Isolation and partial characterization of two distinct DNA topoisomerases from cauliflower inflorescence

DNA topoisomerases which remove superhelical turns in closed circular DNA have been isolated from cauliflower inflorescences using polyethylene glycol fractionation, ammonium sulfate precipitation, and column chromatography on CM-Sephadex or CM-cellulose and DNA-cellulose. Two distinct enzymes, topoisomerase-I and ATP-dependent topoisomerase, were separated clearly by CM-Sephadex or CM-cellulose, and partially characterized using agarose gel electrophoresis with plasmid pBR322 DNA. Topoisomerase-I acts like other eucaryotic DNA topoisomerases in the absence of ATP, is stimulated by spermidine and inhibited by EDTA. The ATP-dependent topoisomerase acts like topoisomerase-I only in the presence of ATP in the reaction medium, is inhibited by spermidine and EDTA, and does not introduce supertwists into closed duplex DNA or produce catenate aggregates under the present reaction conditions.     

HMG17 protein facilitates the DNA catenation reaction catalyzed by DNA topoisomerases

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.     

The topoisomerase II inhibitor teniposide (VM-26) induces apoptosis in unstimulated mature murine lymphocytes.

This study shows that not only concanavalin A-stimulated proliferating lymphocytes but also unstimulated mouse splenic lymphocytes are sensitive to the topoisomerase II (topo II) inhibitor teniposide (VM-26). When unstimulated lymphocytes are pretreated with VM-26 for a 2-h period and are then incubated in drug-free medium, cell viability, as determined by trypan blue exclusion, decreases to 40% of the control by 6 h. The drug-treated cultures show two to three times the level of detergent soluble DNA than the control cultures and agarose gel electrophoresis of the soluble DNA shows the presence of oligonucleosomal-sized fragments, a feature considered to be a hallmark of apoptosis. Phase contrast microscopy, Hoechst staining for DNA, and immunofluorescence microscopy of various nuclear and cytoplasmic antigens (nucleolar fibrillarin, snRNP, ubiquitin, vimentin, tubulin) in the VM-26-treated cells characterize the morphological changes during apoptosis of these cells. The role of topo II as the mediator of the VM-26 effects is supported by pulsed field gel electrophoresis, which shows the typical topo II-induced cleavage of supercoiled DNA into loop-sized 300- and 50-kbp fragments. We conclude that the cancer chemotherapeutic agent VM-26 interacts with topo II and induces apoptosis in unstimulated lymphocytes.     

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation.     

Synthesis and topoisomerase II inhibitory and cytotoxic activity of oxiranylmethoxy- and thiiranylmethoxy-chalcone derivatives

In order to find potential anticancer drug candidate targeting topoisomerases enzyme, we have designed and synthesized oxiranylmethoxy- and thiiranylmethoxy-retrochalcone derivatives and evaluated their pharmacological activity including topoisomerases inhibitory and cytotoxic activity. Of the compounds prepared compound 25 showed comparable or better cytotoxic activity against cancer cell lines tested. Compound 25 inhibited MCF7 (IC₅₀: 0.49 ± 0.21 μM) and HCT15 (IC₅₀: 0.23 ± 0.02 μM) carcinoma cell growth more efficiently than references. In the topoisomerases inhibition test, all the compounds were inactive to topoisomerase I but moderate inhibitors to topoisomerase II enzyme. Especially, compound 25 inhibited topoisomerase II activity with comparable extent to etoposide at 100 μM concentrations. Correlation between cytotoxicity and topoisomerase II inhibitory activity implies that compound 25 can be a possible lead compound for anticancer drug impeding the topoisomerase II function.     

Induction of apoptosis in HT29 human intestinal epithelial cells by the cytotoxic enterotoxin of Aeromonas hydrophila.

The cytotoxic enterotoxin produced by Aeromonas hydrophila is considered to be the main virulence factor in gastrointestinal infections mediated by this pathogen. In this study, we examined the morphological and apoptotic effects of this toxin on HT29 cells, using light and electron microscopy in situ, as well as agarose gel electrophoresis of cell DNA. Cells treated with the cytotoxic enterotoxin became round and lost their polarity as well as their adhesion to each other and to the substrate. Cytoplasmic blebbing and nuclear condensation also occurred. DNA fragmentation was detected by TUNEL labelling and agarose gel electrophoresis. These results show that the cytotoxic enterotoxin of A. hydrophila can induce apoptosis in human intestinal cells in culture.     

Supercondensed structure of plasmid pBR322 DNA in an Escherichia coli DNA topoisomerase II mutant

An unusual structural component, supercondensed pBR322 DNA, has been found in plasmid pBR322 DNA samples isolated from a DNA topoisomerase II mutant of Escherichia coli, SD108 (topA+, gyrB225). The supercondensed pBR322 DNA moved faster than supercoiled pBR322 DNA as a homogeneous band in agrose gels when the DNA samples were analysed by electrophoresis. The mobility of the supercondensed DNA was not substantially affected by chloroquine intercalation. The supercondensed pBR322 DNA migrated as a high density "third DNA band" when the samples were subjected to caesium chloride/ethidium bromide gradient equilibrium centrifugation. The unusual pBR322 DNA visualized by electron microscopy was a globoid-shaped particle. These observations suggest that the pBR322 plasmid can assume a tertiary structure other than a supercoiled or relaxed structure. DNA topoisomerases may be involved in the supercondensation of plasmid DNA and chromosomal DNA.     

Inhibition of Zn(II) Binding Type IA Topoisomerases by Organomercury Compounds and Hg(II)

Type IA topoisomerase activities are essential for resolving DNA topological barriers via an enzyme-mediated transient single strand DNA break. Accumulation of topoisomerase DNA cleavage product can lead to cell death or genomic rearrangement. Many antibacterial and anticancer drugs act as topoisomerase poison inhibitors that form stabilized ternary complexes with the topoisomerase covalent intermediate, so it is desirable to identify such inhibitors for type IA topoisomerases. Here we report that organomercury compounds were identified during a fluorescence based screening of the NIH diversity set of small molecules for topoisomerase inhibitors that can increase the DNA cleavage product of Yersinia pestis topoisomerase I. Inhibition of relaxation activity and accumulation of DNA cleavage product were confirmed for these organomercury compounds in gel based assays of Escherichia coli topoisomerase I. Hg(II), but not As(III), could also target the cysteines that form the multiple Zn(II) binding tetra-cysteine motifs found in the C-terminal domains of these bacterial topoisomerase I for relaxation activity inhibition. Mycobacterium tuberculosis topoisomerase I activity is not sensitive to Hg(II) or the organomercury compounds due to the absence of the Zn(II) binding cysteines. It is significant that the type IA topoisomerases with Zn(II) binding domains can still cleave DNA when interfered by Hg(II) or organomercury compounds. The Zn(II) binding domains found in human Top3α and Top3β may be potential targets of toxic metals and organometallic complexes, with potential consequence on genomic stability and development.     

Gossypol Interferes with Both Type I and Type II Topoisomerase Activities Without Generating Strand Breaks

A considerable number of agents with chemotherapeutic potentials reported over the past years were shown to interfere with the reactions of DNA topoisomerases, the essential enzymes that regulate conformational changes in DNA topology. Gossypol, a naturally occurring bioactive phytochemical is a chemopreventive agent against various types of cancer cell growth with a reported activity on mammalian topoisomerase II. The compounds targeting topoisomerases vary in their mode of action; class I compounds act by stabilizing covalent topoisomerase-DNA complexes resulting in DNA strand breaks while class II compounds interfere with the catalytic function of topoisomerases without generating strand breaks. In this study, we report Gossypol as the interfering agent with type I topoisomerases as well. We also carried out an extensive set of assays to analyze the type of interference manifested by Gossypol on DNA topoisomerases. Our results strongly suggest that Gossypol is a potential class II inhibitor as it blocked DNA topoisomerase reactions with no consequently formed strand breaks.     

Mammalian topoisomerase II activity is modulated by the DNA minor groove binder distamycin in simian virus 40 DNA

DNA topoisomerases II are nuclear enzymes that have been identified recently as targets for some of the most active anticancer drugs. Antitumor topoisomerase II inhibitors such as teniposide (VM-26) produce enzyme-induced DNA cleavage and inhibition of enzyme activity. By adding to such reactions distamycin, a compound whose effects on DNA have been extensively characterized, we investigated the effects of drug binding upon topoisomerase II-mediated DNA cleavage induced by VM-26. We have found a correspondence between distamycin binding (determined by footprinting analysis) and topoisomerase II-mediated cleavage of SV40 DNA (determined by sequencing gel analysis). Distamycin binding potentiated the cleavage of specific sites in the near proximity of distamycin-binding sites (within at least 25 base pairs), which indicates that DNA secondary structure is involved in topoisomerase II-DNA interactions. That distamycin potentiated cleavage only at sites that were recognized in the absence of distamycin and suppressed cleavage directly at distamycin-binding sites indicates that topoisomerase II recognizes DNA on the basis of primary sequence. In addition, distamycin stimulated topoisomerase II-mediated DNA relaxation and antagonized the inhibitory effect of VM-26. These results show that the DNA sequence-specific binding of distamycin produces local and propagated effects in the DNA which markedly affect topoisomerase II activity.     

Role of topoisomerases in cytotoxicity induced by DNA ligand Hoechst-33342 and UV-C in a glioma cell line

DNA ligand Hoechst-33342 significantly enhances UV induced cytotoxicity in human glioma cell lines (BMG-1 & U-87) with supra additive increase in cell death, cytogenetic damage, cell cycle delay, apoptosis and inhibition of PLDR. Cytotoxicity of Hoechst-33342 arises due to its interference in the breakage-rejoining reaction of DNA topoisomerases by stabilization of cleavable complexes. Since topoisomerases have also been implicated in the generation of potentially lethal DNA breaks by interaction with various types of DNA damage including UV induced DNA lesions, we investigated in present studies the role of functional topoisomerases in the synergistic cytotoxicity of Hoechst-33342 and UV in a human glioma cell line (BMG-1). Topoisomerase I activity analyzed by the plasmid relaxation assay, was significantly enhanced upon UV irradiation, implying a possible role of this enzyme in the processing of UV induced lesions. However, this increase in the activity was reduced by >50% in cells incubated with Hoechst-33342 for 1 hr prior to irradiation. Imunoflowcytometric analysis of the chromatin bound topoisomerases I and II levels (cleavable complex) using topoisomerases I and II anti-antibodies showed a good correlation between the induction of apoptosis by Hoechst-33342 and UV and enhancement in the level of topoisomerase II mediated cleavable complexes. Induction of apoptosis was associated with a decline in the level of Bcl2. Taken together, these studies show that supra additive cytotoxic effects of UV-C and Hoechst-33342 in BMG-1 cells are consequences of enhanced stabilization of topo II mediated cleavable complexes and alterations in specific signal transduction pathways of apoptosis, besides the inhibition of topoisomerase mediated repair processes.     

Topoisomerases of kinetoplastid parasites: why so fascinating?

DNA topoisomerases are the key enzymes involved in carrying out high precision DNA transactions inside the cells. However, they are detrimental to the cell when a wide variety of topoisomerase-targeted drugs generate cytotoxic lesions by trapping the enzymes in covalent complexes on the DNA. The discovery of unusual heterodimeric topoisomerase I in kinetoplastid family added a new twist in topoisomerase research related to evolution, functional conservation and their preferential sensitivity to Camptothecin. On the other hand, structural and mechanistic studies on kinetoplastid topoisomerase II delineate some distinguishing features that differentiate the parasitic enzyme from its prokaryotic and eukaryotic counterparts. This review summarizes the recent advances in research in kinetoplastid topoisomerases, their evolutionary significance and the death of the unicellular parasite Leishmania donovani induced by topoisomerase I inhibitor camptothecin.     

Expression, purification and characterization of recombinant mitochondrial topoisomerase II of kinetoplastid Crithidia fasciculata in High-five insect cells

Topoisomerase II of kinetoplastid parasites plays an important role in the replication of unusual networks of kinetoplast DNA (kDNA) and is a very useful target for antiparasitic drugs. In this study, we cloned full-length Crithidia fasciculata mitochondrial topoisomerase II gene into pFastBac-HTc vector and successfully expressed an active recombinant full-length mitochondrial topoisomerase II in Bac-to-Bac baculovirus expression system. A rapid and simple purification strategy was established by incorporating a FLAG-tag at the C-terminus of the protein. The purified recombinant topoisomerase II showed a major single band on SDS-PAGE (>96% purity) and was verified through Western blot analysis. The recombinant full-length mitochondrial topoisomerase II exhibited decatenation, catenation and relaxation activity of type II topoisomerase as well as various sensitivities to a series of known topoisomerase inhibitors. These studies explore new way and lay groundwork to express all other similar full-length kinetoplastid topoisomerases, it will also facilitate further elucidation of X-ray structure, catalysis mechanism of kinetoplastid topoisomerases and design of new antiparasitic drugs targeting kinetoplastid topoisomerases.     

Induction of mammalian DNA topoisomerase I and II mediated DNA cleavage by saintopin, a new antitumor agent from fungus.

Saintopin is an antitumor antibiotic recently discovered in mechanistically oriented screening using purified calf thymus DNA topoisomerases. Saintopin induced topoisomerase I mediated DNA cleavage comparable to that of camptothecin, and topoisomerase II mediated DNA cleavage equipotent to those of 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) or 4'-demethylepipodophyllotoxin 9-(4,6-O-ethylidene-beta-D-glucopyranoside) (VP-16). Treatment of a reaction mixture containing saintopin and topoisomerase I or II with either elevated temperature (65 degrees C) or higher salt concentration (0.5 M NaCl) resulted in a substantial reduction in DNA cleavage, suggesting that the topoisomerase I and II mediated DNA cleavage induced by saintopin is through the mechanism of stabilizing the reversible enzyme-DNA "cleavable complex". Consistent with the cleavable complex formation with both topoisomerases, saintopin inhibited catalytic activities of both topoisomerase I and topoisomerase II. The DNA cleavage intensity pattern induced by saintopin with topoisomerase I was different from that by camptothecin. A difference in cleavage pattern was also detected between saintopin and m-AMSA or VP-16 in topoisomerase II mediated DNA cleavage. DNA unwinding assay using T4 DNA ligase showed that saintopin is a weak DNA intercalator like m-AMSA. Thus, saintopin represents a new class of antitumor agent that can induce both mammalian DNA topoisomerase I and mammalian DNA topisomerase II mediated DNA cleavage.     

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.     

High-throughput fluorescence flow-injection topoisomerase II inhibition assay

A high-throughput fluorescence flow-injection assay is described, suitable for determining the catalytic inhibition of DNA topoisomerase II. The method, which separates high molecular mass trypanosome kinetoplast DNA from its decatenated product by centrifugation, should be useful for the rapid and accurate screening of potential anticancer topoisomerase II inhibitors and the determination of their inhibition constants. Advantages of the flow-injection method over agarose gel electrophoresis and radioactive centrifugation assays are that it is faster, more sensitive, highly linear in its response to product formation, and does not require the production of radioactive trypanosome kinetoplast DNA substrate.