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religation steps
Biochimie 81 (1999) 771−779 © 1999 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS. All rights reserved
The catalytic activities of DNA topoisomerase II are most closely associated with the DNA cleavage/religation steps Denis Scala, Alexandre E. Escargueil, Janine Couprie, Annette K. Larsen* Laboratory of Biology and Pharmacology of DNA Topoisomerases, CNRS UMR 8532, Institut Gustave-Roussy, PR2, 94805 Villejuif cedex, France (Received 20 October 1998; accepted 15 February 1999) Abstract — DNA topoisomerase II regulates the three-dimensional organisation of DNA and is the principal target of many important anticancer and antimicrobial agents. These drugs usually act on the DNA cleavage/religation steps of the catalytic cycle resulting in accumulation of covalent DNA-topoisomerase II complexes. We have studied the different steps of the catalytic cycle as a function of salt concentration, which is a classical way to evaluate the biochemical properties of proteins. The results show that the catalytic activity of topoisomerase II follows a bell-shaped curve with optimum between 100 and 225 mM KCl. No straight-forward correlation exists between DNA binding and catalytic activity. The highest levels of drug-induced covalent DNA-topoisomerase II complexes are observed between 100 and 150 mM KCl. Remarkably, at salt concentrations between 150 mM and 225 mM KCl, topoisomerase II is converted into a drug-resistant form with greatly reduced levels of drug-induced DNA-topoisomerase II complexes. This is due to efficient religation rather than to absence of DNA cleavage as witnessed by relaxation of the supercoiled DNA substrate. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations. Unexpectedly, the addition of DNA stimulates ATP hydrolysis at 100 and 150 mM KCl, but has little or no effect below 100 mM KCl in spite of strong non-covalent DNA binding at these salt concentrations. Therefore, DNA-stimulated ATP hydrolysis appears to be associated with covalent rather than non-covalent binding of DNA to topoisomerase II. Taken together, the results suggest that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II providing a unifying theme for the biological and pharmacological modulation of this enzyme. © 1999 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS topoisomerase II / catalytic activity / topoisomerase inhibitors / covalent DNA-protein complexes / ATP hydrolysis
1. Introduction DNA topoisomerase II is a nuclear enzyme that regulates the topological states of DNA and which is the principal target for a number of important anticancer and antimicrobial agents [1-3]. Topoisomerase II plays an essential role in chromosome condensation and segregation of intertwined sister chromatids during mitosis [4-7] and can relieve DNA supercoiling during transcription and replication [8, 9]. Recent studies suggest that topoisomerase II has the ability to recognize both endogenous and exogenous DNA lesions [10-12] and may play a role in DNA repair [12]. The molecular mechanisms which regulate the catalytic activity of topoisomerase II are poorly understood. Topoisomerase II is phosphorylated by several kinases includ* Correspondence and reprints Abbreviations: SDS, sodium dodecyl sulfate; EDTA, ethylenediamine-tetraacetic acid; EGTA, ethylene glycol-bis(βaminoethyl ether)tetraacetic acid; VP-16, etoposide (4’demethyl-epipodophyllotoxin-9-(4,6-0-ethylene-β-D-glucopyranoside)); CKII, casein kinase II; PKC, protein kinase C.
ing protein kinase C (PKC), casein kinase II (CKII) and cdc2 kinase in vitro as well as in vivo [13-19]. The phosphorylation by CKII was originally shown to stimulate the catalytic activities of both Drosophila and yeast topoisomerase II in vitro [13-16]. However, recent results show that phosphorylation by CKII has no effect on the catalytic activity of purified mouse or human topoisomerase II [20, 21]. Similarly, phosphorylation by cdc2 kinase has no direct effect on the catalytic activities of both yeast and human topoisomerase II [22]. Therefore, it now appears that the relationship between phosphorylation and catalytic activity is more complex than initially thought. The most detailed studies of the rate-limiting step of the catalytic cycle of topoisomerase II have been carried out with the Drosophila enzyme which is stimulated by phosphorylation with CKII or PKC. The Drosophila phosphorylation model showed that the stimulatory effect of phosphorylation could be fully explained by an increase in ATP hydrolysis whereas none of the other steps in the catalytic cycle were affected [13, 16]. These findings led to the general notion that the rate-limiting step in the catalytic cycle of topoisomerase II, which was being regulated in vivo, was ATP hydrolysis.
772 The current uncertainty about the relevance of phosphorylation as a universal way to regulate the catalytic activity of topoisomerase II prompted us to search for an alternative manner to determine which step in the catalytic cycle is most closely associated with the catalytic activity. Variation of salt concentrations is a classical method to evaluate the biochemical properties of proteins. We have used this method to carry out a detailed analysis of the different steps in the catalytic cycle of yeast topoisomerase II. The results show that it is the DNA cleavage/ religation steps that are most closely associated with the enzymatic activities. These findings are not limited to the yeast enzyme, since a close association between DNA cleavage/religation and catalytic activity also is observed for human topoisomerase IIα. Interestingly, these are the same steps which are targeted by both anticancer and antimicrobial agents and which are involved in recognition of DNA lesions, suggesting a unifying theme in the biological and pharmacological modulation of this important class of enzymes.
Scala et al. reaction buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2) containing the indicated concentrations of KCl and were initiated by the addition of topoisomerase II. Except for the decatenation assay, all assays were stopped by the addition of 5 µL of loading buffer (0.05% bromophenol blue, 12.5 mM EDTA and 12.5% sucrose (final concentrations)). The samples were electrophoresed in 1% agarose gels in Tris/borate/EDTA buffer (pH 8). 2.4. Relaxation assay The experimental conditions were as described above except that the reaction buffer also contained 0.5 mM ATP. Different amounts of topoisomerase II (105 to 600 ng, 1 ng corresponding to 3 fmol dimeric topoisomerase II) were incubated with 150 ng of supercoiled pBR322 plasmid DNA in the presence of the indicated concentrations of KCl. The reactions were stopped and the samples submitted to electrophoresis in a 1% agarose gel containing ethidium bromide (0.5 µg/mL) at 7 V/cm for 5 h. 2.5. Decatenation assay
2. Materials and methods 2.1. Materials Supercoiled pBR322 plasmid DNA (> 95% form I) was purchased from Biolabs (Beverly, USA), whereas SV40 plasmid was from Promega (Madison, USA). Highly catenated kinetoplast DNA was purified from trypanosoma Leishmania major generously provided by Dr. Jean-Pierre Dedet (Montpellier, France). Proteinase K, spermidine and ATP were obtained from Sigma. Genistein was purchased from Extrasynthèse Laboratories (Genay, France) while etoposide (VP-16) was a kind gift from Dr. Jerzy Konopa (Gdansk, Poland). 2.2. Enzymes and antibodies Yeast topoisomerase II and human topoisomerase IIα were obtained from Saccharomyces cerevisiae as previously described [23, 24]. Briefly, the two enzymes were overexpressed in yeast from multi-copy expression plasmids kindly provided by Dr. James C. Wang (Harvard University, Boston, USA) and purified by a four-step procedure consisting of yeast disruption, elution from a polyethyleneimine/celite column, ammonium sulphate precipitation and phosphocellulose chromatography. Purified topoisomerase II was dialysed against the storage buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol) and kept at –20 °C. The purified enzyme preparations contained no detectable DNA topoisomerase I activity. 2.3. Topoisomerase II assays All reactions were carried out at 30 °C for the yeast enzyme or at 37 °C for the human enzyme in 15 µL of
Reaction mixtures contained 12 to 255 ng topoisomerase II, 0.5 mM ATP and 220 ng of kinetoplast DNA in 15 µL reaction buffer with the indicated concentrations of KCl. Reactions were stopped by adding 5 µL of 30% glycerol, 1% SDS, 50 mM EDTA and 0.05% bromophenol blue followed by electrophoresis in a 1.2% agarose gel at 5 V/cm for 4 h. 2.6. Binding of topoisomerase II to DNA The binding of topoisomerase II to DNA was determined by an electrophoretic mobility shift assay as previously described [25]. All assays contained 720 ng of topoisomerase II and 150 ng of pBR322 in reaction buffer with the indicated concentrations of KCl. After 5 min incubation at 30 °C the reactions were stopped by the addition of loading buffer without EDTA. Enzyme-bound DNA was separated from free DNA by agarose gel electrophoresis as described above and the DNA visualised under UV light after staining with ethidium bromide. 2.7. Formation of covalent DNA-topoisomerase II complexes Reactions were performed as previously described [26]. Reaction mixtures contained 450 ng topoisomerase II, 150 ng of pBR322 DNA and 0.5 mM ATP. After 15 min of incubation at 30 °C, reactions were terminated by addition of 2 µL of 10% SDS and 5 mg/mL proteinase K followed by incubation at 50 °C for 30 min. After addition of loading buffer, samples were subjected to electrophoresis in 1% agarose gels containing ethidium bromide (0.5 µg/mL) at 2 V/cm for 18 h in Tris/borate/
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Figure 1. Effect of KCl concentration on the catalytic activity of topoisomerase II as measured by relaxation and catenation of supercoiled DNA. Supercoiled pBR322 DNA was incubated with different concentrations of topoisomerase II and the relaxation and/or catenation determined by agarose gel electrophoresis in ethidium bromide-containing gels. DNA FI, supercoiled substrate DNA; DNA FIII, linear DNA; K, catenated DNA; L, linear DNA; S, supercoiled DNA; R, relaxed circular DNA.
EDTA buffer (0.5 µg/mL).
(pH
8.0)
with
ethidium
bromide
2.8. Topoisomerase II-mediated DNA religation SV40 plasmid DNA was linearised by Taq1, labelled with γ-[32P]dCTP by Klenow enzyme (Biolabs, Beverly, USA) and then incubated with BxtX1 restriction enzyme (Boehringer, France). This resulted in the formation of end-labelled DNA with a specific activity of 400 000 cpm/µg. Reaction mixtures contained 40 000 cpm of DNA, 50 ng topoisomerase II and 0.5 mM ATP. Initial DNA cleavage/religation equilibrium was established at 30 °C for 20 min. DNA religation was initiated by rapidly shifting the samples from 30 to 55 °C [27]. Reactions were stopped at various time points by addition of 0.25% SDS and 0.35 µg/µL proteinase K (final concentrations). After 30 min at 50 °C, loading buffer was added followed by electrophoresis in 1% agarose gels containing 0.1% SDS at 2 V/cm for 18 h in Tris/borate/EDTA buffer (pH 8). The gels were then dried, exposed to a Fuji imaging plate type BAS-IIIs and the radioactivity, which corresponds to the intact substrate DNA, determined by phosphoimager Fuji X BAS 1000. 2.9. Hydrolysis of ATP by topoisomerase II ATPase assays were carried out as previously described [28]. Reaction mixtures contained 570 ng topoisomerase II, 150 ng pBR322, 0.5 mM ATP and 20 to 60
nM of γ-[32P]ATP (3000Ci/mmol) per point. After 15 min incubation at 30 °C, 5 µL of loading buffer was added and the samples were immediately analysed by thin layer chromatography on PEI-cellulose sheets (Merck). Radioactive areas corresponding to inorganic phosphate released by ATP hydrolysis were cut out of the chromatograms and quantified by liquid scintillation counting.
3. Results 3.1. The effect of salt concentration on the catalytic activities of topoisomerase II Under standard conditions, catalytic activity of yeast topoisomerase II, as determined by relaxation of supercoiled plasmid DNA, can be detected at salt concentrations between 100 and 225 mM KCl (figure 1, left). A similar bell-shaped curve is observed for human topoisomerase IIα with optimum between 100 and 150 mM KCl (results not shown). These results are not limited to intra-strand DNA passage activities, since decatenation of kinetoplast DNA, which depends on inter-strand DNA passage, provides comparable results (figure 2, left). In contrast, no catalytic activity is observed below 50 mM or above 225 mM KCl by either relaxation or decatenation assays. In the presence of higher enzyme concentrations, decatenation activity can be detected at low salt concentrations (figure 2, right). In contrast, no catalytic activity is
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Figure 2. Effect of KCl concentration on the catalytic activity of topoisomerase II as measured by decatenation. Highly catenated kinetoplast DNA was incubated with different concentrations of topoisomerase II and the liberation of free minicircles determined by agarose gel electrophoresis. K, kinetoplast DNA; M, free minicircles.
observed at salt concentrations above 225 mM KCl even at enzyme concentrations as high as 83 ng/µL (results not shown). Increased enzyme concentrations also result in stimulation of the relaxation of supercoiled DNA at low salt concentrations (figure 1, right). Relaxation is observed starting at 0 mM KCl with optimal activity between 50 and 225 mM KCl. In contrast, no relaxation is observed at salt concentrations above 225 mM KCl. Unexpectedly, part of the DNA is retained in the wells at all salt concentrations below 150 mM. Treatment of samples with proteinase K before loading has no effect on the migration, indicating that the altered electrophoretic migration of the plasmid DNA is not due to DNA-protein aggregation but to catenation of circular DNA molecules. It should be noted that some preparations of topoisomerase II have been reported to contain a non-identified catenation factor that copurifies with topoisomerase II [29-31]. However, this is unlikely to be the case here since we use an overexpression system which allows us to obtain an enzyme preparation purified to homogeneity as judged by silver staining.
3.2. Effect of salt concentration on enzyme-DNA binding An electrophoretic mobility shift assay [25] was employed to determine the influence of salt on the binding of topoisomerase II to DNA. The results show that topoisomerase II retards the migration of supercoiled DNA at all KCl concentrations from 0 to 225 mM KCl (figure 3). An identical DNA binding profile is observed in the absence of Mg2+ ions where only non-covalent binding takes place. DNA binding and catalytic activity are clearly not associated in a straight-forward manner since absence of catalytic activity can be associated with either no (> 225 mM KCl) or strong (< 100 mM KCl) DNA binding, while catalytic activity can be associated with either strong, intermediate or week DNA binding (at 100, 150 and 225 mM KCl, respectively). Furthermore, at elevated enzyme concentrations, topoisomerase II binds to DNA even at salt concentrations higher than 225 mM without a concomitant restoration of the catalytic activity (results not shown).
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Figure 3. Effect of KCl concentration on enzyme-DNA binding. Supercoiled pBR322 DNA (DNA FI) was incubated with topoisomerase II in the absence of ATP and the enzyme-bound DNA separated from free DNA by agarose gel electrophoresis.
The results also show preferential binding of topoisomerase II to supercoiled DNA compared to linear or relaxed circular DNA (figure 3, compare control DNA with DNA in the presence of topoisomerase II at 0 and 225 mM KCl). Therefore, the enzyme’s preferential recognition of supercoiled DNA [25] is observed at all conditions where the enzyme binds to DNA in spite of the differences in effective DNA diameter which exist at different salt concentrations [32]. 3.3. Topoisomerase-mediated DNA cleavage DNA binding is followed by the formation of covalent DNA-topoisomerase complexes which allow doublestranded DNA passage. The level of covalent DNAtopoisomerase complexes (cleavable complexes) can be determined by monitoring the conversion of circular DNA to the linear form after treatment with a strong protein denaturant and proteinase K. To increase the level of cleavable complexes, two different topoisomerase II inhibitors, VP-16 and genistein, were included in the reaction mixture. The cleavable complex formation in the presence of VP-16, which predominantly prevents religation of cleaved DNA [16], follows the same salt profile as previously observed for the catalytic activity between 0 and 150 mM KCl (figure 4). However, only modest levels of cleavable complexes can be detected at 225 mM KCl. The simultaneous presence of relaxed DNA strongly suggests that the low amount of cleavable complexes at this salt concentration is due to a strong religation activity rather than an absence of DNA cleavage. The presence of genistein, which stimulates DNA cleavage but has less effect on DNA religation [16], results in formation of cleavable complexes at all salt concentrations between 0
Figure 4. Effect of KCl concentration on the formation of covalent DNA-protein complexes in the presence of VP-16 or genistein. Supercoiled pBR322 DNA was incubated with topoisomerase II in the presence of VP-16 (50 µM) or genistein (100 µM), treated with SDS and proteinase K and subjected to agarose gel electrophoresis in ethidium bromide-containing gels. DNA FI, supercoiled substrate DNA; N, nicked circular DNA; L, linear DNA; S, supercoiled DNA; R, relaxed circular DNA.
and 100 mM KCl (figure 4). Only modest cleavage is observed at 150 mM KCl and no cleavable complexes are apparent at 225 mM. As for VP-16, the simultaneous presence of relaxed DNA clearly indicates that the low levels of cleavable complexes are not due to absence of DNA cleavage but to a strong religation activity. Taken together, the two experiments suggest that for the yeast enzyme, the DNA cleavage/religation equilibrium varies as a function of the salt concentration. Studies carried out with human topoisomerase IIα give similar results. The highest levels of cleavable complexes are observed at 100 and 150 mM KCl in the presence of VP-16 while no cleavage complex formation can be detected at salt concentrations higher than 100 mM KCl when genistein was present (results not shown). Since all
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Figure 5. Effect of KCl concentration on topoisomerase IImediated DNA religation in the presence of genistein or VP-16. Linear, end-labelled SV40 DNA was incubated with topoisomerase II in the presence of genistein (100 µM) or VP-16 (50 µM) and the DNA religation initiated by shifting the samples from 30 to 55 °C (time 0). Reactions were stopped at various time points as indicated, treated with SDS and proteinase K and analyzed by agarose gel electrophoresis to determine the amounts of intact, religated substrate DNA. The results shown are typical of two independent experiments.
supercoiled DNA is relaxed at 150 mM KCl, the absence of cleavable complexes in the presence of genistein can only be attributed to efficient religation rather than to an absence of DNA cleavage, analogous to what is observed for the yeast enzyme. 3.4. DNA religation The overall levels of cleavable complexes (figure 4) are a function of both DNA cleavage and DNA religation. However, it is possible to uncouple the two reactions at extreme temperatures where religation takes place in the absence of DNA cleavage [26, 33]. We therefore incubated linear end-labelled DNA with topoisomerase II and either etoposide or genistein at 35 mM or 150 mM KCl and followed the rate of religation after a temperature shift to 55 °C. The results (figure 5) show that religation is more efficient at 150 mM than at 35 mM KCl for both drugs. 3.5. ATP hydrolysis The last step in the catalytic cycle of topoisomerase II is ATP hydrolysis which is required for initiation of a new round of catalysis [25, 28]. The ATPase activity is not tightly coupled to DNA strand passage since the enzyme also is able to hydrolyse ATP in the absence of DNA (figure 6). DNA-independent ATP hydrolysis is strongest at low salt concentrations and then gradually decreases with increasing KCl concentrations (figure 6). Unexpect-
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Figure 6. The effect of KCl concentration on topoisomerase II-mediated ATP hydrolysis. Topoisomerase II was incubated for 15 min with radiolabelled ATP (0.5 mM, 3000 Ci/mmol) in the absence (hatched bar) or presence (filled bar) of supercoiled pBR322 DNA and the liberation of inorganic phosphate from ATP was determined by thin layer chromatography. Each point is the average of three independent experiments, Bars, standard deviation.
edly, the addition of DNA stimulates the ATPase activity only at 100 and 150 mM KCl but has little or no effect on ATP hydrolysis at other salt concentrations. While the strong ATPase activities at 100 and 150 mM KCl are associated with catalytic activity, an almost complete dissociation between the two factors is observed at 225 mM KCl where only low levels of ATP hydrolysis are present in spite of a strong catalytic activity. Surprisingly, the salt profile of the DNA-dependent ATPase activity is also different from what was observed for DNA-topoisomerase binding, since strong noncovalent DNA binding is observed for all salt concentrations below 100 mM KCl (figure 3). This suggests that non-covalent binding between DNA and topoisomerase II is not enough to stimulate enzyme-mediated ATP hydrolysis. In contrast, the DNA-dependent ATPase activity is closely related to the DNA cleavage/religation step (figure 4). It is interesting, that the presence of DNA has no effect on ATP hydrolysis at 225 mM KCl where DNA religation is very efficient. It therefore appears that the DNA-dependent stimulation of ATP hydrolysis is associated with the presence of covalent DNA-topoisomerase II complexes.
Catalytic activities of DNA topoisomerase II 4. Discussion Early studies carried out with the Drosophila phosphorylation model suggested that the rate of ATP hydrolysis modulates the overall catalytic activity of DNA topoisomerase II. In contrast, phosphorylation had no detectable effect on any of the other steps in the catalytic cycle. Although thoroughly documented both with respect to phosphorylation by CKII [13] and by PKC [16] these findings were puzzling for several reasons. First, relaxation of negatively supercoiled DNA is not an intrinsically energetically unfavourable reaction as illustrated by the fact that gyrase as well as topoisomerase I can carry out the same reaction in the absence of ATP hydrolysis [1, 34]. It was therefore unexpected that the activity of topoisomerase II should be regulated by the only step in the catalytic cycle which, at least in principle, is not absolutely necessary for its catalytic functions. Second, topoisomerase II is able to hydrolyse ATP at a considerable rate even in the absence of DNA, suggesting that DNA strand passage is not tightly coupled to ATP hydrolysis. This is further supported by the observation that the rate of ATP hydrolysis is never totally inhibited by topoisomerase II inhibitors even at drug concentrations where the catalytic activity is strongly affected [35, 36]. Finally, recent results suggest that the relationship between phosphorylation and catalytic activity is more complex than originally thought [13-16, 20-22]. Together, these findings prompted us to re-examine the catalytic cycle of topoisomerase II by an alternative method. We have chosen variation of salt concentrations, which is a classical method to evaluate biochemical properties of proteins. Our results show that with a standard amount of enzyme, the salt dependence of catalytic activity follows a bell-shaped curve with optimum around 150 mM for the yeast enzyme and between 100 and 150 mM for human topoisomerase IIα. A similar bell-shaped curve has been described for topoisomerase II purified from other sources including rat liver, Drosophila and calf thymus [28, 37, 38]. This salt dependence profile is not limited to DNA relaxation, which relies on intra-strand DNA passage but is also observed for decatenation which relies on interstrand DNA passage. The catalytic activity of topoisomerase II is classically determined at enzyme concentrations between 1 and 10 ng/µL. However, the actual concentrations of topoisomerase II in living cells are much higher. A proliferating eukaryotic cell typically contains 106 topoisomerase II molecules which are contained in a nucleus that is 1.5 to 5 µm in diameter [39]. This corresponds to an average nuclear concentration of topoisomerase II ranging between 500 ng/µL and 20 µg/µL. Therefore, the catalytic activity of topoisomerase II was also determined at higher enzyme concentrations. An increase in enzyme concentrations has two major effects. First, catalytic activity can now be detected at low
777 salt concentrations as determined by both relaxation and decatenation assays. In contrast, no catalytic activity was ever observed at high salt concentrations even at elevated enzyme concentrations, where topoisomerase II is able to bind DNA. Second, catenation activity could be observed at salt concentrations below 150 mM. Usually, topoisomerase II is only able to catenate circular DNA molecules in the presence of DNA condensing agents such as histone H1, spermidine or polyethyleneglycol. However, no such agents were present in our experiments. It has previously been shown, that if only DNA-DNA interactions are taken into account, the probability of catenation decreases with decreasing salt concentrations [40]. Therefore, the driving force must come from the enzyme. Low salt concentrations seem to favour the formation of topoisomerase II multimers at least tetrameric in size [41, 42]. A possible explanation for the catenation activity of topoisomerase II at low salt concentrations is therefore its ability to multimerize under these conditions in combination with the capacity of the enzyme to simultaneous bind to two different double-stranded DNA helixes. Our studies do not show a straight-forward correlation between DNA binding and catalytic activity, since catalytic activity can be associated with either weak (225 mM KCl), intermediate (150 mM) or strong (100 mM) DNA binding. It is interesting, that the two major mitotic functions of topoisomerase II depend on different properties of the enzyme. DNA condensation is mostly based on DNA binding and association with other proteins in the chromosome scaffold [43-45], whereas separation of sister chromatids requires catalytic activity [4-7]. This might explain why DNA binding and catalytic activity are not associated in a straight-forward manner and therefore not likely to be regulated in a co-ordinated fashion. In contrast to DNA binding, DNA cleavage and religation appear to be closely associated with the catalytic activity of topoisomerase II at all salt concentrations studied. It is interesting, that the ability of topoisomerase II to recognise both endogenous and exogenous DNA lesions is based on covalent rather than non-covalent interaction with DNA. In particular, the presence of DNA lesions results in increased DNA cleavage whereas the religation process appears to be unaffected [11]. In contrast, it is the religation step that is targeted by most anticancer and antimicrobial topoisomerase inhibitors in clinical use [3, 16]. It is remarkable that in vitro, it is possible to convert topoisomerase II into a drug-resistant enzyme that does not form drug-stabilised DNA-protein complexes simply by changing the salt concentration. This is not due to an absence of DNA cleavage but rather to a highly efficient religation as witnessed by the complete relaxation of the supercoiled DNA substrate at 225 mM KCl. Although modifications of the internal salt concentrations are not likely involved in the development of resistance to topo-
778 isomerase II-targeted drugs in tumour cells, we would predict that enzyme modifications resulting in increased religation would provide enhanced drug resistance without loss of catalytic activity. This type of topoisomerase II modification has been reported for several drug resistant tumour cell lines, although the underlying molecular mechanism(s) is/are currently unknown [46, 47]. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations and then gradually decreases with increasing salt concentrations. This is clearly very different from the bell-shaped curve observed for the catalytic activity. Interestingly, the presence of DNA increases the rate of ATP hydrolysis at 100 and 150 mM KCl but has little or no effect on ATP hydrolysis at other salt concentrations. Since DNA and topoisomerase II are tightly associated at low salt concentrations, our results strongly suggest that the stimulatory effect of DNA on the rate of ATP hydrolysis is unrelated to non-covalent DNA binding. In contrast, the rate of ATP hydrolysis appears to be closely associated with the cleavage/religation reactions. Cleavable complex formation is strongest between 100 and 225 mM while religation is optimal at 225 mM KCl. Since the presence of DNA has no effect on ATP hydrolysis at 225 mM KCl, it appears that the stimulation of ATP hydrolysis is directly associated with covalent DNAtopoisomerase II binding. This is supported by recent results with mutant yeast topoisomerase II where the absence of the active-site tyrosine (and thereby lack of covalent DNA-topoisomerase II complexes) results in loss of DNA-stimulated ATPase hydrolysis [36]. Taken together, our results show that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II. These findings are not limited to the yeast enzyme, since a close association between DNA cleavage and catalytic activity also is observed for human topoisomerase IIα. Interestingly, these are the same steps which are targeted by both anticancer and antimicrobial agents and which are involved in recognition of DNA lesions suggesting a unifying theme in the biological and pharmacological modulation of this important class of enzymes. Acknowledgments We thank Dr. Andrzej Skladanowski for helpful discussions, and Prof. Jean-Pierre Dedet for generously providing us with trypanosoma kinetoplast DNA. This work was supported in part by Association pour la Recherche sur le Cancer (ARC), Villejuif, France. D.S. is a fellow of Association pour la Recherche sur le Cancer.
References [1] Wang J.C., DNA topoisomerases, Annu. Rev. Biochem. 65 (1996) 635–692.
Scala et al. [2] Corbett A.H., Osheroff N., When good enzymes go bad: conversion of topoisomerase II to a cellular toxin by antineoplastic drugs, Chem. Res. Toxicol. 6 (1993) 585–597. [3] Maxwell A., DNA gyrase as a drug target, Trends Microbiol. 5 (1997) 102–109. [4] Dinardo S., Voelkel K., Sternglanz R., DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication, Proc. Natl. Acad. Sci. USA 81 (1984) 2616–2620. [5] Uemura T., Yanagida M., Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization, EMBO J. 3 (1984) 1737–1744. [6] Newport J., Spann T., Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes, Cell 48 (1987) 219–230. [7] Downes C.S., Mullinger A.M., Johnson R.T., Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis, Proc. Natl. Acad. Sci. USA 88 (1991) 8895–8899. [8] Brill S.J., Dinardo S., Voelkel-Meiman K., Sternglanz R., Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA, Nature 326 (1987) 414–416. [9] Brill S.J., Sternglanz R., Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants, Cell 54 (1988) 403–411. [10] Corbett A.H., Zechiedrich E.L., Lloyd R.S., Osheroff N., Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers, J. Biol. Chem. 266 (1991) 19666–19671. [11] Kingma P.S., Osheroff N., Spontaneous DNA damage stimulates topoisomerase II-mediated DNA cleavage, J. Biol. Chem. 272 (1997) 7488–7493. [12] Eder J.P.J., Chan V.T., Ng S.W., Rizvi N.A., Zacharoulis S., Teicher B.A., Schnipper L.E., DNA topoisomerase II alpha expression is associated with alkylating agent resistance, Cancer Res. 55 (1995) 6109–6116. [13] Corbett A.H., Devore R.F., Osheroff N., Effect of casein kinase II-mediated phosphorylation on the catalytic cycle of topoisomerase II. Regulation of enzyme activity by enhancement of ATP hydrolysis, J. Biol. Chem. 267 (1992) 20513–20518. [14] Cardenas M.E., Dang Q., Glover C.V., Gasser S.M., Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo, EMBO J. 11 (1992) 1785–1796. [15] Bojanowski K., Filhol O., Cochet C., Chambaz E.M., Larsen A.K., DNA topoisomerase II and casein kinase II associate in a molecular complex that is catalytically active, J. Biol. Chem. 268 (1993) 22920–22926. [16] Corbett A.H., Fernald A.W., Osheroff N., Protein kinase C modulates the catalytic activity of topoisomerase II by enhancing the rate of ATP hydrolysis: evidence for a common mechanism of regulation by phosphorylation, Biochemistry 32 (1993) 2090–2097. [17] Wells N.J., Addison C.M., Fry A.M., Ganapathi R., Hickson I.D., Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro, J. Biol. Chem. 269 (1994) 29746–29751. [18] Wells N.J., Fry A.M., Guano F., Norbury C., Hickson I.D., Cell cycle phase-specific phosphorylation of human topoisomerase II alpha. Evidence of a role for protein kinase C, J. Biol. Chem. 270 (1995) 28357–28363. [19] Wells N.J., Hickson I.D., Human topoisomerase II alpha is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase, Eur. J. Biochem. 231 (1995) 491–497. [20] Kimura K., Saijo M., Tanaka M., Enomoto T., Phosphorylationindependent stimulation of DNA topoisomerase II alpha activity, J. Biol. Chem. 271 (1996) 10990–10995.
Catalytic activities of DNA topoisomerase II [21] Redwood C., Davies S.L., Wells N.J., Fry A.M., Hickson I.D., Casein kinase II stabilizes the activity of human topoisomerase II alpha in a phosphorylation-independent manner, J. Biol. Chem. 273 (1998) 3635–3642. [22] Escargueil A.E., Plisov S., Scala D., Borgne A., Meijer L., Gorbsky G.J., Larsen A.K., Formation of molecular complexes between DNA topoisomerase II and p34cdc2-cyclin B stimulates topoisomerase activities independant of phosphorylation, Proc. Am. Assoc. Cancer Res. 39 (1998) 81. [23] Worland S.T., Wang J.C., Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae, J. Biol. Chem. 264 (1989) 4412–4416. [24] Wasserman R.A., Austin C.A., Fisher L.M., Wang J.C., Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast, Cancer Res. 53 (1993) 3591–3596. [25] Osheroff N., Eukaryotic topoisomerase II. Characterization of enzyme turnover, J. Biol. Chem. 261 (1986) 9944–9950. [26] Osheroff N., Zechiedrich E.L., Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: trapping the covalent enzymeDNA complex in an active form, Biochemistry 26 (1987) 4303–4309. [27] Robinson M.J., Osheroff N., Effects of antineoplastic drugs on the post strand-passage DNA cleavage/religation equilibrium of topoisomerase II, Biochemistry 30 (1991) 1807–1813. [28] Osheroff N., Shelton E.R., Brutlag D.L., DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA, J. Biol. Chem. 258 (1983) 9536–9543. [29] Hsieh T., Brutlag D., ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings, Cell 21 (1980) 115–125. [30] Goto T., Wang J.C., Yeast DNA topoisomerase II. An ATPdependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings, J. Biol. Chem. 257 (1982) 5866–5872. [31] Riou G.F., Gabillot M., Barrois M., Breitburd F., Orth G., A type-II DNA topoisomerase and a catenating protein from the transplantable VX2 carcinoma, Eur. J. Biochem. 146 (1985) 483–488. [32] Rybenkov V.V., Cozzarelli N.R., Vologodskii A.V., Probability of DNA knotting and the effective diameter of the DNA double helix, Proc. Natl. Acad. Sci. USA 90 (1993) 5307–5311. [33] Hsiang Y.H., Liu L.F., Evidence for the reversibility of cellular DNA lesions induced by mammalian topoisomerase II poisons, J. Biol. Chem. 264 (1989) 9713–9715.
779 [34] Gellert M., Mizuuchi K., O’Dea M.H., Itoh T., Tomizawa J.I., Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity, Proc. Natl. Acad. Sci. USA 74 (1977) 4772–4776. [35] Robinson M.J., Corbett A.H., Osheroff N., Effects of topoisomerase II-targeted drugs on enzyme-mediated DNA cleavage and ATP hydrolysis: evidence for distinct drug interaction domains on topoisomerase II, Biochemistry 32 (1993) 3638–3643. [36] Hammonds T.R., Maxwell A., The DNA dependence of the ATPase activity of human DNA topoisomerase II alpha, J. Biol. Chem. 272 (1997) 32696–32703. [37] Duguet M., Lavenot C., Harper F., Mirambeau G., DeRecondo A.M., DNA topoisomerases from rat liver: physiological variations, Nucleic Acids Res. 11 (1983) 1059–1075. [38] Schomburg U., Grosse F., Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa, Eur. J. Biochem. 160 (1986) 451–457. [39] Heck M.M., Earnshaw W.C., Topoisomerase II: A specific marker for cell proliferation, J. Cell Biol. 103 (1986) 2569–2581. [40] Rybenkov V.V., Vologodskii A.V., Cozzarelli N.R., The effect of ionic conditions on the conformations of supercoiled DNA. II. Equilibrium catenation, J. Mol. Biol. 267 (1997) 299–311. [41] Vassetzky Y.S., Dang Q., Benedetti P., Gasser S.M., Topoisomerase II forms multimers in vitro: effects of metals, betaglycerophosphate, and phosphorylation of its C-terminal domain, Mol. Cell Biol. 14 (1994) 6962–6974. [42] Lamhasni S., Larsen A.K., Barray M., Monnot M., Delain E., Fermandjian S., Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions, Biochemistry 34 (1995) 3632–3639. [43] Earnshaw W.C., Halligan B., Cooke C.A., Heck M.M., Liu L.F., Topoisomerase II is a structural component of mitotic chromosome scaffolds, J. Cell Biol. 100 (1985) 1706–1715. [44] Adachi Y., Luke M., Laemmli U.K., Chromosome assembly in vitro: topoisomerase II is required for condensation, Cell 64 (1991) 137–148. [45] Bojanowski K., Maniotis A.J., Plisov S., Larsen A.K., Ingber D.E., DNA topoisomerase II can drive changes in higher order chromosome architecture without enzymatically modifying DNA, J. Cell Biochem. 69 (1998) 127–142. [46] Robert J., Larsen A.K., Drug resistance to topoisomerase II inhibitors, Biochimie 80 (1998) 247–254. [47] Larsen A.K., Skladanowski A., Cellular resistance to topoisomerase II-targeted drugs: from drug uptake to cell death, Biochim. Biophys. Acta 1400 (1998) 257–274.
The catalytic activities of DNA topoisomerase II are most closely associated with the DNA cleavage/religation steps Denis Scala, Alexandre E. Escargueil, Janine Couprie, Annette K. Larsen* Laboratory of Biology and Pharmacology of DNA Topoisomerases, CNRS UMR 8532, Institut Gustave-Roussy, PR2, 94805 Villejuif cedex, France (Received 20 October 1998; accepted 15 February 1999) Abstract — DNA topoisomerase II regulates the three-dimensional organisation of DNA and is the principal target of many important anticancer and antimicrobial agents. These drugs usually act on the DNA cleavage/religation steps of the catalytic cycle resulting in accumulation of covalent DNA-topoisomerase II complexes. We have studied the different steps of the catalytic cycle as a function of salt concentration, which is a classical way to evaluate the biochemical properties of proteins. The results show that the catalytic activity of topoisomerase II follows a bell-shaped curve with optimum between 100 and 225 mM KCl. No straight-forward correlation exists between DNA binding and catalytic activity. The highest levels of drug-induced covalent DNA-topoisomerase II complexes are observed between 100 and 150 mM KCl. Remarkably, at salt concentrations between 150 mM and 225 mM KCl, topoisomerase II is converted into a drug-resistant form with greatly reduced levels of drug-induced DNA-topoisomerase II complexes. This is due to efficient religation rather than to absence of DNA cleavage as witnessed by relaxation of the supercoiled DNA substrate. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations. Unexpectedly, the addition of DNA stimulates ATP hydrolysis at 100 and 150 mM KCl, but has little or no effect below 100 mM KCl in spite of strong non-covalent DNA binding at these salt concentrations. Therefore, DNA-stimulated ATP hydrolysis appears to be associated with covalent rather than non-covalent binding of DNA to topoisomerase II. Taken together, the results suggest that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II providing a unifying theme for the biological and pharmacological modulation of this enzyme. © 1999 Société française de biochimie et biologie moléculaire / Éditions scientifiques et médicales Elsevier SAS topoisomerase II / catalytic activity / topoisomerase inhibitors / covalent DNA-protein complexes / ATP hydrolysis
1. Introduction DNA topoisomerase II is a nuclear enzyme that regulates the topological states of DNA and which is the principal target for a number of important anticancer and antimicrobial agents [1-3]. Topoisomerase II plays an essential role in chromosome condensation and segregation of intertwined sister chromatids during mitosis [4-7] and can relieve DNA supercoiling during transcription and replication [8, 9]. Recent studies suggest that topoisomerase II has the ability to recognize both endogenous and exogenous DNA lesions [10-12] and may play a role in DNA repair [12]. The molecular mechanisms which regulate the catalytic activity of topoisomerase II are poorly understood. Topoisomerase II is phosphorylated by several kinases includ* Correspondence and reprints Abbreviations: SDS, sodium dodecyl sulfate; EDTA, ethylenediamine-tetraacetic acid; EGTA, ethylene glycol-bis(βaminoethyl ether)tetraacetic acid; VP-16, etoposide (4’demethyl-epipodophyllotoxin-9-(4,6-0-ethylene-β-D-glucopyranoside)); CKII, casein kinase II; PKC, protein kinase C.
ing protein kinase C (PKC), casein kinase II (CKII) and cdc2 kinase in vitro as well as in vivo [13-19]. The phosphorylation by CKII was originally shown to stimulate the catalytic activities of both Drosophila and yeast topoisomerase II in vitro [13-16]. However, recent results show that phosphorylation by CKII has no effect on the catalytic activity of purified mouse or human topoisomerase II [20, 21]. Similarly, phosphorylation by cdc2 kinase has no direct effect on the catalytic activities of both yeast and human topoisomerase II [22]. Therefore, it now appears that the relationship between phosphorylation and catalytic activity is more complex than initially thought. The most detailed studies of the rate-limiting step of the catalytic cycle of topoisomerase II have been carried out with the Drosophila enzyme which is stimulated by phosphorylation with CKII or PKC. The Drosophila phosphorylation model showed that the stimulatory effect of phosphorylation could be fully explained by an increase in ATP hydrolysis whereas none of the other steps in the catalytic cycle were affected [13, 16]. These findings led to the general notion that the rate-limiting step in the catalytic cycle of topoisomerase II, which was being regulated in vivo, was ATP hydrolysis.
772 The current uncertainty about the relevance of phosphorylation as a universal way to regulate the catalytic activity of topoisomerase II prompted us to search for an alternative manner to determine which step in the catalytic cycle is most closely associated with the catalytic activity. Variation of salt concentrations is a classical method to evaluate the biochemical properties of proteins. We have used this method to carry out a detailed analysis of the different steps in the catalytic cycle of yeast topoisomerase II. The results show that it is the DNA cleavage/ religation steps that are most closely associated with the enzymatic activities. These findings are not limited to the yeast enzyme, since a close association between DNA cleavage/religation and catalytic activity also is observed for human topoisomerase IIα. Interestingly, these are the same steps which are targeted by both anticancer and antimicrobial agents and which are involved in recognition of DNA lesions, suggesting a unifying theme in the biological and pharmacological modulation of this important class of enzymes.
Scala et al. reaction buffer (10 mM Tris-HCl, pH 7.4, 5 mM MgCl2) containing the indicated concentrations of KCl and were initiated by the addition of topoisomerase II. Except for the decatenation assay, all assays were stopped by the addition of 5 µL of loading buffer (0.05% bromophenol blue, 12.5 mM EDTA and 12.5% sucrose (final concentrations)). The samples were electrophoresed in 1% agarose gels in Tris/borate/EDTA buffer (pH 8). 2.4. Relaxation assay The experimental conditions were as described above except that the reaction buffer also contained 0.5 mM ATP. Different amounts of topoisomerase II (105 to 600 ng, 1 ng corresponding to 3 fmol dimeric topoisomerase II) were incubated with 150 ng of supercoiled pBR322 plasmid DNA in the presence of the indicated concentrations of KCl. The reactions were stopped and the samples submitted to electrophoresis in a 1% agarose gel containing ethidium bromide (0.5 µg/mL) at 7 V/cm for 5 h. 2.5. Decatenation assay
2. Materials and methods 2.1. Materials Supercoiled pBR322 plasmid DNA (> 95% form I) was purchased from Biolabs (Beverly, USA), whereas SV40 plasmid was from Promega (Madison, USA). Highly catenated kinetoplast DNA was purified from trypanosoma Leishmania major generously provided by Dr. Jean-Pierre Dedet (Montpellier, France). Proteinase K, spermidine and ATP were obtained from Sigma. Genistein was purchased from Extrasynthèse Laboratories (Genay, France) while etoposide (VP-16) was a kind gift from Dr. Jerzy Konopa (Gdansk, Poland). 2.2. Enzymes and antibodies Yeast topoisomerase II and human topoisomerase IIα were obtained from Saccharomyces cerevisiae as previously described [23, 24]. Briefly, the two enzymes were overexpressed in yeast from multi-copy expression plasmids kindly provided by Dr. James C. Wang (Harvard University, Boston, USA) and purified by a four-step procedure consisting of yeast disruption, elution from a polyethyleneimine/celite column, ammonium sulphate precipitation and phosphocellulose chromatography. Purified topoisomerase II was dialysed against the storage buffer (50 mM Tris-HCl, pH 7.4, 100 mM KCl, 1 mM EDTA, 1 mM EGTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol) and kept at –20 °C. The purified enzyme preparations contained no detectable DNA topoisomerase I activity. 2.3. Topoisomerase II assays All reactions were carried out at 30 °C for the yeast enzyme or at 37 °C for the human enzyme in 15 µL of
Reaction mixtures contained 12 to 255 ng topoisomerase II, 0.5 mM ATP and 220 ng of kinetoplast DNA in 15 µL reaction buffer with the indicated concentrations of KCl. Reactions were stopped by adding 5 µL of 30% glycerol, 1% SDS, 50 mM EDTA and 0.05% bromophenol blue followed by electrophoresis in a 1.2% agarose gel at 5 V/cm for 4 h. 2.6. Binding of topoisomerase II to DNA The binding of topoisomerase II to DNA was determined by an electrophoretic mobility shift assay as previously described [25]. All assays contained 720 ng of topoisomerase II and 150 ng of pBR322 in reaction buffer with the indicated concentrations of KCl. After 5 min incubation at 30 °C the reactions were stopped by the addition of loading buffer without EDTA. Enzyme-bound DNA was separated from free DNA by agarose gel electrophoresis as described above and the DNA visualised under UV light after staining with ethidium bromide. 2.7. Formation of covalent DNA-topoisomerase II complexes Reactions were performed as previously described [26]. Reaction mixtures contained 450 ng topoisomerase II, 150 ng of pBR322 DNA and 0.5 mM ATP. After 15 min of incubation at 30 °C, reactions were terminated by addition of 2 µL of 10% SDS and 5 mg/mL proteinase K followed by incubation at 50 °C for 30 min. After addition of loading buffer, samples were subjected to electrophoresis in 1% agarose gels containing ethidium bromide (0.5 µg/mL) at 2 V/cm for 18 h in Tris/borate/
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Figure 1. Effect of KCl concentration on the catalytic activity of topoisomerase II as measured by relaxation and catenation of supercoiled DNA. Supercoiled pBR322 DNA was incubated with different concentrations of topoisomerase II and the relaxation and/or catenation determined by agarose gel electrophoresis in ethidium bromide-containing gels. DNA FI, supercoiled substrate DNA; DNA FIII, linear DNA; K, catenated DNA; L, linear DNA; S, supercoiled DNA; R, relaxed circular DNA.
EDTA buffer (0.5 µg/mL).
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2.8. Topoisomerase II-mediated DNA religation SV40 plasmid DNA was linearised by Taq1, labelled with γ-[32P]dCTP by Klenow enzyme (Biolabs, Beverly, USA) and then incubated with BxtX1 restriction enzyme (Boehringer, France). This resulted in the formation of end-labelled DNA with a specific activity of 400 000 cpm/µg. Reaction mixtures contained 40 000 cpm of DNA, 50 ng topoisomerase II and 0.5 mM ATP. Initial DNA cleavage/religation equilibrium was established at 30 °C for 20 min. DNA religation was initiated by rapidly shifting the samples from 30 to 55 °C [27]. Reactions were stopped at various time points by addition of 0.25% SDS and 0.35 µg/µL proteinase K (final concentrations). After 30 min at 50 °C, loading buffer was added followed by electrophoresis in 1% agarose gels containing 0.1% SDS at 2 V/cm for 18 h in Tris/borate/EDTA buffer (pH 8). The gels were then dried, exposed to a Fuji imaging plate type BAS-IIIs and the radioactivity, which corresponds to the intact substrate DNA, determined by phosphoimager Fuji X BAS 1000. 2.9. Hydrolysis of ATP by topoisomerase II ATPase assays were carried out as previously described [28]. Reaction mixtures contained 570 ng topoisomerase II, 150 ng pBR322, 0.5 mM ATP and 20 to 60
nM of γ-[32P]ATP (3000Ci/mmol) per point. After 15 min incubation at 30 °C, 5 µL of loading buffer was added and the samples were immediately analysed by thin layer chromatography on PEI-cellulose sheets (Merck). Radioactive areas corresponding to inorganic phosphate released by ATP hydrolysis were cut out of the chromatograms and quantified by liquid scintillation counting.
3. Results 3.1. The effect of salt concentration on the catalytic activities of topoisomerase II Under standard conditions, catalytic activity of yeast topoisomerase II, as determined by relaxation of supercoiled plasmid DNA, can be detected at salt concentrations between 100 and 225 mM KCl (figure 1, left). A similar bell-shaped curve is observed for human topoisomerase IIα with optimum between 100 and 150 mM KCl (results not shown). These results are not limited to intra-strand DNA passage activities, since decatenation of kinetoplast DNA, which depends on inter-strand DNA passage, provides comparable results (figure 2, left). In contrast, no catalytic activity is observed below 50 mM or above 225 mM KCl by either relaxation or decatenation assays. In the presence of higher enzyme concentrations, decatenation activity can be detected at low salt concentrations (figure 2, right). In contrast, no catalytic activity is
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Figure 2. Effect of KCl concentration on the catalytic activity of topoisomerase II as measured by decatenation. Highly catenated kinetoplast DNA was incubated with different concentrations of topoisomerase II and the liberation of free minicircles determined by agarose gel electrophoresis. K, kinetoplast DNA; M, free minicircles.
observed at salt concentrations above 225 mM KCl even at enzyme concentrations as high as 83 ng/µL (results not shown). Increased enzyme concentrations also result in stimulation of the relaxation of supercoiled DNA at low salt concentrations (figure 1, right). Relaxation is observed starting at 0 mM KCl with optimal activity between 50 and 225 mM KCl. In contrast, no relaxation is observed at salt concentrations above 225 mM KCl. Unexpectedly, part of the DNA is retained in the wells at all salt concentrations below 150 mM. Treatment of samples with proteinase K before loading has no effect on the migration, indicating that the altered electrophoretic migration of the plasmid DNA is not due to DNA-protein aggregation but to catenation of circular DNA molecules. It should be noted that some preparations of topoisomerase II have been reported to contain a non-identified catenation factor that copurifies with topoisomerase II [29-31]. However, this is unlikely to be the case here since we use an overexpression system which allows us to obtain an enzyme preparation purified to homogeneity as judged by silver staining.
3.2. Effect of salt concentration on enzyme-DNA binding An electrophoretic mobility shift assay [25] was employed to determine the influence of salt on the binding of topoisomerase II to DNA. The results show that topoisomerase II retards the migration of supercoiled DNA at all KCl concentrations from 0 to 225 mM KCl (figure 3). An identical DNA binding profile is observed in the absence of Mg2+ ions where only non-covalent binding takes place. DNA binding and catalytic activity are clearly not associated in a straight-forward manner since absence of catalytic activity can be associated with either no (> 225 mM KCl) or strong (< 100 mM KCl) DNA binding, while catalytic activity can be associated with either strong, intermediate or week DNA binding (at 100, 150 and 225 mM KCl, respectively). Furthermore, at elevated enzyme concentrations, topoisomerase II binds to DNA even at salt concentrations higher than 225 mM without a concomitant restoration of the catalytic activity (results not shown).
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Figure 3. Effect of KCl concentration on enzyme-DNA binding. Supercoiled pBR322 DNA (DNA FI) was incubated with topoisomerase II in the absence of ATP and the enzyme-bound DNA separated from free DNA by agarose gel electrophoresis.
The results also show preferential binding of topoisomerase II to supercoiled DNA compared to linear or relaxed circular DNA (figure 3, compare control DNA with DNA in the presence of topoisomerase II at 0 and 225 mM KCl). Therefore, the enzyme’s preferential recognition of supercoiled DNA [25] is observed at all conditions where the enzyme binds to DNA in spite of the differences in effective DNA diameter which exist at different salt concentrations [32]. 3.3. Topoisomerase-mediated DNA cleavage DNA binding is followed by the formation of covalent DNA-topoisomerase complexes which allow doublestranded DNA passage. The level of covalent DNAtopoisomerase complexes (cleavable complexes) can be determined by monitoring the conversion of circular DNA to the linear form after treatment with a strong protein denaturant and proteinase K. To increase the level of cleavable complexes, two different topoisomerase II inhibitors, VP-16 and genistein, were included in the reaction mixture. The cleavable complex formation in the presence of VP-16, which predominantly prevents religation of cleaved DNA [16], follows the same salt profile as previously observed for the catalytic activity between 0 and 150 mM KCl (figure 4). However, only modest levels of cleavable complexes can be detected at 225 mM KCl. The simultaneous presence of relaxed DNA strongly suggests that the low amount of cleavable complexes at this salt concentration is due to a strong religation activity rather than an absence of DNA cleavage. The presence of genistein, which stimulates DNA cleavage but has less effect on DNA religation [16], results in formation of cleavable complexes at all salt concentrations between 0
Figure 4. Effect of KCl concentration on the formation of covalent DNA-protein complexes in the presence of VP-16 or genistein. Supercoiled pBR322 DNA was incubated with topoisomerase II in the presence of VP-16 (50 µM) or genistein (100 µM), treated with SDS and proteinase K and subjected to agarose gel electrophoresis in ethidium bromide-containing gels. DNA FI, supercoiled substrate DNA; N, nicked circular DNA; L, linear DNA; S, supercoiled DNA; R, relaxed circular DNA.
and 100 mM KCl (figure 4). Only modest cleavage is observed at 150 mM KCl and no cleavable complexes are apparent at 225 mM. As for VP-16, the simultaneous presence of relaxed DNA clearly indicates that the low levels of cleavable complexes are not due to absence of DNA cleavage but to a strong religation activity. Taken together, the two experiments suggest that for the yeast enzyme, the DNA cleavage/religation equilibrium varies as a function of the salt concentration. Studies carried out with human topoisomerase IIα give similar results. The highest levels of cleavable complexes are observed at 100 and 150 mM KCl in the presence of VP-16 while no cleavage complex formation can be detected at salt concentrations higher than 100 mM KCl when genistein was present (results not shown). Since all
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Figure 5. Effect of KCl concentration on topoisomerase IImediated DNA religation in the presence of genistein or VP-16. Linear, end-labelled SV40 DNA was incubated with topoisomerase II in the presence of genistein (100 µM) or VP-16 (50 µM) and the DNA religation initiated by shifting the samples from 30 to 55 °C (time 0). Reactions were stopped at various time points as indicated, treated with SDS and proteinase K and analyzed by agarose gel electrophoresis to determine the amounts of intact, religated substrate DNA. The results shown are typical of two independent experiments.
supercoiled DNA is relaxed at 150 mM KCl, the absence of cleavable complexes in the presence of genistein can only be attributed to efficient religation rather than to an absence of DNA cleavage, analogous to what is observed for the yeast enzyme. 3.4. DNA religation The overall levels of cleavable complexes (figure 4) are a function of both DNA cleavage and DNA religation. However, it is possible to uncouple the two reactions at extreme temperatures where religation takes place in the absence of DNA cleavage [26, 33]. We therefore incubated linear end-labelled DNA with topoisomerase II and either etoposide or genistein at 35 mM or 150 mM KCl and followed the rate of religation after a temperature shift to 55 °C. The results (figure 5) show that religation is more efficient at 150 mM than at 35 mM KCl for both drugs. 3.5. ATP hydrolysis The last step in the catalytic cycle of topoisomerase II is ATP hydrolysis which is required for initiation of a new round of catalysis [25, 28]. The ATPase activity is not tightly coupled to DNA strand passage since the enzyme also is able to hydrolyse ATP in the absence of DNA (figure 6). DNA-independent ATP hydrolysis is strongest at low salt concentrations and then gradually decreases with increasing KCl concentrations (figure 6). Unexpect-
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Figure 6. The effect of KCl concentration on topoisomerase II-mediated ATP hydrolysis. Topoisomerase II was incubated for 15 min with radiolabelled ATP (0.5 mM, 3000 Ci/mmol) in the absence (hatched bar) or presence (filled bar) of supercoiled pBR322 DNA and the liberation of inorganic phosphate from ATP was determined by thin layer chromatography. Each point is the average of three independent experiments, Bars, standard deviation.
edly, the addition of DNA stimulates the ATPase activity only at 100 and 150 mM KCl but has little or no effect on ATP hydrolysis at other salt concentrations. While the strong ATPase activities at 100 and 150 mM KCl are associated with catalytic activity, an almost complete dissociation between the two factors is observed at 225 mM KCl where only low levels of ATP hydrolysis are present in spite of a strong catalytic activity. Surprisingly, the salt profile of the DNA-dependent ATPase activity is also different from what was observed for DNA-topoisomerase binding, since strong noncovalent DNA binding is observed for all salt concentrations below 100 mM KCl (figure 3). This suggests that non-covalent binding between DNA and topoisomerase II is not enough to stimulate enzyme-mediated ATP hydrolysis. In contrast, the DNA-dependent ATPase activity is closely related to the DNA cleavage/religation step (figure 4). It is interesting, that the presence of DNA has no effect on ATP hydrolysis at 225 mM KCl where DNA religation is very efficient. It therefore appears that the DNA-dependent stimulation of ATP hydrolysis is associated with the presence of covalent DNA-topoisomerase II complexes.
Catalytic activities of DNA topoisomerase II 4. Discussion Early studies carried out with the Drosophila phosphorylation model suggested that the rate of ATP hydrolysis modulates the overall catalytic activity of DNA topoisomerase II. In contrast, phosphorylation had no detectable effect on any of the other steps in the catalytic cycle. Although thoroughly documented both with respect to phosphorylation by CKII [13] and by PKC [16] these findings were puzzling for several reasons. First, relaxation of negatively supercoiled DNA is not an intrinsically energetically unfavourable reaction as illustrated by the fact that gyrase as well as topoisomerase I can carry out the same reaction in the absence of ATP hydrolysis [1, 34]. It was therefore unexpected that the activity of topoisomerase II should be regulated by the only step in the catalytic cycle which, at least in principle, is not absolutely necessary for its catalytic functions. Second, topoisomerase II is able to hydrolyse ATP at a considerable rate even in the absence of DNA, suggesting that DNA strand passage is not tightly coupled to ATP hydrolysis. This is further supported by the observation that the rate of ATP hydrolysis is never totally inhibited by topoisomerase II inhibitors even at drug concentrations where the catalytic activity is strongly affected [35, 36]. Finally, recent results suggest that the relationship between phosphorylation and catalytic activity is more complex than originally thought [13-16, 20-22]. Together, these findings prompted us to re-examine the catalytic cycle of topoisomerase II by an alternative method. We have chosen variation of salt concentrations, which is a classical method to evaluate biochemical properties of proteins. Our results show that with a standard amount of enzyme, the salt dependence of catalytic activity follows a bell-shaped curve with optimum around 150 mM for the yeast enzyme and between 100 and 150 mM for human topoisomerase IIα. A similar bell-shaped curve has been described for topoisomerase II purified from other sources including rat liver, Drosophila and calf thymus [28, 37, 38]. This salt dependence profile is not limited to DNA relaxation, which relies on intra-strand DNA passage but is also observed for decatenation which relies on interstrand DNA passage. The catalytic activity of topoisomerase II is classically determined at enzyme concentrations between 1 and 10 ng/µL. However, the actual concentrations of topoisomerase II in living cells are much higher. A proliferating eukaryotic cell typically contains 106 topoisomerase II molecules which are contained in a nucleus that is 1.5 to 5 µm in diameter [39]. This corresponds to an average nuclear concentration of topoisomerase II ranging between 500 ng/µL and 20 µg/µL. Therefore, the catalytic activity of topoisomerase II was also determined at higher enzyme concentrations. An increase in enzyme concentrations has two major effects. First, catalytic activity can now be detected at low
777 salt concentrations as determined by both relaxation and decatenation assays. In contrast, no catalytic activity was ever observed at high salt concentrations even at elevated enzyme concentrations, where topoisomerase II is able to bind DNA. Second, catenation activity could be observed at salt concentrations below 150 mM. Usually, topoisomerase II is only able to catenate circular DNA molecules in the presence of DNA condensing agents such as histone H1, spermidine or polyethyleneglycol. However, no such agents were present in our experiments. It has previously been shown, that if only DNA-DNA interactions are taken into account, the probability of catenation decreases with decreasing salt concentrations [40]. Therefore, the driving force must come from the enzyme. Low salt concentrations seem to favour the formation of topoisomerase II multimers at least tetrameric in size [41, 42]. A possible explanation for the catenation activity of topoisomerase II at low salt concentrations is therefore its ability to multimerize under these conditions in combination with the capacity of the enzyme to simultaneous bind to two different double-stranded DNA helixes. Our studies do not show a straight-forward correlation between DNA binding and catalytic activity, since catalytic activity can be associated with either weak (225 mM KCl), intermediate (150 mM) or strong (100 mM) DNA binding. It is interesting, that the two major mitotic functions of topoisomerase II depend on different properties of the enzyme. DNA condensation is mostly based on DNA binding and association with other proteins in the chromosome scaffold [43-45], whereas separation of sister chromatids requires catalytic activity [4-7]. This might explain why DNA binding and catalytic activity are not associated in a straight-forward manner and therefore not likely to be regulated in a co-ordinated fashion. In contrast to DNA binding, DNA cleavage and religation appear to be closely associated with the catalytic activity of topoisomerase II at all salt concentrations studied. It is interesting, that the ability of topoisomerase II to recognise both endogenous and exogenous DNA lesions is based on covalent rather than non-covalent interaction with DNA. In particular, the presence of DNA lesions results in increased DNA cleavage whereas the religation process appears to be unaffected [11]. In contrast, it is the religation step that is targeted by most anticancer and antimicrobial topoisomerase inhibitors in clinical use [3, 16]. It is remarkable that in vitro, it is possible to convert topoisomerase II into a drug-resistant enzyme that does not form drug-stabilised DNA-protein complexes simply by changing the salt concentration. This is not due to an absence of DNA cleavage but rather to a highly efficient religation as witnessed by the complete relaxation of the supercoiled DNA substrate at 225 mM KCl. Although modifications of the internal salt concentrations are not likely involved in the development of resistance to topo-
778 isomerase II-targeted drugs in tumour cells, we would predict that enzyme modifications resulting in increased religation would provide enhanced drug resistance without loss of catalytic activity. This type of topoisomerase II modification has been reported for several drug resistant tumour cell lines, although the underlying molecular mechanism(s) is/are currently unknown [46, 47]. In the absence of DNA, ATP hydrolysis is strongest at low salt concentrations and then gradually decreases with increasing salt concentrations. This is clearly very different from the bell-shaped curve observed for the catalytic activity. Interestingly, the presence of DNA increases the rate of ATP hydrolysis at 100 and 150 mM KCl but has little or no effect on ATP hydrolysis at other salt concentrations. Since DNA and topoisomerase II are tightly associated at low salt concentrations, our results strongly suggest that the stimulatory effect of DNA on the rate of ATP hydrolysis is unrelated to non-covalent DNA binding. In contrast, the rate of ATP hydrolysis appears to be closely associated with the cleavage/religation reactions. Cleavable complex formation is strongest between 100 and 225 mM while religation is optimal at 225 mM KCl. Since the presence of DNA has no effect on ATP hydrolysis at 225 mM KCl, it appears that the stimulation of ATP hydrolysis is directly associated with covalent DNAtopoisomerase II binding. This is supported by recent results with mutant yeast topoisomerase II where the absence of the active-site tyrosine (and thereby lack of covalent DNA-topoisomerase II complexes) results in loss of DNA-stimulated ATPase hydrolysis [36]. Taken together, our results show that it is the DNA cleavage/religation steps that are most closely associated with the catalytic activities of topoisomerase II. These findings are not limited to the yeast enzyme, since a close association between DNA cleavage and catalytic activity also is observed for human topoisomerase IIα. Interestingly, these are the same steps which are targeted by both anticancer and antimicrobial agents and which are involved in recognition of DNA lesions suggesting a unifying theme in the biological and pharmacological modulation of this important class of enzymes. Acknowledgments We thank Dr. Andrzej Skladanowski for helpful discussions, and Prof. Jean-Pierre Dedet for generously providing us with trypanosoma kinetoplast DNA. This work was supported in part by Association pour la Recherche sur le Cancer (ARC), Villejuif, France. D.S. is a fellow of Association pour la Recherche sur le Cancer.
References [1] Wang J.C., DNA topoisomerases, Annu. Rev. Biochem. 65 (1996) 635–692.
Scala et al. [2] Corbett A.H., Osheroff N., When good enzymes go bad: conversion of topoisomerase II to a cellular toxin by antineoplastic drugs, Chem. Res. Toxicol. 6 (1993) 585–597. [3] Maxwell A., DNA gyrase as a drug target, Trends Microbiol. 5 (1997) 102–109. [4] Dinardo S., Voelkel K., Sternglanz R., DNA topoisomerase II mutant of Saccharomyces cerevisiae: topoisomerase II is required for segregation of daughter molecules at the termination of DNA replication, Proc. Natl. Acad. Sci. USA 81 (1984) 2616–2620. [5] Uemura T., Yanagida M., Isolation of type I and II DNA topoisomerase mutants from fission yeast: single and double mutants show different phenotypes in cell growth and chromatin organization, EMBO J. 3 (1984) 1737–1744. [6] Newport J., Spann T., Disassembly of the nucleus in mitotic extracts: membrane vesicularization, lamin disassembly, and chromosome condensation are independent processes, Cell 48 (1987) 219–230. [7] Downes C.S., Mullinger A.M., Johnson R.T., Inhibitors of DNA topoisomerase II prevent chromatid separation in mammalian cells but do not prevent exit from mitosis, Proc. Natl. Acad. Sci. USA 88 (1991) 8895–8899. [8] Brill S.J., Dinardo S., Voelkel-Meiman K., Sternglanz R., Need for DNA topoisomerase activity as a swivel for DNA replication for transcription of ribosomal RNA, Nature 326 (1987) 414–416. [9] Brill S.J., Sternglanz R., Transcription-dependent DNA supercoiling in yeast DNA topoisomerase mutants, Cell 54 (1988) 403–411. [10] Corbett A.H., Zechiedrich E.L., Lloyd R.S., Osheroff N., Inhibition of eukaryotic topoisomerase II by ultraviolet-induced cyclobutane pyrimidine dimers, J. Biol. Chem. 266 (1991) 19666–19671. [11] Kingma P.S., Osheroff N., Spontaneous DNA damage stimulates topoisomerase II-mediated DNA cleavage, J. Biol. Chem. 272 (1997) 7488–7493. [12] Eder J.P.J., Chan V.T., Ng S.W., Rizvi N.A., Zacharoulis S., Teicher B.A., Schnipper L.E., DNA topoisomerase II alpha expression is associated with alkylating agent resistance, Cancer Res. 55 (1995) 6109–6116. [13] Corbett A.H., Devore R.F., Osheroff N., Effect of casein kinase II-mediated phosphorylation on the catalytic cycle of topoisomerase II. Regulation of enzyme activity by enhancement of ATP hydrolysis, J. Biol. Chem. 267 (1992) 20513–20518. [14] Cardenas M.E., Dang Q., Glover C.V., Gasser S.M., Casein kinase II phosphorylates the eukaryote-specific C-terminal domain of topoisomerase II in vivo, EMBO J. 11 (1992) 1785–1796. [15] Bojanowski K., Filhol O., Cochet C., Chambaz E.M., Larsen A.K., DNA topoisomerase II and casein kinase II associate in a molecular complex that is catalytically active, J. Biol. Chem. 268 (1993) 22920–22926. [16] Corbett A.H., Fernald A.W., Osheroff N., Protein kinase C modulates the catalytic activity of topoisomerase II by enhancing the rate of ATP hydrolysis: evidence for a common mechanism of regulation by phosphorylation, Biochemistry 32 (1993) 2090–2097. [17] Wells N.J., Addison C.M., Fry A.M., Ganapathi R., Hickson I.D., Serine 1524 is a major site of phosphorylation on human topoisomerase II alpha protein in vivo and is a substrate for casein kinase II in vitro, J. Biol. Chem. 269 (1994) 29746–29751. [18] Wells N.J., Fry A.M., Guano F., Norbury C., Hickson I.D., Cell cycle phase-specific phosphorylation of human topoisomerase II alpha. Evidence of a role for protein kinase C, J. Biol. Chem. 270 (1995) 28357–28363. [19] Wells N.J., Hickson I.D., Human topoisomerase II alpha is phosphorylated in a cell-cycle phase-dependent manner by a proline-directed kinase, Eur. J. Biochem. 231 (1995) 491–497. [20] Kimura K., Saijo M., Tanaka M., Enomoto T., Phosphorylationindependent stimulation of DNA topoisomerase II alpha activity, J. Biol. Chem. 271 (1996) 10990–10995.
Catalytic activities of DNA topoisomerase II [21] Redwood C., Davies S.L., Wells N.J., Fry A.M., Hickson I.D., Casein kinase II stabilizes the activity of human topoisomerase II alpha in a phosphorylation-independent manner, J. Biol. Chem. 273 (1998) 3635–3642. [22] Escargueil A.E., Plisov S., Scala D., Borgne A., Meijer L., Gorbsky G.J., Larsen A.K., Formation of molecular complexes between DNA topoisomerase II and p34cdc2-cyclin B stimulates topoisomerase activities independant of phosphorylation, Proc. Am. Assoc. Cancer Res. 39 (1998) 81. [23] Worland S.T., Wang J.C., Inducible overexpression, purification, and active site mapping of DNA topoisomerase II from the yeast Saccharomyces cerevisiae, J. Biol. Chem. 264 (1989) 4412–4416. [24] Wasserman R.A., Austin C.A., Fisher L.M., Wang J.C., Use of yeast in the study of anticancer drugs targeting DNA topoisomerases: expression of a functional recombinant human DNA topoisomerase II alpha in yeast, Cancer Res. 53 (1993) 3591–3596. [25] Osheroff N., Eukaryotic topoisomerase II. Characterization of enzyme turnover, J. Biol. Chem. 261 (1986) 9944–9950. [26] Osheroff N., Zechiedrich E.L., Calcium-promoted DNA cleavage by eukaryotic topoisomerase II: trapping the covalent enzymeDNA complex in an active form, Biochemistry 26 (1987) 4303–4309. [27] Robinson M.J., Osheroff N., Effects of antineoplastic drugs on the post strand-passage DNA cleavage/religation equilibrium of topoisomerase II, Biochemistry 30 (1991) 1807–1813. [28] Osheroff N., Shelton E.R., Brutlag D.L., DNA topoisomerase II from Drosophila melanogaster. Relaxation of supercoiled DNA, J. Biol. Chem. 258 (1983) 9536–9543. [29] Hsieh T., Brutlag D., ATP-dependent DNA topoisomerase from D. melanogaster reversibly catenates duplex DNA rings, Cell 21 (1980) 115–125. [30] Goto T., Wang J.C., Yeast DNA topoisomerase II. An ATPdependent type II topoisomerase that catalyzes the catenation, decatenation, unknotting, and relaxation of double-stranded DNA rings, J. Biol. Chem. 257 (1982) 5866–5872. [31] Riou G.F., Gabillot M., Barrois M., Breitburd F., Orth G., A type-II DNA topoisomerase and a catenating protein from the transplantable VX2 carcinoma, Eur. J. Biochem. 146 (1985) 483–488. [32] Rybenkov V.V., Cozzarelli N.R., Vologodskii A.V., Probability of DNA knotting and the effective diameter of the DNA double helix, Proc. Natl. Acad. Sci. USA 90 (1993) 5307–5311. [33] Hsiang Y.H., Liu L.F., Evidence for the reversibility of cellular DNA lesions induced by mammalian topoisomerase II poisons, J. Biol. Chem. 264 (1989) 9713–9715.
779 [34] Gellert M., Mizuuchi K., O’Dea M.H., Itoh T., Tomizawa J.I., Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity, Proc. Natl. Acad. Sci. USA 74 (1977) 4772–4776. [35] Robinson M.J., Corbett A.H., Osheroff N., Effects of topoisomerase II-targeted drugs on enzyme-mediated DNA cleavage and ATP hydrolysis: evidence for distinct drug interaction domains on topoisomerase II, Biochemistry 32 (1993) 3638–3643. [36] Hammonds T.R., Maxwell A., The DNA dependence of the ATPase activity of human DNA topoisomerase II alpha, J. Biol. Chem. 272 (1997) 32696–32703. [37] Duguet M., Lavenot C., Harper F., Mirambeau G., DeRecondo A.M., DNA topoisomerases from rat liver: physiological variations, Nucleic Acids Res. 11 (1983) 1059–1075. [38] Schomburg U., Grosse F., Purification and characterization of DNA topoisomerase II from calf thymus associated with polypeptides of 175 and 150 kDa, Eur. J. Biochem. 160 (1986) 451–457. [39] Heck M.M., Earnshaw W.C., Topoisomerase II: A specific marker for cell proliferation, J. Cell Biol. 103 (1986) 2569–2581. [40] Rybenkov V.V., Vologodskii A.V., Cozzarelli N.R., The effect of ionic conditions on the conformations of supercoiled DNA. II. Equilibrium catenation, J. Mol. Biol. 267 (1997) 299–311. [41] Vassetzky Y.S., Dang Q., Benedetti P., Gasser S.M., Topoisomerase II forms multimers in vitro: effects of metals, betaglycerophosphate, and phosphorylation of its C-terminal domain, Mol. Cell Biol. 14 (1994) 6962–6974. [42] Lamhasni S., Larsen A.K., Barray M., Monnot M., Delain E., Fermandjian S., Changes of self-association, secondary structure, and biological activity properties of topoisomerase II under varying salt conditions, Biochemistry 34 (1995) 3632–3639. [43] Earnshaw W.C., Halligan B., Cooke C.A., Heck M.M., Liu L.F., Topoisomerase II is a structural component of mitotic chromosome scaffolds, J. Cell Biol. 100 (1985) 1706–1715. [44] Adachi Y., Luke M., Laemmli U.K., Chromosome assembly in vitro: topoisomerase II is required for condensation, Cell 64 (1991) 137–148. [45] Bojanowski K., Maniotis A.J., Plisov S., Larsen A.K., Ingber D.E., DNA topoisomerase II can drive changes in higher order chromosome architecture without enzymatically modifying DNA, J. Cell Biochem. 69 (1998) 127–142. [46] Robert J., Larsen A.K., Drug resistance to topoisomerase II inhibitors, Biochimie 80 (1998) 247–254. [47] Larsen A.K., Skladanowski A., Cellular resistance to topoisomerase II-targeted drugs: from drug uptake to cell death, Biochim. Biophys. Acta 1400 (1998) 257–274.