Topoisomerase function during replication-independent chromatin assembly in yeast.

DNA topoisomerases I and II are the two major nuclear enzymes capable of relieving torsional strain in DNA. Of these enzymes, topoisomerase I plays the dominant role in relieving torsional strain during chromatin assembly in cell extracts from oocytes, eggs, and early embryos. We tested if the topoisomerases are used differentially during chromatin assembly in Saccharomyces cerevisiae by a combined biochemical and pharmacological approach. As measured by plasmid supercoiling, nucleosome deposition is severely impaired in assembly extracts from a yeast mutant with no topoisomerase I and a temperature-sensitive form of topoisomerase II (strain top1-top2). Expression of wild-type topoisomerase II in strain top1-top2 fully restored assembly-driven supercoiling, and assembly was equally efficient in extracts from strains expressing either topoisomerase I or II alone. Supercoiling in top1-top2 extract was rescued by adding back either purified topoisomerase I or II. Using the topoisomerase II poison VP-16, we show that topoisomerase II activity during chromatin assembly is the same in the presence and absence of topoisomerase I. We conclude that both topoisomerases I and II can provide the DNA relaxation activity required for efficient chromatin assembly in mitotically cycling yeast cells.     

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.     

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.     

Characterization of a potent catenation activity of HeLa cell nuclei

Using an assay which measures catenation of a supercoiled DNA template, we have characterized and quantitated a potent activity identified in crude fractions of HeLa cell nuclei. Catenation requires Mg-ATP and a DNA-condensing agent, polyvinyl alcohol. A filter-binding or agarose gel assay can be used to quantitate activity. In this reaction, DNA topoisomerase I relaxes the input supercoiled DNA to provide DNA topoisomerase II, a strongly favored template for catenation. DNA topoisomerase II preferentially catenates relaxed DNA over supercoiled DNA by a factor of 100. One molecule of DNA topoisomerase II is able to catenate about 20 circles of relaxed DNA/min at 30 degrees C but only 0.16 circle of supercoiled DNA/min at 30 degrees C. The purified HeLa topoisomerase I can also catenate DNA under these assay conditions, yet in an ATP-independent fashion. It is much less efficient than topoisomerase II; one molecule of topoisomerase I catenates only about 3.8 X 10(-3) molecules of supercoiled DNA/min at 30 degrees C with a DNA template containing 5% nicked circles. This remarkable difference between the two enzymes allows quantitation of DNA topoisomerase II activity seen in the presence of excess topoisomerase I. Unlike Escherichia coli topoisomerase I (omega), catenation by the HeLa topoisomerase I is not stimulated by gapped circles.     

Patterns of strongly protein-associated simian virus 40 DNA replication intermediates resulting from exposures to specific topoisomerase poisons

Exposure of infected CV-1 cells to specific type I and type II topoisomerase poisons caused strong protein association with distinct subsets of simian virus 40 (SV40) DNA replication intermediates. On the basis of the known specificity and mechanisms of action of these drugs, the proteins involved are assumed to be the respective topoisomerases. Camptothecin, a topoisomerase I poison, caused strong protein association with form II (relaxed circular) and form III (linear) viral genomes and replication intermediates having broken DNA replication forks but not with form I (superhelical) viral DNA or normal late replication intermediates which were present. In contrast, type II topoisomerase poisons caused completely replicated forms and late viral replication forms to be tightly bound to protein--some to a greater extent than others. Different type II topoisomerase inhibitors caused distinctive patterns of protein association with the replication intermediates present. Both intercalating and nonintercalating type II topoisomerase poisons caused a small amount of form I (superhelical) SV40 DNA to be protein-associated in vivo. The protein complex with form I viral DNA was entirely drug-dependent and strong, but apparently noncovalent. The protein associated with form I DNA may represent a drug-stabilized "topological complex" between type II topoisomerase and SV40 DNA.     

Interleukin-2 induces the activities of DNA topoisomerase I and DNA topoisomerase II in HuT 78 cells

The induction by interleukin-2 of DNA topoisomerase I and DNA topoisomerase II activities in the human T cell line HuT 78 was investigated. HuT 78 cells were treated with 1000 U of interleukin-2/ml, and extracts of the HuT 78 nuclei were prepared over a 24 h period. The extracts were assayed quantitatively for the activities of DNA topoisomerase I and DNA topoisomerase II. Three concomitant, transient increases of 3- to 11-fold in the specific activities of both DNA topoisomerase I and DNA topoisomerase II were observed following treatment with IL-2 at 0.5, 4, and 10 h after treatment with interleukin-2. The specific activities of both enzymes returned to base-line values after each of these transient increases. These results reveal that the activities of DNA topoisomerase I and DNA topoisomerase II are highly regulated in HuT 78 cells upon treatment with IL-2.     

New insight for fluoroquinophenoxazine derivatives as possibly new potent topoisomerase I inhibitor

Fluoroquinolones, represented by ciproxacin and norfloxacin, are well-known clinical antimicrobial agents, and their phenyl ring expanded quinophenoxazines are reported as possible antitumor active compounds. These quinophenoxazines are known to inhibit DNA topoisomerase II essential for cell replication cycle. But there were no reports for topoisomerase I inhibition study for these compounds. In this report, we have prepared a few quinophenoxazine analogues and tested their topoisomerases I and II inhibitory activities and cytotoxicity. From the result, we found that quinophenoxazine analogues possessed strong topoisomerase I inhibitory capacity as well as topoisomerase II inhibition. Among the compounds prepared, A-62176 analogues showed strong topoisomerases I and II inhibitory activities. Interestingly, compound 8 missing the 3-aminopyrrolidine moiety at C2 position has similar potent inhibitory capacity against topoisomerases I and II at higher concentrations (20 and 10 microM, respectively). But compound 8 inhibited topoisomerase I function more selectively at lower concentration, 2 microM. Our observation might strongly implicate that fluoroquinophenoxazines can be developed as efficient topoisomerase I inhibitor with the elaborate modification.     

Topoisomerase activity is associated with purified SV40 T antigen.

Purified SV40 T antigen has been assayed for topoisomerase activity. The ability to relax negatively-supercoiled SV40 DNA was found in preparations of T antigen purified either from human 293 cells infected with Ad5-SVR111 virus or from insect Sf9 cells infected with recombinant baculovirus 941T. The T antigen-associated relaxing activity was stimulated by MgCl2 and was not dependent on ATP, suggesting that it is not due to cellular topoisomerase II. The topoisomerase activity was immunoprecipitated by a monoclonal antibody specific for T antigen, but not by a control monoclonal antibody. In addition, immunoblotting of purified T antigen from human 293 cells with antihuman topoisomerase I and anti-human topoisomerase II antibodies failed to detect cellular topoisomerases I or II. Sedimentation analysis of purified T antigen revealed that the topoisomerase activity co-sedimented with the hexameric form of T antigen at 23S. The topoisomerase activity is, therefore, either inherent to T antigen or due to a cellular topoisomerase I tightly bound to, and co-purifying with, T antigen.     

The involvement of topoisomerases and DNA polymerase I in the mechanism of induced thermal and radiation resistance in yeast

Either an ionizing radiation exposure or a heat shock is capable of inducing both thermal tolerance and radiation resistance in yeast. Yeast mutants, deficient in topoisomerase I, in topoisomerase II, or in DNA polymerase I, were used to investigate the mechanism of these inducible resistances. The absence of either or both topoisomerase activities did not prevent induction of either heat or radiation resistance. However, if both topoisomerase I and II activities were absent, the sensitivity of yeast to become thermally tolerant (in response to a heat stress) was markedly increased. The absence of only topoisomerase I activity (top1) resulted in the constitutive expression of increased radiation resistance equivalent to that induced by a heat shock in wild-type cells, and the topoisomerase I-deficient cells were not further inducible by heat. This heat-inducible component of radiation resistance (or its equivalent constitutive expression in top1 cells) was, in turn, only a portion of the full response inducible by radiation. The absence of polymerase I activity had no detectable effect on either response. Our results indicate that the actual systems that confer resistance to heat or radiation are independent of either topoisomerase activity or DNA polymerase function, but suggest that topoisomerases may have a regulatory role during the signaling of these mechanisms. The results of our experiments imply that maintenance of correct DNA topology prevents induction of the heat-shock response, and that heat-shock induction of a component of the full radiation resistance in yeast may be the consequence of topoisomerase I inactivation.     

Studies on DNA polymerases and topoisomerases in archaebacteria

We have isolated DNA polymerases and topoisomerases from two thermoacidophilic archaebacteria: Sulfolobus acidocaldarius and Thermoplasma acidophilum. The DNA polymerases are composed of a single polypeptide with molecular masses of 100 and 85 kDa, respectively. Antibodies against Sulfolobus DNA polymerase did not cross react with Thermoplasma DNA polymerase. Whereas the major DNA topoisomerase activity in S. acidocaldarius is an ATP-dependent type I DNA topoisomerase with a reverse gyrase activity, the major DNA topoisomerase activity in T. acidophilum is a ATP-independent relaxing activity. Both enzymes resemble more the eubacterial than the eukaryotic type I DNA topoisomerase. We have found that small plasmids from halobacteria are negatively supercoiled and that DNA topoisomerase II inhibitors modify their topology. This suggests the existence of an archaebacterial type II DNA topoisomerase related to its eubacterial and eukaryotic counterparts. As in eubacteria, novobiocin induces positive supercoiling of halobacterial plasmids, indicating the absence of a eukaryotic-like type I DNA topoisomerase that relaxes positive superturns.     

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.     

An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity

Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli, typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP-dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.     

Discrete Localization of Different DNA Topoisomerases in HeLa and K562 Cell Nuclei and Subnuclear Fractions

Monoclonal antibodies raised against DNA topoisomerase I and against topoisomerase II α and β isoforms, which have been previously demonstrated to be highly specific and capable of detecting cell cycle-related variations of the topoisomerase II isoforms (Negri et al., 1992, Exp. Cell Res. 200, 452-459), have been utilized for a fine subcellular localization. Immunocytochemistry by confocal and electron microscopy have been used for a topological and quantitative evaluation of the fine distribution of the different topoisomerases in HeLa and K562 cells. Topoisomerase I and topoisomerase II α are present both in the nucleoplasm and in the nucleolus, though at different relative ratios, while topoisomerase II β is exclusively present at the nucleolar level. This is further confirmed by immunoblotting and immunocytochemical quantitative evaluations performed on purified nuclear matrix fractions obtained from K562 cells. In fact, the amount of topoisomerase I and topoisomerase II α present in the whole cell nuclei is partly lost in isolated nuclei but, while topoisomerase I is further significantly reduced in nuclear matrix preparations, the topoisomerase II α content is only slightly decreased. On the other hand, the great majority of topoisomerase II β is retained in the nuclear matrix and can be detected exclusively in association with the nucleolar remnant. These results are consistent with specific functional roles hypothesized for the different topiosomerase types.     

Iridoids as DNA topoisomerase I poisons

The discovery of new topoisomerase I inhibitors is necessary since most of the antitumor drugs are targeted against type II and only a very few can specifically affect type I. Topoisomerase poisons generate toxic DNA damage by stabilization of the covalent DNA-topoisomerase cleavage complex and some have therapeutic efficacy in human cancer. Two iridoids, aucubin and geniposide, have shown antitumoral activities, but their activity against topoisomerase enzymes has not been tested. Here it was found that both compounds are able to stabilize covalent attachments of the topoisomerase I subunits to DNA at sites of DNA strand breaks, generating cleavage complexes intermediates so being active as poisons of topoisomerase I, but not topoisomerase II. This result points to DNA damage induced by topoisomerase I poisoning as one of the possible mechanisms by which these two iridoids have shown antitumoral activity, increasing interest in their possible use in cancer chemoprevention and therapy.     

Comparison of effects of fostriecin, novobiocin, and camptothecin, inhibitors of DNA topoisomerases, on DNA replication and repair in human cells.

Fostriecin causes a delayed inhibition of replicative DNA synthesis in human cells, consistent with a role for DNA topoisomerase II (its target enzyme) at a late stage in replication. Fostriecin does not inhibit UV-induced excision repair. The less specific inhibitor novobiocin blocks repair in permeabilised cells given a low dose of UV, presumably through a mechanism other than the inhibition of topoisomerase II. Its effect cannot be accounted for by a depletion of the ATP required for incision. Camptothecin, an inhibitor of DNA topoisomerase I, blocks replicative DNA synthesis immediately but incompletely, suggesting a participation of topoisomerase I at the replication fork, but it, too, has no influence on DNA repair. We thus find no evidence for involvement of either topoisomerase I or II in the response of cells to UV damage.