distributive manner depending on reaction conditions. The anti-tumor drug, camptothecin, induces significant breakage by the enzyme on purified DNA molecules unless destabilized by the addition of KCl. The tobacco topoisomerase I can catalyze the formation of stable nucleosomes on circular DNA templates, suggesting a role for the enzyme in chromatin assembly.