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.     

Identification of the yeast TOP3 gene product as a single strand-specific DNA topoisomerase.

The TOP3 gene of the yeast Saccharomyces cerevisiae was postulated to encode a DNA topoisomerase, based on its sequence homology to Escherichia coli DNA topoisomerase I and the suppression of the poor growth phenotype of top3 mutants by the expression of the E. coli enzyme (Wallis, J.W., Chrebet, G., Brodsky, G., Golfe, M., and Rothstein, R. (1989) Cell 58, 409-419). We have purified the yeast TOP3 gene product to near homogeneity as a 74-kDA protein from yeast cells lacking DNA topoisomerase I and overexpressing a plasmid-borne TOP3 gene linked to a phosphate-regulated yeast PHO5 gene promoter. The purified protein possesses a distinct DNA topoisomerase activity: similar to E. coli DNA topoisomerases I and III, it partially relaxes negatively but not positively supercoiled DNA. Several experiments, including the use of a negatively supercoiled heteroduplex DNA containing a 29-nucleotide single-stranded loop, indicate that the activity has a strong preference for single-stranded DNA. A protein-DNA covalent complex in which the 74-kDa protein is linked to a 5' DNA phosphoryl group has been identified, and the nucleotide sequences of 30 sites of DNA-protein covalent complex formation have been determined. These sequences differ from those recognized by E. coli DNA topoisomerase I but resemble those recognized by E. coli DNA topoisomerase III. Based on these results, the yeast TOP3 gene product can formally be termed S. cerevisiae DNA topoisomerase III. Analysis of supercoiling of intracellular yeast plasmids in various DNA topoisomerase mutants indicates that yeast DNA topoisomerase III has at most a weak activity in relaxing negatively supercoiled double-stranded DNA in vivo, in accordance with the characteristics of the purified enzyme.     

High mobility group protein 1 preferentially conserves torsion in negatively supercoiled DNA

HMG 1 is known to bind to a variety of DNAs and to unwind nicked and closed circular DNA. We now report evidence that it has a significantly higher unwinding angle on negatively supercoiled DNA than on the other torsional forms. The degree of unwinding observed on nicked circular DNA depends on the purity of the HMG 1 preparation used. HMG 1 from CM-Sephadex has an unwinding angle of 28.8 degrees, compared to 7.2 degrees for the purer preparation obtained from Mono S, suggesting that contaminating strand-separating activity is removed by the additional purification step. The subsequent studies on closed circular forms of DNA were all performed using the purer HMG 1. After preincubation of highly negatively supercoiled DNA (sigma = -0.040) with HMG 1, the DNA-protein mixture was relaxed with Escherichia coli topoisomerase I. At molar ratios of less than 100:1 (HMG 1 to DNA), negatively supercoiled DNA displays a dose-dependent change in the linking number, indicating an unwinding angle of 57.6 degrees. HMG 1 protects 50% of highly negatively supercoiled DNA from E. coli topoisomerase I at a molar ratio of 100:1, and protects all supercoils at a molar ratio of 200:1, indicating saturation of the DNA at this concentration. HMG 1 also protects highly negatively supercoiled DNA from calf thymus topoisomerase I, with an apparent unwinding angle of 57.6 degrees. Moderately negatively supercoiled DNA (sigma = -0.018), but not moderately positively supercoiled DNA (sigma = +0.011), competes for the protective effect of HMG 1 on highly negatively supercoiled DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transition protein 4 from boar late spermatid nuclei is a topological factor that stimulates DNA-relaxing activity of topoisomerase I

Transition protein 4 (TP4) from boar late spermatid nuclei, having higher affinity for double-stranded DNA and a local melting activity of DNA, stimulated SV40 DNA-relaxing activity of eukaryotic topoisomerase I at TP4/DNA molar ratios of 6.6–11. A TP4-spermidine mixture stimulated the activity of topoisomerase I much more than spermidine alone, but no more than TP4 alone, and poly-l-arginine did not. These results suggest that TP4 contributes to the chromatin reorganization in the late spermatid nuclei from nucleosomal-type structure with negatively supercoiled DNA to nucleoprotamine structure with no supercoiled DNA.     

Purification and characterization of a novel ATP-independent type I DNA topoisomerase from a marine methylotroph

DNA topoisomerase is involved in DNA repair and replication. In this study, a novel ATP-independent 30-kDa type I DNA topoisomerase was purified and characterized from a marine methylotroph, Methylophaga sp. strain 3. The purified enzyme composed of a single polypeptide was active over a broad range of temperature and pH. The enzyme was able to relax only negatively supercoiled DNA. Mg(2+) was required for its relaxation activity, while ATP gave no effect. The enzyme was clearly inhibited by camptothecin, ethidium bromide, and single-stranded DNA, but not by nalidixic acid and etoposide. Interestingly, the purified enzyme showed Mn(2+)-activated endonuclease activity on supercoiled DNA. The N-terminal sequence of the purified enzyme showed no homology with those of other type I enzymes. These results suggest that the purified enzyme is an ATP-independent type I DNA topoisomerase that has, for the first time, been characterized from a marine methylotroph.     

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.     

On the mechanism of topoisomerase I inhibition by camptothecin: evidence for binding to an enzyme-DNA complex

Camptothecin, a cytotoxic antitumor compound, has been shown to produce protein-linked DNA breaks mediated by mammalian topoisomerase I. We have investigated the mechanism by which camptothecin disrupts DNA processing by topoisomerase I and have examined the effect of certain structurally related compounds on the formation of a DNA-topoisomerase I covalent complex. Enzyme-mediated cleavage of supercoiled plasmid DNA in the presence of camptothecin was completely reversed upon the addition of exogenous linear DNA or upon dilution of the reaction mixture. Camptothecin and topoisomerase I produced the same amount of cleavage from supercoiled DNA or relaxed DNA. In addition, the alkaloid decreased the initial velocity of supercoiled DNA relaxation mediated by catalytic quantities of topoisomerase I. Inhibition occurred under conditions favoring processive catalysis as well as under conditions favoring distributive catalysis. By use of [3H]camptothecin and an equilibrium dialysis assay, the alkaloid was shown to bind reversibly to a DNA-topoisomerase I complex, but not to isolated enzyme or isolated DNA. These results are consistent with a model in which camptothecin reversibly traps an intermediate involved in DNA unwinding by topoisomerase I and thereby perturbs a set of equilibria, resulting in increased DNA cleavage. By examining certain compounds that are structurally related to camptothecin, it was found that the 20-hydroxy group, which has been shown to be essential for antitumor activity, was also necessary for stabilization of the covalent complex between DNA and topoisomerase I. In contrast, no such correlation existed for UV-light-induced cleavage of DNA by Cu(II)-camptothecin derivatives.     

Characterization of topoisomerase I and II activities in nuclear extracts during callogenesis in immature embryos of Zea mays

We have characterized the topoisomerase I and II activities in nuclear extracts from immature embryos of Zea mays and the effect of the treatment with 2,4-dichlorophenoxyacetic acid (2,4-D) and abscisic acid (ABA). These extracts were shown to be essentially devoid of protease and nuclease activities and they were tested for their ability to relax supercoiled DNA, unknotting P4 DNA and catenate circular duplex DNA under catalytic conditions. Unknotting and catenation reactions are strictly magnesium- and ATP-dependent, but not the relaxation of circular supercoiled DNA allowing the detection of both topoisomerase I and II activities. Two cytotoxic drugs, camptothecin, a plant alkaloid that inhibits cukaryotic topoisomerase I, and epipodophyllotoxin VM-26 (teniposide) that inhibits topoisomerase II, have been assayed in our extracts showing similar inhibitory effects on topoisomerase enzymes. Alkaline phosphatase treatment of nuclear extracts abolishes both topoisomerase activities. Nuclear extracts from embryos treated with 2,4-D showed 200% increase on topoisomerase II activity as compared with untreated ones, but only residual activity was detected in ABA-treated embryos. Nuclear extracts from hormone-treated and untreated embryos showed similar topoisomerase I activity with deviations of less than 25%. These differences are discussed in terms of possible post-translational modifications of the enzymes associated with the increase in proliferation activity of calli.     

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.     

The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs

Collisions with DNA tracking systems are critical for the conversion of transient topoisomerase-DNA cleavage complexes to permanent strand breaks. Since DNA is overwound ahead of tracking systems, cleavage complexes most likely to produce permanent strand breaks should be formed between topoisomerases and positively supercoiled molecules. Therefore, the ability of human topoisomerase IIalpha and IIbeta and topoisomerase I to cleave positively supercoiled DNA was assessed in the absence or presence of anticancer drugs. Topoisomerase IIalpha and IIbeta maintained approximately 4-fold lower levels of cleavage complexes with positively rather than negatively supercoiled DNA. Topoisomerase IIalpha also displayed lower levels of cleavage with overwound substrates in the presence of nonintercalative drugs. Decreased drug efficacy was due primarily to a drop in baseline (i.e., nondrug) cleavage, rather than an altered interaction with the enzyme-DNA complex. Similar results were seen for topoisomerase IIbeta, but the effects of DNA geometry on drug-induced scission were somewhat less pronounced. With both topoisomerase IIalpha and IIbeta, intercalative drugs displayed greater relative cleavage enhancement with positively supercoiled DNA. This appeared to result from negative effects of high concentrations of intercalative agents on underwound DNA. In contrast to the type II enzymes, topoisomerase I maintained approximately 3-fold higher levels of cleavage complexes with positively supercoiled substrates and displayed an even more dramatic increase in the presence of camptothecin. These findings suggest that the geometry of DNA supercoils has a profound influence on topoisomerase-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either topoisomerase IIalpha or IIbeta.     

DNA topoisomerase III from extremely thermophilic archaebacteria. ATP-independent type I topoisomerase from Desulfurococcus amylolyticus drives extensive unwinding of closed circular DNA at high temperature.

A second type I topoisomerase was purified from the extremely thermophilic archaebacterium Desulfurococcus amylolyticus. In contrast to the previously described reverse gyrase from this organism, the novel enzyme designated as Dam topoisomerase III is an ATP-independent relaxing topoisomerase. It is a monomer with Mr 108,000, as determined by electrophoresis under denaturing conditions and by size exclusion chromatography. Dam topoisomerase III, like other bacterial type I topoisomerases, absolutely requires Mg2+ for activity and is specific for single-stranded DNA. At 60-80 degrees C, it relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA. At 95 degrees C, the enzyme unwinds both positively and negatively supercoiled substrates and produces extensively unwound form I* and I** DNA. The peculiarities of DNA topoisomerization at high temperatures are discussed.     

核多角体病毒感染昆虫细胞的DNA拓扑异构酶性质的研究

核多角体病毒(Nuclear polyhedrosis virus)的核酸是双股环状DNA,在病毒颗粒中呈超螺旋状态。超螺旋DNA复制时,一般皆有超螺旋解旋过程。为了解这一机制,本文报道了从NPV感染的家蚕中肠组织中分离细胞核,经过羟基磷灰石、磷酸纤维素柱层析,ssDNA-纤维素亲和层析,纯化了DNA拓扑异构酶I,SDS-PAGE测定分子量为47kd,最适Mg~(++)浓度约为5mM。AcNPV感染的TN368细胞DNA拓扑异构酶I总活力较正常细胞酶活力高1～3倍,且活力的提高与病毒增殖平行。讨论了昆虫细胞DNA拓扑异构酶I的性质及其与NPV复制的关系。     

DNA chirality-dependent stimulation of topoisomerase IV activity by the C-terminal AAA+ domain of FtsK

We have studied the stimulation of topoisomerase IV (Topo IV) by the C-terminal AAA+ domain of FtsK. These two proteins combine to assure proper chromosome segregation in the cell. Stimulation of Topo IV activity was dependent on the chirality of the DNA substrate: FtsK stimulated decatenation of catenated DNA and relaxation of positively supercoiled [(+)ve sc] DNA, but inhibited relaxation of negatively supercoiled [(−)ve sc] DNA. The DNA translocation activity of FtsK was not required for stimulation, but was required for inhibition. DNA chirality did not affect any of the activities of FtsK, suggesting that FtsK possesses an inherent Topo IV stimulatory activity that is presumably mediated by protein–protein interactions, the stability of Topo IV on the DNA substrate dictated the effect observed. Inhibition occurs because FtsK can strip distributively acting topoisomerase off (−)ve scDNA, but not from either (+)ve scDNA or catenated DNA where the enzyme acts processively. Our analyses suggest that FtsK increases the efficiency of trapping of the transfer segment of DNA during the catalytic cycle of the topoisomerase.     

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as ‘topological poisons of topoisomerase I’.     

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.     

The MukB-ParC Interaction Affects the Intramolecular,Not Intermolecular,Activities of Topoisomerase IV

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.     

Pea chloroplast topoisomerase I: purification,characterization, and role in replication

A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl₂. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.     

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.     

Differential modulation by spermidine of reactions catalyzed by type 1 prokaryotic and eukaryotic topoisomerases

In the absence of DNA aggregation, spermidine inhibited the relaxation of negatively supercoiled DNA by Escherichia coli topoisomerase I at concentrations of the polyamine normally found intracellularly. Spermidine also curtailed the cleavage of negatively supercoiled ColE1 DNA by the enzyme in the absence of Mg2+. On the contrary, knotting of M13 single-stranded DNA circles catalyzed by topoisomerase I was stimulated by the polyamine. Relaxation of supercoiled DNA by eukaryotic type 1 topoisomerases, such as calf thymus topoisomerase I and wheat germ topoisomerase, was significantly stimulated by spermidine in the same range of concentrations that inhibited the prokaryotic enzyme. In reactions catalyzed by S1 nuclease, the polyamine enhanced the digestion of single-stranded DNA and inhibited the nicking of negatively supercoiled DNA. These results suggest that spermidine modifies the supercoiled duplex substrate in these reactions by modulating the degree of single strandedness.