Eukaryotic DNA topoisomerase I reaction is topology dependent.

The effects of supercoiling on the topoisomerization reaction by eukaryotic DNA topoisomerases I have been analyzed. The systems used were: DNA topoisomerase I from wheat germ, chicken erythrocyte and calf thymus on a 2.3 kb DNA fragment which encompasses the immunoglobulin kappa-light chain (L kappa) promoter of the mouse plasmacytoma MPC11; S. cerevisiae DNA topoisomerase I on a 2.2 kb DNA fragment from the same organism which encompasses the regulatory and the coding region of the ADH II gene; wheat germ DNA topoisomerase I on the plasmid pUC18. It was found in every system that lack of torsional stress prevents topoisomerization of the substrate. A simple regulatory model of DNA topoisomerase I function, based on topological considerations, is presented.     

Eukaryotic topoisomerase II. Characterization of enzyme turnover

While the binding of adenyl-5'-yl imidodiphosphate (App(NH)p) to Drosophila melanogaster topoisomerase II induces a double-stranded DNA passage reaction, its nonhydrolyzable beta,gamma-imidodiphosphate bond prevents enzyme turnover (Osheroff, N., Shelton, E. R., and Brutlag, D. L. (1983) J. Biol. Chem. 258, 9536-9543). Therefore, this ATP analog was used to characterize the interactions between Drosophila topoisomerase II and DNA which occur after DNA strand passage but before enzyme turnover. In the presence of App(NH)p, a stable post-strand passage topoisomerase II-nucleic acid complex is formed when circular DNA substrates are employed. Although noncovalent in nature, these complexes are resistant to increases in ionic strength and show less than 5% dissociation under salt concentrations (greater than 500 mM) that disrupt 95% of the enzyme-DNA interactions formed in the absence of App(NH)p or under a variety of other conditions that do not support DNA strand passage. These results strongly suggest that the process of enzyme turnover not only regenerates the active conformation of topoisomerase II but also confers upon the enzyme the ability to disengage from its nucleic acid product. Experiments with linear DNA molecules indicate that after strand passage has taken place, topoisomerase II may be able to travel along its DNA substrate by a linear diffusion process that is independent of enzyme turnover. Further studies demonstrate that the regeneration of the enzyme's catalytic center does not require enzyme turnover, since topoisomerase II can cleave double-stranded DNA substrates after strand passage has taken place. Finally, while the 2'-OH and 3'-OH of ATP are important for its interaction with Drosophila topoisomerase II, neither are required for turnover.     

Biochemical characterization of topoisomerase I purified from avian erythrocytes.

A type I topoisomerase has been purified from avian erythrocyte nuclei. The most pure fraction contains one major polypeptide of Mr = 105,000 (80% of total) and several minor ones of lower molecular weight. Active forms of the topoisomerase were identified by covalently binding the enzyme to 32P-DNA, digesting with nuclease and detecting 32P labeled peptides by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Topoisomerase activity, as measured by the ability to covalently bind DNA, is associated with the following peptides: Mr = 105, 83, 54 and 30,000. The similar chromatographic properties of the various forms of topoisomerase suggests a common structural identity as previously proposed for the HeLa topoisomerase I (Liu, L.F. and Miller, K.G. (1981) Proc. Natl. Acad. Sci. USA 78, 3487-3491). The avian enzyme is similar to other eucaryotic type I DNA topoisomerases in that it covalently binds double and single stranded DNA forming an enzyme linked to the 3'-phosphoryl end and after binding to single stranded DNA it can transfer the single stranded donor DNA to an acceptor DNA possessing 5'-OH end groups. The binding site size of topoisomerase on DNA has also been determined using micrococcal nuclease to digest unprotected DNA in the native enzyme/DNA complex. The enzyme blocks access to the helix over a span of 25 bp. These findings are discussed in light of the distribution and function of topoisomerase I in chromatin.     

Mode of action of topoisomerase II-targeting agents at a specific DNA sequence. Uncoupling the DNA binding, cleavage and religation events.

Methods of uncoupling the DNA binding, cleavage and religation reactions of topoisomerase II were employed to investigate the influence of topoisomerase II-directed drugs on the individual steps in the enzyme's catalytic cycle. A special DNA substrate containing a major topoisomerase II interaction site, which can be cleaved by the enzyme in the absence of any concomitant religation, was used to examine the effect of topoisomerase II-directed agents upon the DNA cleavage reaction. The experiment demonstrated that the topoisomerase II targeting agent Ro 15-0216 stimulates the DNA cleavage reaction extensively, whereas the traditional topoisomerase II inhibitor, mAMSA, has only a minor effect on this reaction. Topoisomerase II trapped in the cleavage complexes can religate to the 3' hydroxyl end of another DNA strand. Using this religation assay, it was demonstrated that the major effect of mAMSA is an inhibition of the enzyme's religation reaction, whereas Ro 15-0216 has no effect on this reaction. Recently, considerable attention has been given to drugs preventing topoisomerase II from introducing DNA cleavages. In the present paper the initial non-covalent DNA binding reaction of topoisomerase II was investigated under conditions excluding enzyme-mediated DNA cleavage. This demonstrated that the anthracycline, aclarubicin, prevents topoisomerase II from performing its initial non-covalent DNA binding reaction and thereby abolishes the DNA cleavage reaction of the enzyme. The results presented here demonstrate that profound differences exist in the mode of action of different agents targeting topoisomerase II, and that the enzyme can be affected by such agents at both its DNA binding, cleavage and religation subreactions.     

Human autoantibody to topoisomerase II

The rheumatic diseases are characterized by the production of autoantibodies that are usually directed against components of the cell nucleus. In this communication, we describe autoantibodies that recognize DNA topoisomerase II (anti-topoII) present in the serum of a patient with systemic lupus erythematosus. Several lines of evidence indicate that this antibody recognizes topoisomerase II. First, it binds to the native enzyme in soluble extracts prepared from isolated chromosomes and effectively depletes such extracts of active enzyme. Second, the serum binds to topoisomerase II in immunoblots of mitotic chromosomes and chromosome scaffolds. Finally, the antiserum binds strongly to a fusion protein encoded by a cloned cDNA and expressed in Escherichia coli that (based on immunological evidence) represents the carboxy-terminal portion of chicken topoisomerase II. Autoantibodies such as the one described here may provide useful reagents for the study of human topoisomerase II.     

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.     

Human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for DNA ligation. Use of 5' covalently activated oligonucleotide substrates to study enzyme mechanism

Despite the importance of topoisomerase II-mediated DNA ligation to the essential physiological functions of the enzyme, the mechanistic details of this important reaction are poorly understood. Because topoisomerase II normally does not release cleaved DNA molecules prior to ligation, it is not known whether all of the nucleic acid specificity of its cleavage/ligation cycle is embodied in DNA cleavage or whether ligation also contributes specificity to the enzyme. All currently available ligation assays require that topoisomerase II cleave the initial DNA substrate before rejoining can be monitored. Consequently, it has been impossible to examine the specificity of DNA ligation separately from that of scission. To address this issue, a cleavage-independent topoisomerase II DNA ligation assay was developed. This assay utilizes a nicked oligonucleotide whose 5'-phosphate terminus at the nick has been activated by covalent attachment to the tyrosine mimic, p-nitrophenol. Human topoisomerase IIalpha and enzymes with active-site mutations that abrogated cleavage activity ligated the activated nick by catalyzing the direct attack of the terminal 3'-OH on the activated 5'-phosphate. Results with different DNA sequences indicate that human topoisomerase IIalpha possesses an intrinsic nucleic acid specificity for ligation that parallels its specificity for DNA cleavage.     

Escherichia coli DNA topoisomerase III: purification and characterization of a new type I enzyme

A new topoisomerase capable of relaxing negatively supercoiled DNA in Escherichia coli has been identified during chromatography on novobiocin-Sepharose. A simple and reproducible purification procedure is described to obtain this enzyme, called topoisomerase III (topo III), in a homogeneous form. The protein is a single polypeptide with a molecular weight of 74 000 +/- 2000 and is a type I topoisomerase, changing the linking number of DNA circles in steps of one. It is present in deletion strains lacking the topA gene and further differs from the well-studied topoisomerase I (omega protein; Eco topo I) in (1) its requirement for K+ in addition to Mg2+ to exhibit optimal activity and (2) its affinity to novobiocin-Sepharose. Positively supercoiled DNA is not relaxed during exposure to the enzyme. Topo III has no ATPase activity, and ATP does not show any discernible effect on the reduction of superhelical turns. The purified topoisomerase has no supercoiling activity and is unaffected by high concentrations of oxolinic acid and novobiocin in the relaxing reaction. Single-stranded DNA and spermidine strongly inhibit the topoisomerase activity.     

Localization of topoisomerase II in mitotic chromosomes

In the preceding article we described a polyclonal antibody that recognizes cSc-1, a major polypeptide component of the chicken mitotic chromosome scaffold. This polypeptide was shown to be chicken topoisomerase II. In the experiments described in the present article we use indirect immunofluorescence and immunoelectron microscopy to examine the distribution of topoisomerase II within intact chromosomes. We also describe a simple experimental protocol that differentiates antigens that are interspersed along the chromatin fiber from those that occupy restricted domains within the chromosome. These experiments indicate that the distribution of the enzyme appears to be independent of the bulk chromatin. Our data suggest that topoisomerase II is bound to the bases of the radial loop domains of mitotic chromosomes.     

Furanocoumarins: Novel topoisomerase I inhibitors from Ruta graveolens L.

Topoisomerase I inhibitors from Ruta graveolens are reported for the first time. Potent topoisomerase I inhibitory activity from in vitro culture extracts R. graveolens were observed. Stabilization of DNA–topoisomerase covalent complex was observed in all the tested extracts. The mechanism of topoisomerase inhibition was determined by preincubation studies. The irreversible topoisomerase I mediated relaxation of plasmid in enzyme–substrate preincubation study, indicated that the observed inhibitory activity of extract constituents was not mediated through conformational changes in the DNA. Furthermore, the affinity of inhibitors with the enzyme was tested by enzyme–extract preincubation study. Increase in inhibition of topoisomerase activity and promotion of DNA–enzyme complex was observed after enzyme–extract preincubation. The activity could be assigned to furanocoumarins—psoralen, bergapten and xanthotoxin, identifying them as novel, potent topoisomerase I inhibitors.     

Processing of nucleopeptides mimicking the topoisomerase I-DNA covalent complex by tyrosyl-DNA phosphodiesterase

Tyrosyl-DNA phosphodiesterase-1 (Tdp1) is the only known enzyme to remove tyrosine from complexes in which the amino acid is linked to the 3′-end of DNA fragments. Such complexes can be produced following DNA processing by topoisomerase I, and recent studies in yeast have demonstrated the importance of TDP1 for cell survival following topoisomerase I-mediated DNA damage. In the present study, we used synthetic oligodeoxynucleotide–peptide conjugates (nucleopeptides) and recombinant yeast Tdp1 to investigate the molecular determinants for Tdp1 activity. We find that Tdp1 can process nucleopeptides with up to 13 amino acid residues but is poorly active with a 70 kDa fragment of topoisomerase I covalently linked to a suicide DNA substrate. Furthermore, Tdp1 was more effective with nucleopeptides with one to four amino acids than 15 amino acids. Tdp1 was also more effective with nucleopeptides containing 15 nt than with homolog nucleopeptides containing 4 nt. These results suggest that DNA binding contributes to the activity of Tdp1 and that Tdp1 would be most effective after topoisomerase I has been proteolyzed in vivo.     

Cleavage of DNA by mammalian DNA topoisomerase II

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.     

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.     

Purification and characterization of yeast topoisomerase I

Yeast topoisomerase I (Mr = 76,000) has been purified to 80% homogeneity using a combination of ion exchange, gel filtration, and DNA-cellulose chromatography. The enzyme was characterized with respect to its ability to relax supercoiled DNA and to catenate nicked circular DNA. Yeast topoisomerase I will remove both positive and negative turns in DNA supercoils in the absence of ATP and magnesium ion. The products of the catenating activity of the enzyme were examined on agarose gels and in the electron microscope. These analyses indicate that yeast topoisomerase I will generate large catenated DNA networks which appear to rearrange to multimeric linear structures upon long incubation time.     

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.     

ATP-independent DNA topoisomerase II as potential drug target in trypanosomes

Two categories of trypanosomal type II topoisomerases have been isolated from trypanosomes: one is unique since it is able to realize DNA topoisomerization reactions in the absence of ATP, in contrast to the other enzyme and mammalian topoisomerase II. The biochemical properties of ATP-independent topoisomerase II from Trypanosoma cruzi are described in this report. The enzyme can decatenate trypanosome kinetoplast DNA networks, catenate supercoiled DNA molecules, unknot P4 phage DNA, and cleave double-stranded DNA. The enzyme is inhibited by various classes of drugs and is more sensitive than mammalian topoisomerase II. Therefore, trypanosome ATP-independent topoisomerase II provides a potential target for chemotherapy.     

Role of the divalent cation in topoisomerase II mediated reactions

The effects of magnesium ions on interactions between Drosophila melanogaster topoisomerase II and its substrates were assessed by a number of kinetic and binding assays. Results indicated that the divalent cation plays two distinct functions in promoting enzyme-substrate interactions. One class of magnesium ions participates directly in enzyme-mediated DNA cleavage reactions. A second class of magnesium ions participates directly in topoisomerase II mediated ATPase reactions and functions by providing the enzyme with a magnesium-ATP substrate. In contrast, the divalent cation did not affect the quaternary structure of the enzyme, was not required for the site-specific binding of topoisomerase II to DNA, and did not affect the enzyme's ability to discern the topological state of its nucleic acid substrate.     

Cloning and characterization of Drosophila topoisomerase IIIbeta. Relaxation of hypernegatively supercoiled DNA

We cloned cDNA encoding Drosophila DNA topoisomerase III. The top3 cDNA encodes an 875-amino acid protein, which is nearly 60% identical to mammalian topoisomerase IIIbeta enzymes. Similarity between the Drosophila protein and the topoisomerase IIIbetas is particularly striking in the carboxyl-terminal region, where all contain eight highly conserved CXXC motifs not found in other topoisomerase III enzymes. We therefore propose the Drosophila protein is a member of the beta-subfamily of topoisomerase III enzymes. The top3beta gene is a single-copy gene located at 5 E-F on the X chromosome. P-element insertion into the 5'-untranslated region of this gene affects topoisomerase IIIbeta protein levels, but not the overall fertility and viability of the fly. We purified topoisomerase IIIbeta to near homogeneity and observed relaxation activity only with a hypernegatively supercoiled substrate, but not with plasmid DNA directly isolated from bacterial cells. Despite this difference in substrate preference, the degree of relaxation of the hypernegatively supercoiled substrate is comparable to relaxation of plasmid DNA by other type I enzymes. Drosophila topoisomerase IIIbeta forms a covalent linkage to 5' DNA phosphoryl groups, and the DNA cleavage reaction prefers single-stranded substrate over double-stranded, suggesting an affinity of this enzyme for DNA with non-double-helical structure.     

Yeast DNA topoisomerase II. An ATP-dependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings

An activity from the yeast Saccharomyces cerevisiae, initially noted for its catalysis of aggregation of covalently closed double-stranded DNA rings in the presence of ATP, has been identified as a type II DNA topoisomerase and is designated yeast DNA topoisomerase II. The formation of the DNA aggregate, which has been shown to be a network of DNA rings that are topologically interlocked, requires the presence of a yeast DNA-binding protein in addition to the topoisomerase. In the absence of the binding protein, yeast DNA topoisomerase II catalyzes decatenation and unknotting of duplex DNA rings and the relaxation of negatively or positively supercoiled DNA. All reactions are ATP-dependent and require Mg(II). Similar to other eukaryotic and phage T4-type II DNA topoisomerases, the yeast enzyme does not catalyze DNA supercoiling under the assay conditions employed. The activity is not sensitive to the gyrase inhibitor nalidixic acid, oxolinic acid, or novobiocin. Coumermycin inhibits the activity, however, at a concentration as low as 5 microgram/ml.