THE JOURNAL OF BIOLOGICAL CHEMISTRY

Printed in U. S. A. Vol. 257. No. 10, Issue of May 25. pp. 5866-5872. 1982

Yeast DNA Topoisomerase I1 AN ATP-DEPENDENT TYPE I1 TOPOISOMERASE THAT CATALYZES THE CATENATION, DECATENATION, UNKNOTTING, AND RELAXATION OF DOUBLE-STRANDED DNA RINGS*

(Received for publication, November 24, 1981)

Tadaatsu Goto and James C. Wang From the Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138

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

DNA topoisomerases have been subject to intensive studies in recent years (for reviews, see Refs. 1-5). They can be divided into two classes. The type I enzymes are those that break and rejoin DNA strands one at a time; the type I1 enzymes are those that break and rejoin both strands of a duplex DNA more-or-less in concert (6, 7). In bacteria, the major type I activity is exemplified by Escherichia coli DNA topoisomerase I, an enzyme that was first reported in 1971 as the w protein (8). The major type I1 activity is DNA gyrase, which was discovered in 1976 as an ATP-dependent enzyme that catalyzes the negative supercoiling of DNA (9). Bacterial topoisomerases that are distinct from these two enzymes have not yet been reported. Several phage coded enzymes, however, have been identified and studied extensively (for reviews, see Refs. 1-5). In eukaryotic organisms, the major type I activity is exemplified by the mouse enzyme that was f i s t reported in 1972 (10). Type I1 DNA topoisomerases in eukaryotic orga- nisms were found only recently (11-13). Catalytically, all eukaryotic type I1 enzymes are similar to the type I1 enzyme coded by phage T4 (14) in terms of their ATP dependence and their inability to supercoil DNA, at least when assayed under the conditions employed. These enzymes are detectable by their relaxation of supercoiled DNA, catenation or decate- nation of double-stranded DNA rings, and unknotting of duplex DNA rings containing topological knots. All of these

* This work has been supported by a grant from the United States Public Health Service (GM 24544). The costs of publication of this article were defrayed in part by the payment of page charges. This

ance with 18 U.S.C. Section 1734 solely to indicate this fact. article must therefore be hereby marked “advertisement” in accord-

reactions have also been demonstrated for DNA gyrase, and a derivative of gyrase lacking part of the B subunit (for reviews, see Refs. 2 and 3).

Although much progress has been made on the enzymolog- ical and mechanistic aspects of topoisomerases, studies on the functional aspects of these enzymes have been essentially limited to the prokaryotic ones. For E. coli DNA gyrase, the identification of the subunits A and B as the targets of the antibiotics nalidixic acid and coumermycin, and the availabil- ity of mutants in these genes, have led to a rapid accumulation of information on the in vivo roles ,of this enzyme (2, 3). For E. coli DNA topoisomerase I, its structural gene has recently been identified and a preliminary account of the plausible roles of this enzyme has appeared (15-17).

Because of the lack of information on the biological func- tions of the eukaryotic DNA topoisomerases, we have initiated studies of such enzymes of the lower eukaryote Saccharomy- ces cereuisiae. This organism offers a number of advantages for such studies: it is readily available in large quantities for biochemical work, and it can be subject to detailed genetic analysis. With the rapid development of in vitro recombinant DNA methodology for this organism, close interplay between biochemical and genetic analysis is now possible.

In terms of the enzymology of yeast DNA topoisomerases, the presence of a type I activity with catalytic characteristics similar to those of higher eukaryotic DNA topoisomerases has been reported (18). We have now purified this type I enzyme, to homogeneity, which will be referred to as yeast DNA topoisomerase I. During the purification of yeast DNA topo- isomerase I, an activity that catalyzes the aggregation of DNA rings in an ATP-dependent fashion was detected. This activity has been purified and shown to be a type I1 topoisomerase, and will be referred to as yeast DNA topoisomerase 11. The detailed purification procedures for these enzymes will be published elsewhere. In the present communication, we report the identification of the DNA aggregate formed by the yeast type I1 enzyme as topologically interlocked networks of DNA rings, and our studies on the purified yeast type I1 DNA topoisomerase.

EXPERIMENTAL PROCEDURES

Materials-Restriction endonucleases, pancreatic DNase I, and E. coli DNA polymerase I were purchased from commercial sources including New England Biolabs, Worthington, Boehringer Mannheim, Miles, and Bethesda Research Laboratories. E . coli and Micrococcus luteus DNA topoisomerase I were the preparations previously re- ported (19.20). T4 DNA ligase and calf thymus DNA topoisomerase I were prepared in this laboratory by Drs. P. Martens and K. Java- herian, respectively. Electrophoresis grade agarose, heparin agarose, aminopentyl agarose, and hydroxylapatite were purchased from Be- thesda Research Laboratories. CsCl was purchased from Harshaw Chemical Co., Solon, OH, ethidium bromide from Calbiochem, and all other chemicals were analytical grade or higher grades from commercial sources.

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Phage and plasmid DNAs were prepared by conventional methods; density gradient centrifugation in CsC1-ethidium (21) was employed to purify the covalently closed form. Radioactive labeling of phage PM2 DNA was done by nick translation of the unlabeled DNA with E. coli DNA polymerase I in the presence of a labeled and three unlabeled deoxynucleoside triphosphates. T4 DNA ligase and ATP were also added to the mixture in order to obtain the labeled DNA in the covalently closed form. Positively supercoiled PM2 DNA was prepared by relaxing the DNA at 0 "C with calf thymus DNA topoisomerase I in the yeast topoisomerase assay mixture described below. The DNA was phenol-extracted and then dialyzed exhaus- tively. At higher temperatures in the yeast topoisomerase assay mixture, the DNA becomes slightly positively supercoiled because of the temperature coefficient of the average helix screw of DNA (22-24).

Assays-As mentioned in the introduction, the yeast type I1 to- poisomerase activity was first detected during the purification of the yeast type I topoisomerase as an activity that forms aggregates of duplex DNA rings. Therefore, the assay mixture employed initially was the one used for the assay of yeast DNA topoisomerase I. Each assay mixture contained, in 20 pl, 0.2 pg of closed circular DNA, 20 mM Tris-HCI, pH 7.4.50 mM KCI, 10 m MgCL, 0.1 mM Nan EDTA, 1 mM 2-mercaptoethanol, 0.2 m ATP, and 50 pg/ml of gelatin. As the purification of the activity proceeded, the requirement of a yeast DNA-binding protein DBP I for the aggregation was uncovered and a stimulatory effect of yeast DNA topoisomerase I was noted when supercoiled DNA was the substrate (see "Results" and "Discussion"). Hence, the assay mixture was supplemented with approximately 0.2 pg of DBP I and purified yeast DNA topoisomerase I was also added when supercoiled DNA was used as the substrate.

Assays employing the unknotting of knotted phage P4 DNA rings were carried out as described (25). The assay mixture was the same as the one described above, except that the KC1 concentration was raised to 150 m, and DBP I and topoisomerase I were omitted.

For either assay, incubation was carried out at 30 "C for 30 min to 6 h, and the reaction was stopped by the addition of a mixture containing sodium dodecyl sulfate, Ficoll, and xylene cyanol and bromphenol blue as tracking dyes. The final mixture, containing approximately 1% sodium dodecyl sulfate and 5% Ficoll, was then loaded in a sample well of a horizontal or vertical slab agarose gel (0.7%) in TBE buffer (50 mM Tris base, 50 m boric acid, and 1 mM Na2EDTA). Electrophoresis was at a field around 2 V/cm. When the xylene cyanol dye reached about one-third of the full length of the gel, the run was stopped and the gel was stained in 1 pg/ml of ethidium bromide before photography. In cases where ethidium was included in the gel and electrophoresis buffer, the ethidium-staining step was omitted (26).

Other Techniques-Density gradient centrifugation in CsC1-ethid- ium bromide for the identification of the catenated species was carried out at 35,000 rpm and 20 "C for 48 h, in an SW 50.1 rotor, as described (21). The nitrocellulose tube containing about 5 ml of solution was dripped through a hole punctured at i t s bottom, and each fraction collected was extracted with 1-butanol and then dialyzed against 10 mM Tris.HC1, pH 8, 0.1 mM NaZEDTA. Electron microscopy was done according to the aqueous spreading procedure of Davis et al. (27).

Purification of Yeast DNA Topoisomerase Z and ZZ and DNA- binding Protein Z-Detailed documentation on the purification of these proteins will be presented elsewhere. Most of the preparations used in this work were obtained from kilogram quantities of commer- cial bakers' yeast (Federal Yeast). In one preparation, S. cereuisiae strain D22 (a ade;), obtained from Dr. Bernard Dujon, Harvard University, was grown in a 200-liter fermentor, and cells were pelleted by centrifugation and processed immediately. Disruption of the cells was carried out by grinding with glass beads in a Dyno-Mill cell disrupter (Willy A. Bachofen AG Maschinenfabrik, Basel, Switzer- land). Upon removal of the cell debris by centrifugation, the lysate was subject to precipitation with Polymin P, back extraction of the precipitate, fractionation by precipitation with (NH&SO,, and batch- wise adsorption to phosphocellulose. In one preparation, the eluent from phosphocellulose was further purified by chromatography suc- cessively on carboxymethyl cellulose, heparin agarose, and single- stranded DNA agarose. The last column separates pure DBP I from topoisomerase activities. In a separate preparation, the phosphocel- lulose eluent was purified by chromatography successively on heparin agarose, hydroxylapatite, double-stranded DNA cellulose, and ami- nopentyl agarose. The last column separates yeast DNA topoisom- erases I and 11. Unless specified otherwise, the experiments reported here were carried out with one of the DNA topoisomerase I1 prepa-

rations from commercial yeast that gives two bands of roughly equal intensities upon electrophoresis in a sodium dodecyl sulfate-polyacryl- amide gel and staining with Coomassie blue. The apparent molecular weights of these two species were estimated to be approximately 150,000 and 160,000 from their electrophoretic mobilities. The purity of the preparation with the freshly grown yeast appears to approach homogeneity, and only the 150,000-dalton band is present. Repetition of the experiments with this preparation revealed no significant differences.

RESULTS

Catenation ofDouble-stranded DNA Rings by Yeast DNA Topoisomerase ZZ-The yeast type I1 DNA topoisomerase was first detected as an activity that promotes the aggregation of double-stranded rings into giant networks (Fig. 1). Because of the size of the network, it barely enters 0.7% agarose gel upon electrophoresis; therefore, i ts formation can be readily monitored by the accumulation of the input DNA on the top of the gel.

Several lines of evidence suggest that this aggregation in- volves the topological linking of DNA rings. First, no such network is formed if linear DNA is used as the substrate. Second, with a circular DNA substrate, the network that is formed is resistant to protein denaturants including phenol, dodecyl sulfate, and proteinase K, but is readily converted to monomeric linear DNA when it is digested with a restriction enzyme that cleaves each monomeric ring once (results not shown).

To elucidate further the nature of the linkage among mem- bers of the network, we focused on a dimeric species that is produced early in the aggregation reaction. Fig. 2 depicts the electrophoretic patterns of samples incubated with the en- zyme in the presence of different amounts of ATP. At a low ATP concentration of 20 p ~ , a distinct species with an elec- trophoretic mobility slightly higher than that of the nicked circular monomer is formed. The mobility of the species suggests that it is a covalently closed dimer. The formation of this species as well as the much larger aggregate requires Mg(I1) and another yeast protein in addition to the topoisom- erase. This protein turns out to be a DNA-binding protein, which we will describe in a later section.

The assignment of the species formed at low ATP concen- tration as the covalently closed dimer received further support

. . .. ,

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FIG. 1. An electron micrograph showing part of a network of DNA rings formed by incubating phage PM2 DNA with yeast DNA topoisomerase 11. The Parlodion-coated grid after sampling was first stained with uranyl acetate, shadowed with plati- num-palladium, and viewed and photographed with a Philips 300 electron microscope.

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FIG. 2. Dependence of the DNA network formation on ATP concentration. Reaction mixtures containing phage I'M2 DNA were incubated with a yeast activity in the absence or presence of varying amounts of ATP, and then analyzed by electrophoresis in a 0.7% agarose gel. Ethidium bromide was present in the gel as well as the electrophoresis buffer at a concentration of 0.5 pg/ml. The leftmost lane contained the control in which the yeast activity was omitted. The bulk of the DNA was in the negatively supercoiled form, and a small amount of the nicked monomeric rings migrated as a slower band. The second to the tenth lanes, from left to right, contained samples incubated with the yeast activity in the presence of 0, 2, 5, 10, 20, 50, 100, 200, and 500 p~ ATP, respectively. A distinct band that migrates slightly faster than the nicked monomers can be seen in samples incubated with 20 p~ or higher concentrations of ATP. The formation of the aggregated DNA network is evident in samples run in the rightmost four lanes: a band that barely enters the gel can be clearly seen. The fraction containing the yeast activity used in these samples also contained the ATP-independent yeast DNA to- poisomerase I activity, which relaxes the input supercoiled DNA even in the absence of ATP (lane Z ) , and hence increases the mobility of the DNA in the presence of excess ethidium (compare the patterns of samples run in lanes 1 and 2).

when we examined whether extensive homology in nucleotide sequences is required for its formation. Fig. 3 depicts the results of such an experiment. Samples of "2P-labeled phage PM2 DNA with or without added unlabeled double-stranded replicative form of $X 174 DNA were incubated with the yeast enzyme as described in the figure legend. In the absence of +X 174 DNA, the formation of the putative dimeric PM2 DNA is evident (lane 2). The addition of 4X 174 DNA yielded a new species; the position of which is marked by an arrow in Fig. 3. With covalently closed duplex $X 174 DNA, PM2 DNA, and the putative dimeric PM2 DNA as markers, the electrophoretic mobility of this new species suggest that it is a heterodimer containing one $X 174 DNA and one PM2 DNA ring, both in the covalently closed form. In agreement with this assignment, it is found that the band disappears if the sample is digested with either Xho I, which cleaves $X 174 but not PM2 DNA (lane 6), or with HindIII, which cleaves PM2 but not +X 174 DNA (lane 7).

In the heterodimer-formation experiment, 4x174 and PM2 DNA were present a t equal weights in the reaction mixture, and therefore the molar concentration of the former was higher than the latter. In spite of this higher concentration, the 9X174-PM2 heterodimer band is much fainter than the PM2 homodimer band. This lower intensity is partly due to the fact that only PM2 DNA is radioactively labeled. In addition, the sue of the DNA affects the extent of catenation. Incubation mixtures identical with those described in the legend to Fig. 3 but containing only relaxed @X 174 DNA or simian virus 40 DNA gave significantly lower amounts of homodimers compared with incubation mixtures containing only PM2 DNA at the same nucleotide concentrations (results not shown). Although these results were not analyzed quan-

titatively, qualitatively it appears that the two factors de- scribed above are sufficient to account for the lower intensity of the heterodimer band.

The covalently closed nature of the dimeric species is fur- ther established by two experiments. In one, a PM2 DNA sample containing the dimeric species was analyzed by two- dimensional gel electrophoresis. Following electrophoresis of the sample ig an edge lane of a slab gel a t neutral pH and in the presence of 0.5 p g / d of ethidium bromide, the gel was equilibrated with an alkaline buffer and electrophoresis in the second dimension was then carried out. The nicked monomer that was present in the PM2 DNA sample used yielded two

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FIG. 3. An autoradiogram demonstrating the formation of a heterodimer between 32P-labeled PM2 DNA and unlabeled co- valently closed double-stranded phage @X 174 DNA. Incubation of the DNA mixtures (containing approximately 0.2 pg of each DNA) with the yeast DNA topoisomerases and the yeast DNA-binding protein was carried out at 30 "C for 6 h as described under "Experimental Procedures," except that the ATP concentration was 20 p ~ . Restriction endonucleases were then added to the appropriate samples to be specified below and incubation was continued at 37 "C for 30 min. The samples were then analyzed by electrophoresis in a 0.7% agarose gel containing TBE buffer plus 0.5 pg/ml of ethidium bromide. The gel was dried and autoradiographed. Numbering from left to right, lane 1 contained the ,''2P-labeled PM2 DNA without treatment with the yeast activity. The predominant species are closed and nicked monomeric rings, and a small amount of linear monomers in between the two major species is also present. A faint band immediately below the nicked monomer band is discernible on the original autoradiogram, and is most likely the covalently closed di- meric catenanes that amount to several per cent by mass in PM2 DNA (28). Treatment of PM2 with the yeast activity in the presence of 20 p~ ATP increases the amount of the dimeric catenane band (lane 2). The sample run in lane 3 was treated with the yeast activity and then the restriction endonuclease HindIII, which cleaves PM2 into seven fragments, all but two had cleared the gel. The sample run in lane 4 was a control that contained a mixture of "P-labeled PM2 DNA and unlabeled +X 174 DNA, but was incubated in the absence of the yeast enzyme. Incubation of this sample with the yeast activity gives a new species (lane 5) at a position indicated by an arrow on the left side of the autoradiogram. This new species disappears if prior to electrophoresis the mixture is digested with the restriction endonuclease Xho I, which cleaves @X 174 DNA but not PM2 DNA (lane 6). or with HindIII, which cleaves PM2 DNA but not @X 174 DNA (lane 7).

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FIG. 4. Analysis of fractions collected from CsCI-ethidium bromide density gradient centrifugation by electrophoresis in an ethidium-containing agarose gel. Approximately 30 pg of PM2 DNA was incubated with yeast DNA topoisomerase I1 and a yeast DNA-binding protein in the presence of 20 p~ ATP. CsCl and ethidium bromide were then added according to the procedure de- scribed (21) and the mixture was centrifuged at 35,000 rpm for 48 h in a Beckman SW 50.1 rotor. Fractions were collected, butanol-ex- tracted, and dialyzed, and approximately one-tenth of each fraction was loaded on the gel. The leftmost and rightmost lanes show the pattern of a PM2 DNA control that had been treated with M. luteus DNA topoisomerase I. The middle eight lanes, from left to right, contained fractions with decreasing buoyancy in the CsC1-ethidium bromide density gradient. See text for further description.

species in the second dimension: a faster migrating linear single strand and a slower migrating circular single strand. In contrast, the dimeric species yielded a single discrete species in the second dimension. This shows that the components of the dimeric species cannot be dissociated by alkali treatment that can separate complementary strands in a nicked circular DNA.

In a second experiment, the reaction mixture after incuba- tion with the yeast topoisomerase was first subjected to CsCI- ethidium bromide density gradient centrifugation. Fractions collected from such a gradient were extracted with 1-butanol to remove ethidium, dialyzed, and then examined by gel electrophoresis. The results are shown in Fig. 4. The leftmost and rightmost lanes contained a control sample that had been treated with M. luteus DNA topoisomerase I. We have shown previously that the bacterial type I topoisomerase can catalyze the formation of dimeric catenanes between a nicked mono- meric ring and a covalently closed monomeric ring (band 1 in the figure) and between two nicked monomeric rings (band 2). Lanes 1 through 8 contained fractions collected from the CsC1-ethidium gradient. Clearly, the distribution of the di- meric species formed by the yeast topoisomerase coincides with that of the covalently closed monomer, indicating that this species is covalently closed.

The experiments described in this section leave little doubt that the yeast enzyme can promote the topological linking of covalently closed double-stranded DNA rings. This in turn suggests that the enzyme is a type I1 topoisomerase. Fig. 4 also indicates that two other dimeric species are formed by the yeast enzyme as well. The band that peaks in fractions run in lanes 6 and 7, where nicked DNA is expected, has a mobility identical with band 2 of the control, and is most likely a catenated dimer between two nicked monomers. The band that peaks in the fraction run in lane 4, where a cate- nated dimer between a nicked and a covalently closed ring is expected, has a mobility identical with band 1 of the control and is most likely identical with band 1. Faint bands with mobilities lower than those of the various dimers are also noticeable in the corresponding fractions and are presumably

catenanes of higher molecular weights. They were not char- acterized further.

Catenation of DNA by Yeast DNA Topoisomerase ZZ Re- quires a Yeast DNA-binding Protein DBP Z-The require- ment of a yeast DNA-binding protein for the aggregation reaction was uncovered during the course of purification of the principles needed for this reaction. The activity that promotes the reaction disappeared after chromatography on a single-stranded DNA-agarose column (see “Experimental Procedures”) and could be recovered only by combining dif- ferent pools. Further purification of one of these pools yielded a homogeneous protein factor with a peptide weight of 30,000 from its electrophoretic mobility in a sodium dodecyl sulfate- polyacrylamide gel. The combination of this protein and highly purified yeast DNA topoisomerase I1 is sufficient for the linking of DNA rings into giant networks. This protein binds strongly to both single- and double-stranded DNA, and hence, we have tentatively designated it yeast DNA-binding protein I or DBP I for short. Whether DBP I is identical with one of the known yeast DNA-binding proteins is uncertain. Its molecular weight, however, appears to distinguish it from the 20,000-dalton DNA-binding protein from yeast mitochon- dria (29) and the 36,000-dalton DNA-binding protein reported by Chang et al. (30).

We considered the possibility that DBP I might be a subunit of the topoisomerase, and came to the conclusion that this is highly unlikely, because of the various DNA topoisomeriza- tion reactions that do not require DBP I. These reactions will be described below.

Yeast DNA Topoisomerase ZZ Can Uncatenate the DNA Network in the Absence of DBP Z-It is found that if the DNA network formed in the presence of DBP I is first deproteinized and then treated again with the topoisomerase in the absence of DBP I, dissolution of the network occurs and monomeric rings are released. The decatenation reaction requires ATP and proceeds efficiently only at KC1 concentra- tions higher than 150 m~ (results not shown).

This experiment strongly suggests that the role of DBP I in the network-forming reaction is to favor the aggregation of DNA molecules and therefore shift the equilibrium toward catenation. For all the topoisomerases studied to date, a t moderate DNA concentrations, a “condensing agent” is needed for catenane formation. This agent can be a protein (12), or a polycation such as spermidine (31). With the yeast topoisomerase studied here, we have not yet found conditions under which spermidine can substitute DBP I in the catena- tion reaction.

The Yeast Topoisomerase Can Relax Supercoiled DNA and Unknot Knotted Duplex DNA Rings-During the earlier stage of purification of yeast DNA topoisomerase 11, its catal- ysis of relaxation of supercoiled DNA could not be monitored due to the proficiency of the contaminating type I topoisom- erase in this reaction. Once the type I1 enzyme is purified free of the type I activity, its relaxation of either positively or negatively supercoiled DNA can be readily demonstrated. Fig. 5 depicts the results of such an experiment.

Three sets of samples were analyzed on the gel. From left to right, lanes 1 to 5 depict the electrophoretic patterns of the negatively supercoiled PM2 DNA after incubating with the yeast enzyme for 0,20,40,60, and 90 min, respectively. Lanes 6 to 10 contained samples of a slightly positively supercoiled PM2 DNA after incubating with the same amount of enzyme for time intervals that correspond to samples run in lanes 1 to 5. The final set of five samples (lanes 11 to 15) correspond to the other two sets with the two DNAs mixed together before the addition of the enzyme. Although the difference in the degrees of superhelicity of the positively and negatively su-

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percoiled DNAs make a quantitative comparison of the rates impractical, qualitatively, the rates are clearly comparable.

To test whether the linking number of the DNA changes in steps of two in the relaxation reaction, as one would expect for a type I1 DNA topoisomerase-catalyzed reaction (6,32), a single negatively supercoiled topoisomer was isolated (24) and treated with the enzyme. The linking number of this topoiso- mer is indeed found to change in steps of 2 (result not shown).

We have also tested the unknotting of knotted duplex DNA rings by the yeast type I1 topoisomerase. It has been shown recently that the DNA extracted from tailess phage P2 or P4 capsids is in the form of a complex knotted ring in which two single chain breaks are present as a result of the joining of the cohesive ends of the DNA molecule (25, 33). Several DNA topoisomerases have been tested with this substrate. All type I1 enzymes, but not type I enzymes, are capable of unknotting this DNA even after its treatment with DNA ligase to convert it to the covalently closed form. The yeast DNA topoisomer- ase 11, as expected, converts the knotted phage P4 DNA to simple rings without knots. As shown in Fig. 6, treatment of knotted phage P4 DNA with the yeast enzymes converts the faster migrating knotted species to a distinct band with an electrophoretic mobility identical with that of nicked circular P4 DNA without knot. If the knotted P4 DNA was first treated with DNA ligase to convert it to the covalently closed form, the subsequent treatment with the yeast type I1 enzyme gives a ladder of topoisomers characteristic of simple cova- lently closed DNA rings with a distribution in their linking numbers.

The Salt Dependence of Yeast DNA Topoisomerase-cata- lyzed Reactions-As mentioned earlier, yeast DNA topoisom- erase I1 requires Mg(I1) for its activity. Substitution of Mn(I1) for Mg(I1) results in a reduction of activity by a t least a factor of 10, as assayed by the unknotting reaction. As shown in Fig. 7, the unknotting reaction is strongly dependent on KC1 concentration in the medium, with a sharp optimum in salt concentration in between 150 and 200 m. The range of KC1 concentration in which yeast DNA topoisomerase I1 can effi- ciently relax supercoiled DNA or uncatenate linked rings is also narrowly defined; the salt dependence profies for these reactions resemble closely that of the unknotting reaction (data not shown). On the other hand, the catenation reaction shows a much broader salt dependence profile, with the opti- mal KC1 concentration around 100 to 150 m~ (data not shown).

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FIG. 5. Relaxation of negatively and positively supercoiled PM2 DNA by yeast DNA topoisomerase 11. Each incubation mixture contained 0.2 pg of DNA in 20 pl of 20 mM Tris- HCI, pH 7.4, 150 mM KCI, 10 mM MgC12,O.l mM Na?EDTA, 1 m~ 2-mercaptoeth- anol, 0.2 m~ ATP, and 50 pg/ml of gelatin. Identical amounts of purified yeast DNA topoisomerase I1 were added to the mixtures kept on ice, and the samples were immediately incubated at 30 “C for the time intervals specified in the text. Stopping of the reaction and gel electrophoresis of the samples were done as described under “Experimental Procedures.”

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FIG. 6. Unknotting of knotted P4 DNA. Knotted P4 DNA was extracted from phage P4 capsids without tails as described previously (25). From left to right, lane I contained the untreated DNA. The two faint sharp bands are the slower migrating nicked rings and the faster migrating linear species. The broad smear that migrated ahead of the nicked rings contains rings with knots of varying degrees of complexity (25,31). Treatment of this DNA with purified yeast DNA topoisomerase I1 converts all the knotted species to simple nicked rings (lane 2). Note that the treatment has no effect on the linear species. Lane 3 shows the knotted phage P4 DNA after treatment with DNA ligase. Subsequent incubation of the ligase treated sample with yeast DNA topoisomerase I1 converts the knotted species to a group of bands that migrated with mobilities of those expected for topoisomers obtained by the covalent closure of nicked rings without knots at the ligase treatment conditions (lune 4).

Yeast DNA Topoisomerase 11 is Strongly Inhibited by Coumermycin-Bacterial gyrases are inhibited by the anti- biotics novobiocin and coumermycin at concentrations as low as a few tenth pg/ml. Inhibition of the type I1 topoisomerases from Drosophila and human cells occurs only at much higher levels of these drugs (12, 13).

For the yeast type I1 enzyme, novobiocin is not an effective inhibitor; a concentration of 300 pg/ml of the drug is needed to reduce the relaxation activity of the enzyme by a factor of 2. Coumermycin is, however, a potent inhibitor of the yeast enzyme. Strong inhibition of the relaxation reaction is evident at a drug concentration as low as 5 pg/ml. As expected, the catenation activity of the yeast enzyme follows a similar pattern of inhibition as the relaxation activity. We have ob- served no inhibitory effect when the gyrase inhibitors nalidixic and oxolinic acids were tested with yeast DNA topoisomerase 11.

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FIG. 7. Dependence of the yeast DNA topoisomerase 11-cat- alyzed unknotting reaction on KC1 concentration. The leftmost lane contained the untreated knotted P4 DNA control. Samples run in lanes 2 to 9 were incubated with 50,75, 100, 125, 150, 175,200, and 225 mM KCI, respectively, under otherwise standard unknotting assay conditions.

DISCUSSION

The yeast DNA topoisomerase I1 resembles the eukaryotic type I1 DNA topoisomerases from Xenopus, Drosophila, and HeLa, and the phage T4 DNA topoisomerase, in terms of its strict ATP dependence in catalyzing the transient breakage and passage of duplex DNA and its inability to catalyze the supercoiling of DNA. The yeast enzyme reported here appears to be of nuclear origin. Upon fractionation of nuclei, cytoplas- mic proteins, and mitochondria by the procedure of Ide and Saunders (34), the DNA topoisomerase I1 activity is found mainly in the nuclei fractions (results to be detailed else- where). It has been previously reported that the catalytic characteristics of yeast DNA topoisomerase I also resemble those of other eukaryotic type I topoisomerases (18). Thus, the lower eukaryote yeast appears to be a suitable system for studying the biological functions of the DNA topoisomerases in eukaryotic organisms.

The classification of the yeast ATP-dependent DNA topo- isomerase as a type I1 enzyme is fumly established. The decatenation, unknotting, and relaxation reactions that are catalyzed by the enzyme are most likely different manifesta- tions of the same basic reaction: the transient breakage of a duplex DNA and the passage of a double-stranded DNA segment through this gate (6, 31). The very similar salt de- pendence profdes for these reactions are entirely consistent with this view.

The catenation reaction is more complicated. At the DNA concentration employed for the assays, catenation of mono- meric DNA rings is expected to be thermodynamically unfa- vorable (35). The possibility that the utilization of ATP can be coupled to the linkage of the rings, and therefore overcome the intrinsic unfavorable entropic term, is ruled out by the decatenation reaction described under “Results.” Thus, cate- nation a t ordinary DNA concentrations is driven by agents that can bring separate DNA molecules into close proximity (12,31). In the case of the reaction described in this work, the yeast DNA-binding protein DBP I presumably fiis such a role. The requirement of a protein factor for the catenation of duplex DNA rings by the type I1 Drosophila DNA topoisom- erase has been reported previously (12). The salt dependence profde for the catenation reaction differs from those of the other reactions the topoisomerase catalyzes. This is most likely a resuit of the participation of the binding protein in this reaction. Studies on how DBP I facilitates the catenation

reaction and the in vivo roles of this protein are yet to be carried out, however.

It has been reported previously that relaxed circular DNA appears to be a better substrate than supercoiled DNA in the catenation reaction catalyzed by E. coli DNA gyrase in the presence of spermidine (31). We have also observed that the formation of the aggregated DNA network by yeast DNA topoisomerase I1 and DBP I is faster with relaxed DNA than with negatively supercoiled DNA (results not shown). Several possibilities can be raised to interpret this difference. The larger hydrodynamic volume of the relaxed DNA ring may make it a more favorable substrate for catenation; the super- helicity of the DNA may affect its interaction with the enzyme and the processivity of the reaction; the DBP I may interact differently with supercoiled and relaxed DNA. This superhel- icity dependence of the catenation reaction sometimes gives the false impression during the early stages of the purification of a type I1 enzyme that a type I enzyme is also required, since fractions that also contain the type I enzyme may show higher catenation activities because of the efficient relaxation of the input DNA by this enzyme. Thus, the unknotting reaction is more advantageous for assays of type I1 DNA topoisomerases during purification.

The steep salt dependence of the yeast topoisomerase I1 catalyzed reaction is striking. This may reflect the displace- ment of a large number of cations when the enzyme binds to DNA (36). If the steep salt dependence is a general property of the eukaryotic type I1 DNA topoisomerases, then the blockage of decatenation of interlocked S V 40 DNA in vivo by hypertonic shock (37) may be related to the inhibition of the type I1 enzyme resulting from an increase in intracellular salt concentration.

The sensitivity of the yeast DNA topoisomerase I1 to inhi- bition by coumermycin is of interest. Although inhibition of other type I1 DNA topoisomerases by coumermycin has been observed before (12), the yeast enzyme is much more sensitive toward inhibition by this drug. It should be worthwhile to explore the possible use of this drug in vitro or in vivo for the elucidation of the mechanistic and functional aspects of the yeast enzyme.

Acknowledgments-We thank Professor Leroy Liu for communi- cating to us the details of the unknotting assay with knotted phage P4 DNA prior to the publication of the procedure, and Dr. Michael Kotewicz for his assistance during the early phase of the purification of the yeast enzymes.

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T Goto and J C Wangdouble-stranded DNA rings.

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

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