Proc. Natl. Acad. Sci. USA Vol. 83, pp. 7152-7156, October 1986 Biochemistry ATP-independent type II from trypanosomes (DNA cleavage/) SETHA DOUC-RASY, ALAIN KAYSER, JEAN-FRANrOIS Riou, AND GuY RIou* Laboratoire de Pharmacologie Clinique et Molculaire, Institut Gustave Roussy, 94805 Villejuif Cedex, France Communicated by William Trager, June 24, 1986

ABSTRACT We have characterized in Trypanosoma cruzi type II topoisomerase, but its activity is not ATP-dependent a DNA topoisomerase capable of decatenating complex (9). Such a type II has also been characterized in trypanosomal kinetoplast DNA networks in the absence of Trypanosoma equiperdum. ATP. The enzymatic activity requires Mg2' and K+. Using a The involved in DNA metabolism may offer defined DNA topoisomer we showed that the linking number targets for , especially in rapidly proliferating changes by steps of 2, which characterizes the enzyme as a type cells. Some trypanocidal drugs have been shown to inhibit II topoisomerase. The enzyme can catenate supercoiled DNA the in vitro activity of trypanosomal (10, 11). molecules, unknot DNA, and cleave double-stranded DNA. Other drugs have now been tested in vitro before attempting The enzyme has no ATPase activity. The native enzyme has an to selectively inhibit the growth of the parasite in its host. Mr of about 200,000. Crude extracts and partially purified fractions contain an aggregating factor that can substitute MATERIALS AND METHODS spermidine in catenating reactions. Because of the presence of this factor, the kinetoplast DNA can only be decatenated by Materials. Hydroxylapatite was prepared according to the purified fractions. The enzyme is inhibited by certain drugs and method of Bernardi (12), and DNA-cellulose was prepared provides a potential target for chemotherapy. Such an enzyme according to the method of Alberts and Herrick (13). was also characterized in Trypanosoma equiperdum. Phosphocellulose was purchased from Whatman. Calf thy- mus topoisomerase II was prepared as described by Miller et The topology of DNA inside cells is governed by DNA al. (14). Circular DNA was the replicative form of bacterio- topoisomerases implicated in such fundamental processes as phage fd, simian virus 40 DNA, or the pUC13 plasmid, replication, segregation of replicas, and gene transcription purified by CsCl/ethidium bromide centrifugation. kDNA [see reviews by Gellert (1) and by Wang (2)]. There are two was extracted from T. cruzi (15). Knotted DNA was prepared classes of topoisomerases according to their mechanisms of from tailless capsids of bacteriophage P4, according to the action. The type I enzymes break and reseal one strand of method of Liu et al. (16). The 32P-labeled single topoisomers DNA changing the linking number in steps of 1 whereas the were prepared by A. Prunell according to Shore et al. (17). type II enzymes break and reseal both strands in a concerted Drugs. derivatives were provided by J. B. Le manner changing the linking number in steps of 2. In Pecq and dimers were provided by B. P. Roques. , the role of type I topoisomerases could be to Coumermycin A2 was a gift of J. C. Wang. Hydroxystilbam- relax, whereas that ofthe type II could be to supercoil DNA, idine and clorobiocin were purchased from Rhone Poulenc thus permitting a correct balance only when both types are (Vitry, France). was purchased from Sigma, present in the cell (3). The DNA gyrase that is a type II nalidixic acid was from Winthrop (Longvic, France), oxolinic topoisomerase of bacteria can introduce negative supercoils acid was from Substancia (Courbevoie, France), Lampit (a into DNA in vitro and in vivo by utilizing the energy from the 5-nitrofurane derivative) was from Bayer, and 4'-(9-acridi- hydrolysis of ATP (1). Eukaryotic cells contain also nylamino)methanesulfon-m-anisidide (mAMSA) was a gift of topoisomerases potentially capable of modifying torsional B. Baguley (University of Auckland Medical School, stress in DNA (4). In vitro, both types of topoisomerase can Auckland, New Zealand). remove torsional stress from DNA but cannot introduce Purification of the Topoisomerase. T. cruzi, Tehuantepec supercoils. It was recently demonstrated that topoisomerase strain, was cultured in vitro and collected in the exponential II assumes an essential function in yeast (5) and in mamma- growth phase (15). The cells (7 g) were washed in a solution lian cells (6). The eukaryotic type II topoisomerases are of 50 mM Tris, pH 7.5/0.5 mM EDTA, resuspended in 80 ml known to be ATP-dependent enzymes. However, their ATP of the same buffer, and disrupted in a Potter homogenizer requirement remains unexplained. The trypanosomal en- (3000 rpm, 10 strokes). The extract was centrifuged (10,000 zyme characterized in this report is not ATP-dependent. x g for 30 min). All of the different steps of purification were Trypanosomes have special characteristics, such as the carried out at 0-4°C. As described by Miller et al. (14), the existence of a cell cycle through various hosts and a con- pellet was resuspended in 45 ml of a solution containing 50 spicuous abundance of kinetoplast DNA (kDNA). The me- mM Tris (pH 7.5), 4 mM EDTA, and 10 mM mercapto- tabolism of kDNA has been studied by biochemical methods ethanol. An equal volume of 2 M NaCl in the same buffer was and electron microscopy (7). DNA topoisomerases have been added; then 45 ml of 18% (wt/vol) polyethylene glycol (PEG implicated in the replication ofkDNA consisting in thousands 6000) in the same buffer with 1 M NaCl was also added. After of interlocked circular molecules. We have previously de- stirring for 40 min, the mixture was centrifuged (10,000 x g, scribed a type I enzyme in Trypanosoma cruzi (8). In this 30 min). The supernatant was loaded on a hydroxylapatite paper we describe an enzyme that is capable of decatenating column (1.5 x 7 cm) equilibrated with a solution of 1 M the complex kDNA network of trypanosomes and catenating NaCl/50 mM Tris, pH 7.5/10 mM mercaptoethanol/1 mM closed circular DNA molecules. This enzyme actually is a Abbreviations: kDNA, kinetoplast DNA; PhMeSO2F, phenylmeth- The publication costs of this article were defrayed in part by page charge ylsulfonyl fluoride; mAMSA, 4'-(9-acridinylamino)methanesulfon- payment. This article must therefore be hereby marked "advertisement" m-anisidide. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom correspondence should be addressed. 7152 Downloaded by guest on September 29, 2021 Biochemistry: Douc-Rasy et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7153 phenylmethylsulfonyl fluoride (PhMeSO2F)/6% PEG. The precipitated with ethanol and electrophoresed in a 4% column was washed with 0.2 M potassium phosphate (pH 7.0) acrylamide gel. The gel was dried and autoradiographed. in solution A (20% glycerol/10 mM mercaptoethanol/l mM PhMeSO2F). The proteins were eluted with 70 ml of a linear RESULTS gradient (0.2-0.8 M) of potassium phosphate in the same solution. The relaxation activity eluted between 0.35 and 0.55 Decatenation of kDNA Networks. DNA topoisomerases M. The pooled fractions were diluted 1:2.5 and loaded on a were purified from T. cruzi. The purification steps were a phosphocellulose column (1 x 4 cm) equilibrated with 0.2 M PEG precipitation followed by chromatographic fraction- potassium phosphate in solution A. The column was washed ations on hydroxylapatite, phosphocellulose, and DNA- with 0.2 M potassium phosphate and developed stepwise with cellulose. A purified enzyme was able to decatenate kDNA 5 ml of0.3, 0.4, 0.5, and 0.6 M buffer. The relaxation activity networks. However, the decatenating activity was not found eluted between 0.3 and 0.5 M. The pooled fractions were either in the PEG supernatant or in the hydroxylapatite dialyzed for 4 hr against solution B (40 mM Tris, pH 7.5/20% fractions, which were the first purification steps (Table 1). glycerol/10 mM mercaptoethanol/1 mM PhMeSO2F) con- A sample of the DNA-cellulose fraction (62 ng of protein) taining 0.1 M KCl and then loaded on a single-stranded dispersed the kDNA minicircles in the absence of ATP (Fig. DNA-cellulose column (0.9 x 2.5 cm) equilibrated with the 1A, lane 3). ATP did not stimulate the activity of a sample same buffer. The column was washed with the same buffer containing a more diluted enzyme. The liberated minicircles and eluted stepwise with 3 ml of 0.2, 0.3, 0.4, 0.6, and 1.0 M appeared unbroken when their migration rate was compared KCl in solution B. The relaxation activity eluted in a bimodal with that of minicircles liberated by calf thymus topoisom- fashion between 0.25 and 0.30 M and between 0.4 and 0.6 M. erase II in the presence of0.5 mM ATP (lane 2). Three bands, The former fraction was protected by addition of serum containing nicked, relaxed, and slightly supercoiled albumin (1 mg/ml) and dialyzed against a solution containing minicircles, appeared in the electrophoretic pattern. By 100 mM Tris (pH 7.5), 50% glycerol, 10 mM mercapto- varying the composition of the reaction mixture, we found ethanol, and 0.5 mM EDTA. This preparation contained the that decatenation required Mg2' (Fig. 1A, lane 4). KCl also topoisomerase studied in this paper. The size of the native was required, 80 mM being the optimal concentration (not enzyme was measured by using the gel filtration column shown). Decatenation was less effective when NaCl was (Sephacryl S-300) and the 20-40% glycerol gradient centrif- added instead of KCl (not shown). The optimal enzymatic ugation of the phosphocellulose fraction as described in activity occurred between pH 7.0 and 7.5. legend ofFig. 4. The decatenating fraction that sedimented at The decatenating activity was presumably masked in crude 9 S was used for the determination ofthe type ofthe enzyme. extracts and in hydroxylapatite fractions (Table 1) by the It was free of topoisomerase I, which sediments at 4.3 S. presence of an inhibitor (20-24). To test this hypothesis, the Assays ofTopoisomerase Activities. Relaxation was assayed "hydroxylapatite fraction" was added to purified topoisom- in 20-1.l samples containing 10mM Tris (pH 7.5), 50mM KCl, erase II from mammalian cells: the decatenation was inhib- 10 mM MgCl2, 0.5 mM dithiothreitol, 0.5 mM EDTA, 15 uig ited (Fig. 2). Furthermore, the hydroxylapatite fraction was of albumin per ml, and about 0.1 ,ug of supercoiled DNA. able to catenate individual circular molecules (Fig. 1B). Incubation was at 37TC, 30 min. Decatenation was assayed in Therefore, an aggregating factor was present with the cate- the relaxation mixture, using a KCl concentration of 80 mM nating enzyme in the hydroxylapatite fraction. After chro- and kDNA from T. cruzi as substrate (0.1 ,ug). One unit matography on phosphocellulose, a peak of decatenating decatenated half of the kDNA in 30 min at 370C. The activity was separated from a peak of aggregating factor, the decatenation mixture for mammalian topoisomerase II as- latter eluting at a higher phosphate concentration (0.8 M). says contained 10 mM Tris (pH 7.9), 10 mM MgCl2, 100 mM Change in Linking Number of Single Topoisomers. The KCl, 1 mM ATP, 0.5 mM EDTA, and 15 ,ug of albumin per critical experiment was to relax-in the absence of ATP-a ml. Unknotting ofP4 DNA was assayed under the conditions defined topoisomer using the decatenating fraction from the of decatenation of trypanosome enzyme. Catenation was glycerol gradient that was free of topoisomerase I contami- assayed like relaxation, but in the absence of KCl. When nation. It was done with a radioactive topoisomer 506 base phosphocellulose fractions were assayed, spermidine (5 mM) pairs in size. Taking the relaxed topoisomer as a reference, was added as a condensing agent. Double-stranded DNA linking number differences (ALk) will characterize each band cleavage was performed in the relaxation mixture. The of the patterns. Unreacted topoisomers are presented in Fig. substrate was pUC13 DNA (0.2 ,ug). After 10 min of incu- 3 (lanes 1 and 4). Their respective ALk are -3 and -2. The bation at 37°C, the reaction was stopped by the addition of 5 ALk = -3 substrate was transformed into ALk = -1 ,ul of 5% NaDodSO4, 4 mg of proteinase K per ml, 0.02% topoisomer by the decatenating enzyme (lane 2). By contrast, bromophenol blue, and 25% glycerol. The incubation was action of trypanosome topoisomerase I transforms the same continued for 45 min at 50°C. The reversion of the double- substrate into the complete 'series of more relaxed topoiso- stranded cleavage by saline was performed as described (18, mers: i.e., ALk = -2 (faint band), ALk = -1, and the more 19). prominent ALk = 0 (lane 3). The ALk = -2 substrate was Electrophoresis. The reactions were stopped by addition of transformed into ALk = 0 without appearance of a ALk = -1 4 1,u of a solution containing NaDodSO4 (0.1%), glycerol band (lane 5). Lanes 2 and 5 show the step of 2 in linking (50%), and bromophenol blue (0.05%). The DNA was ana- lyzed in 1% agarose slab gels. The buffer contained 36 mM Table 1. Purification steps of the decatenating enzyme Tris base, 30 mM NaH2PO4, and 1 mM Na3EDTA (pH 7.7) Decatenating Specific (20). The electrical field consisted in 3 V cm'1 for 2 hr. The Protein, activity, activity, DNA was stained with ethidium bromide (10 mgl-1) and the Step mg units units/mg gel was photographed under UV light using a Polaroid camera PEG supernatant (135 ml) 37 None (film 665) and a red filter. Relaxation of a Single 32P-Labeled Topoisomer. The relax- Hydroxylapatite fraction (30 ml) 2.85 None ation mixture contained 1500 cpm of the pBR322-derived Phosphocellulose topoisomer and 0.2 ,ug of unlabeled pBR322. The reaction 1.6 x 2 104 was stopped by addition of NaDodSO4 to a final concentra- fraction (18 ml) 0,8 104 x tion of 1%. The solution was made up to 1 M NaCl and then DNA-cellulose fraction 0.12 3.9 x 3.2 x 104 extracted with chloroform/isoamyl alcohol (24:1). DNA was (5 ml) 103 Downloaded by guest on September 29, 2021 7154 Biochemistry: Douc-Rasy et al. Proc. Natl. Acad Sci. USA 83 (1986)

A B 1 2 3 4 5 1 2 3 4 5

kDNA_ c aten a ted,

1"+ 11 _ mCI +11 -2 - 1.45 1.09 -0.74 -3

0.36

FIG. 3. Change in linking number by trypanosome topoisomer- FIG. 1. (A) Decatenation of T. cruzi kDNA networks. Lane 1, ases. The 32P-labeled single topoisomers were obtained by circular- kDNA (control). Lane 2, with 50 ng of calf thymus topoisomerase II ization in the presence of a suitable concenmration of ethidium and 0.5 mM ATP; minicircle (mC) topoisomers are liberated. Lane bromide of the 506-base-pair Hinfl restriction fragment of pBR322. 3, with 60 ng of trypanosomal enzyme (DNA-cellulose fraction); no DNA in lane 1 is referred to as the ALk -3 topoisomer (the fastest ATP was added. Lane 4, like lane 3, but without Mg addition. Lane component). It was transformed by topoisomerase II ('1 unit of a 5, DNA size markers; kDNA minicircles digested by Hae III (15). (B) glycerol gradient fraction, 30-min incubation) into ALk = -1. No Catenation of fd phage DNA molecules. Lane 1, fd DNA (control). ALk = 0 (relaxed topoisomer, the slowest component) appeared Lane 2, with 200 ng of trypanosomal enzyme (hydroxylapatite (lane 2). For comparison, the same topoisomer was transformed by fraction); no ATP addition. topoisomerase I (:1 unit, 5-min incubation) (lane 3). A faint ALk = -2 band is visible. ALk = -1 is more abundant and ALk = 0 is much more abundant. DNA in lane 4 is the ALk = -2 topoisomer that number change which characterizes relaxation by a type II identifies the band just cited in lane 3. It was transformed by topoisomerase. The decatenating enzyme will be called T. topoisomerase II (=1 unit, 30-min incubation) into ALk = 0 without cruzi topoisomerase II. appearance ofa ALk = -1 band (lane 5). Lanes 2 and 5 show the step Physical Characteristics of the Enzyme. The topoisomerase of 2, which characterizes relaxation by type II topoisomerase. The II (phosphocellulose was on a band appearing between 0 and -1 was probably due to radiolysis fraction) chromatographed products. Electrophoresis was carried out at room temperature in a calibrated Sephacryl column, which indicated a Stokes radius 4% polyacrylamide gel with a Tris acetate/EDTA buffer (40 mM of 54 A for the molecule (Fig. 4B). In a 20-40% glycerol Tris-HCl/20 mM sodium acetate/2 mM EDTA, pH 7.8). gradient, the enzyme sedimented at 9 S. The aggregating factor sedimented at about 2.0 S (Fig. 4A). Assuming a partial specific volume of 0.725 cm3/g, an evaluation carried out reported, the enzyme fully catenated supercoiled DNA according to Siegel and Monty (26) gave a native Mr of molecules in the absence of ATP (8, 11). 200,000 and a frictional ratio (f/fo) of 1.4 for the enzyme. In Unknotting. Unknotting is analogous with decatenation. NaDodSO4/polyacrylamide gels, the purified enzyme either However, knotted P4 DNA is an open molecule, whereas from the glycerol gradient fraction or from the gel filtration kDNA is constituted of closed circles. Nevertheless, fraction showed faint bands ranging from 110,000 to 50,000. unknotting by the T. cruzi enzyme did not depend on the Catenation. We have shown above that the hydroxylapatite presence of ATP (Fig. 5). As for the decatenation reaction, fraction contained a masked decatenating activity and that Mg2+ and K+ were required (80-100 mM, lanes 4 and 5). only more purified fractions were able to decatenate kDNA. Neither the PEG supernatant nor the hydroxylapatite frac- When KCl was excluded from the reaction mixture and a tion was able to unknot P4 phage DNA molecules. condensing agent was added, the purified fractions became Inhibition of Catenation and Decatenation. About 2 units of able to catenate enzyme was added to reaction mixtures containing DNA and circular DNA (Fig. 1B). When used in variable concentrations of drug. Inhibition of decatenation suitable concentrations, spermidine (5 mM) and histone H1 was shown by the disappearance of the minicircle bands. (0.05 ,uM) allowed DNA condensation (8). Mg2+ was requirpd Inhibition of catenation was demonstrated by the persistence but could be replaced by Mn2+ or Ca2+. Above 50 mM, KCI of free DNA molecules. Several drugs, known for their and NaCl were inhibitory (data not shown). As previously antitumoral properties, trypanocidal action, or inhibitory effect on DNA gyrase activity, were tested. Coumermycin 1 2 3 4 5 A2 and clorobiocin (27) were rather good inhibitors of the D N k A , catenating and decatenating reactions (at concentration of about 20 ,M). These effects were analogous to the inhibition of type II topoisomerase from Drosophila by coumermycin Al (28). In contrast, nalidixic and oxolinic acids (.2000 AM) and novobiocin (800 ,uM) were poor inhibitors. The trypanocidal drugs berenil and hydroxystilbamidine were inhibitory at concentrations of 30 ,M and 100 ,uM, respec- mC-_ tively. Intercalating drugs, such as ellipticine derivatives (11), were potent inhibitors (1.5-3 ,uM). Double-Stranded DNA Cleavage. As prokaryotic type II topoisomerases (29-31), enzymes of eukaryotic origin have FIG. 2. Inhibition of decatenation by the "hydroxylapatite frac- been implicated in double-stranded DNA cleavage reactions tion." Lane 1, kDNA (control). Lane 2, with calf thymus (18, 19). Topoisomerase and DNA form tight complexes, topoisomerase II (2 units). Lanes 3-5, with calf thymus which, upon NaDodSO4 treatment, result in DNA breaks. topoisomerase II and trypanosomal enzyme (hydroxylapatite frac- When the enzyme was used in a stoichiometric concentra- tion) at 100, 200, and 400 ng, respectively. mC, minicircle. tion, the linear form of the circular substrate could be Downloaded by guest on September 29, 2021 Biochemistry: Douc-Rasy et al. Proc. Natl. Acad. Sci. USA 83 (1986) 7155

Catal A B 1 0O- 100- TG 01) 0<

Cu CO) co co enzyme tCatal 5 a) 50[- zI 0 enzyme C') Aggregating -J' Myo factor Chymo I II 10 20 0.5 1.0 Fraction No (Kav) 1/3 FIG. 4. Sedimentation coefficient (A) and Stokes radius determinations (B). (A) Topoisomerase (phosphocellulose fraction, about 600 units) was applied to a preformed linear 20-40% glycerol gradient containing 0.1 M KCI, 50 mM Tris-HCI (pH 7.9), 1 mM PhMeSO2F, 0.5 mM dithiothreitol, and 10 mM mercaptoethanol. Centrifugation was carried out at 40C for 24 hr at 47,000 rpm in a Beckman SW 50.1 rotor. Fractions of 180 ,ul were collected from the bottom of the tube, and 2-1.l samples were assayed for decatenating activity. Markers of known sedimentation coefficients, purchased from Pharmacia, were used in separate tubes: beef liver catalase (Catal) (11.3 S, 1 mg), aldolase (Aldo) (7.35 S, 0.5 mg), bovine serum albumin (BSA) (4.35 S, 1 mg), and chymotrypsinogen A (Chymo) (2.54 S 0.5 mg). (B) Topoisomerase (phosphocellulose fraction, about 8000 units) was applied to a Sephacryl S-300 gel column (100 x 1.6 cm) equilibrated in 1 M NaCI/15 mM sodium phosphate, pH 7.1/0.5 M dithiothreitol/0.1 mM EDTA and eluted with a flow rate of 10 ml/hr. The topoisomerase was located on the basis ofits decatenating activity. Standard proteins of known Stokes radius, purchased from Bio-Rad, were used to calibrate the column: thyroglobulin (TG) (85 A), beef liver catalase (Catal) (52 A), ovalbumin (Oval) (30.5 A), and myoglobin (Myo) (22 A). Blue dextran and phenol red were used to measure the void and'included volumes, respectively. The relative elution, Kay, was calculated for each protein and (K0V)J13 was plotted against the Stokes radius (25).

visualized on electrophoretic gels. The cleavage was highly ATP for all catalytic activities. However, during the purifti- stimulated by mAMSA addition to the reaction mixture (32). cation ofthe enzymes, some oftheir native properties may be The trypanosomal enzyme produced a low proportion of lost due to proteolytic degradation or to a resolution of their linearized molecules (Fig. 6, lane 3), unless mAMSA (lane 4) supramolecular structure (5). Type I topoisomerases were was added to the reaction mixtures. The cleavage reaction known not to require ATP. However, an enzyme from the was reversed by 0.5 M NaCl addition (lane 5). The reaction archaebacterium Sulfolobus acidocaldarius was recently was not stimulated by addition of ATP (not shown), unlike shown to perform relaxation and positive supercoiling in the the cleavage generated by other eukaryotic enzymes (18, 33). presence of ATP by changing the linking number in steps of 1 (34). DISCUSSION The trypanosomal extracts (PEG supernatant), as well as the enzyme obtained after hydroxylapatite chromatography, Topoisomerases have been extracted from cells of various are capable of catenating supercoiled circular DNA mole- origins. Their properties have been investigated in vitro. cules. When this reaction was reported earlier (8), it was Their in vivo activities are less well known. In addition to the stated that ATP addition was not required. The extracts fundamental property that defines the type I or II enzymes, contained a factor that prevented the reverse reaction of other properties have been recognized as being shared by decatenation. We have now shown that T. cruzi contains a topoisomerases of similar origins. For example, type II topoisomerases from eukaryotic cells are known to require 1 2 3 4 5 1 2 3 4 5

11+10 = Unknotted _ Knotted

FIG. 6. Stimulation of double-stranded DNA cleavage by mAMSA. Lane 1, pUC13 DNA (control). Lane 2, with EcoRI. The endonuclease cleaved DNA at a unique site and linearized (III) the FIG. 5. Unknotting of P4 phage DNA. Lane 1, knotted DNA different forms of the substrate (I, lo, and II). Lane 3, with (control). Lane 2, with calf thymus topoisomerase (2 units). Lanes trypanosomal enzyme (20 units). Lane 4, same as lane 3, with 5 ,4M 3-5, trypanosomal enzyme (60 ng); no ATP addition; KCI concen- mAMSA. Lane 5, same as lane 4 and after 10 min of incubation, with trations of 50, 80, and 100 mM. 0.5 M NaCl. Downloaded by guest on September 29, 2021 7156 Biochemistry: Douc-Rasy et al. Proc. Natl. Acad. Sci. USA 83 (1986) decatenating enzyme that is a type II topoisomerase. A R. Acad. Sci. Ser. 3 302, 283-286. similar enzyme was extracted from Trypanosoma 10. Riou, G., Gabillot, M., Douc-Rasy, S. & Kayser, A. (1984) in equiperdum (unpresented data). However, the type II Molecular Biology of Host Parasite Interactions, eds. topoisomerases so far isolated from vertebrates (14), as well Agabian, N. & Eisen, H. (Liss, New York), pp. 279-289. 11. Douc-Rasy, S., Kayser, A. & Riou, G. (1984) EMBO J. 3, as from insects (20), yeast (21), protozoa such as Plasmodium 11-16. berghei (35), and even the trypanosomatid Crithidia 12. Bernardi, G. (1971) Methods Enzymol. 21, 95-139. fasciculata (23), depend on the presence of ATP for their 13. Alberts, B. & Herrick, G. (1971) Methods Enzymol. 21, activity. Furthermore, the Crithidia enzyme has an optimal 198-217. KCl concentration of about 140 mM, which is inhibitory for 14. Miller, K. G., Liu, L. F. & Englund, P. T. (1981) J. Biol. our enzyme. When tested by the method of Arai et al. (36), Chem. 256, 9334-9339. the latter enzyme has no ATPase activity, in contrast with 15. Riou, G. & Yot, P. (1977) Biochemistry 16, 2390-2396. that of Crithidia. The trypanosomal topoisomerase, which 16. Liu, L. F., Davis, J. L. & Calendar, R. (1981) Nucleic Acids does not require ATP, is different from that of Res. 9, 3979-3989. probably 17. Shore, D., Langowski, J. & Baldwin, R. L. (1981) Proc. Natl. Crithidia. The same ATP-independent enzyme was found in Acad. Sci. USA 78, 4833-4837. either nuclei or whole cells. It would be interesting to find out 18. Liu, L. F., Rowe, T. C., Yang, L., Tewey, K. M. & Chen, whether our topoisomerase is also present in mitochondria. G. L. (1983) J. Biol. Chem. 258, 15365-15370. A more promising approach may be to further characterize 19. Sander, M. & Hsieh, T. (1983) J. Biol. Chem. 258, 8421-8428. the protein moiety ofthe kDNA-protein complex revealed by 20. Hsieh, T. & Brutlag, D. (1980) Cell 21, 115-125. CsCl centrifugation experiments (37). It is tempting to pro- 21. Goto, T. & Wang, J. C. (1982) J. Biol. Chem. 257, 5866-5872. pose that a topoisomerase may be associated with kDNA. 22. Krasnow, M. & Cozzarelli, N. R. (1982) J. Biol. Chem. 257, Such a situation would be analogous to that observed in 2687-2693. mitotic scaffolds II has 23. Shlomai, J. & Zadok, A. (1983) Nucleic Acids Res. 11, (38). Topoisomerase 4019-4034. been found only in of cycling cells and, 24. Riou, G., Gabillot, M., Barrois, M., Breitburd, F. & Orth, G. similarly, the trypanosomal protein may be bound to kDNA (1985) Eur. J. Biochem. 146, 483-488. only in growing cultures. It should be noted that Castora et 25. Andrews, P. (1970) Methods Biochem. Anal. 18, 1-50. al. (39) have obtained an enzyme preparation from rat liver 26. Siegel, L. M. & Monty, K. J. (1966) Biochim. Biophys. Acta mitochondria capable of catenating circular DNA in the 112, 346-362. absence of ATP. 27. Ninet, L., Benazet, F., Charpentie, Y., Dubost, M., Florent, We have been able to stimulate double-stranded DNA J., Mancy, D., Preud'homme, J., Threlfall, T. L, Vuillemin, cleavage by addition of mAMSA or ellipticine to the reaction B., Wright, D. E., Abraham, A., Cartier, M., De Chezelles, N., Godard, C. & Theilleux, J. (1972) C. R. Acad. Sci. Ser. 3 mixtures. This suggested involvement of a type II 275, 455-458. topoisomerase. The type I trypanosomal topoisomerase al- 28. Osheroff, N., Shelton, E. R. & Brutlag, D. L. (1983) J. Biol. ready described (8) differed in its sensitivity to various drugs Chem. 258, 9536-9543. from the present enzyme. The type II enzyme is more 29. Gellert, M., Mizuuchi, K., O'Dea, M. H., Itoh, T. & sensitive to ellipticines-with the exception of dimethylhy- Tomizawa, J. (1977) Proc. Natl. Acad. Sci. USA 74, droxyellipticinium (40)-than the topoisomerase I. The in- 4772-4776. hibitory concentrations are about the same as those of a 30. Sugino, A., Peebles, C. L., Kreuzer, K. N. & Cozzarelli, typical type II topoisomerase (41). Since trypanosomes N. R. (1977) Proc. Natl. Acad. Sci. USA 74, 4767-4771. proliferate quite rapidly in host tissues, the activity of their 31. Tse, Y. C., Kirkegaard, K. & Wang, J. C. (1980) J. Biol. topoisomerase may be ofa comparatively high level, as found Chem. 255, 5560-5565. 32. Nelson, E. M., Tewey, K. M. & Liu, L. F. (1984) Proc. Natl. in regenerating rat liver (42) and in tumor cells (24). The Acad. Sci. USA 81, 1361-1365. topoisomerase, therefore, represents a potential target for 33. Udvardy, A., Schedl, P., Sarider, M. & Hsieh, T. (1985) Cell trypanocidal agents that can either stimulate double-stranded 40, 933-941. DNA cleavage or inhibit the catalytic activity. It might be an 34. Forterre, P., Mirambeau, G., Jaxel, C., Nadal, M. & Duguet, appropriate system to select new drugs. M. (1985) EMBO J. 4, 2123-2128. 35. Riou, J. F., Gabillot, M., Philippe, M., Schrevel, J. & Riou, G. The critical experiment of relaxation by steps of a defined (1986) Biochemistry 25, 1471-1479. topoisomer was done in the laboratory ofA. Prunell (Institut Jacques 36. Arai, K., Yasuda, S. & Kornberg, A. (1981) J. Biol. Chem. 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