THE JOURNAL OF BIOLOGICAL CHEMISTRY VoI. 256, No. 11, Issue of June 10, pp. 5860-5865, 1981 Printed in U S.A.

Purification and Characterization of Wheat Germ DNA Topoisomerase I (Nicking-Closing Enzyme)*

(Received for publication, October 20, 1980)

William S. DynanS, Jerome J. Jendrisakfj, Dayle A. Hager, and Richard R. Burgess1 From the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

Wheat germ contains an enzyme capable of removing supercoils from circular DNA. We have purified this enzyme using Polymin P fractionation, ammonium sul- fate precipitation, and chromatography on Bio-Rex 70 and phenyl-Sepharose. Renaturation after electropho- resis on sodium dodecyl sulfate-polyacrylamide gels shows that topoisomerase activity is associated with a polypeptide with a M, = about 111,000. The enzyme is similar to other eukaryotic type I DNA topoisomerases (nicking-closing enzymes) by the following criteria: it is capable of increasing or decreasing the topological linking number of covalently closed DNA substrate; it is capable of restoring an equilibrium distribution of linking numbers to DNA substrate with a single unique linking number; and it does not require magnesium ion or ATP for activity.

An enzyme activity from mammalian cells that removes positive and negative superhelical turns from DNA was first described by Champoux and Dulbecco (1972). Similar en- zymes have been purified to near homogeneity from human tissue culture cells (Keller, 1975a) and from rat liver (Mc- Conaughy and Champoux, 1976). The enzyme appears to be widespread among eukaryotes, having been found in Drosoph- ila (Baase and Wang, 1974), Xenopus (Mattoccia et al., 1976), and sea urchins (Poccia et QL, 1978) in addition to various mammalian cells and tissues. The presence of this activity in plants has not previously been reported.

The properties of enzymes that interconvert topological isomers of DNA have been described in two recent reviews (Champoux, 1978, and Wang and Liu, 1979). Following the recommendations of the latter reviewers, we have here adopted the term DNA topoisomerase I in place of earlier designations such as nicking-closing enzyme, relaxation pro- tein, or w protein.

In general, type I eukaryotic DNA topoisomerases do not require magnesium ion or ATP for activity. The mechanism by which they remove supercoils from closed circular DNA is believed to be the introduction of a transient swivel during the concerted breakage and rejoining of one of the two strands of double helical DNA, Consistent with this mechanism, the distribution of topological linking numbers in the fully relaxed

* This work was supported by Grants CA-07175 and CA-23076 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. + These studies were in partial fulfillment of the requirements for a Ph.D. degree. Present address, Department of Biochemistry, Uni- versity of California, Berkeley, CA 94720.

Q Present address, Department of Botany, University of Minnesota, St. Paul, MN 55108.

7 To whom reprint requests should be directed.

product is the same as that generated when open circular DNA is closed by DNA ligase.

DNA topoisomerase has been used to generate DNA with different amounts of supercoiling for studying in vitro tran- scription, (Dyan and Burgess, 1981, accompanying paper) for generating standards with which to count the number of supercoils in naturally occurring DNA (Keller, 1975133, for studying the effect of ion type and concentration on DNA conformation (Anderson and Bauer, 1978), and for in vitro chromatin reconstitution (Germond et al., 1979).

We describe in this paper a convenient method for purifying large amounts of DNA topoisomerase from wheat germ. The method involves a series of low speed centrifugations followed by chromatography on Bio-Rex 70 and phenyl-Sepharose. Phenyl-Sepharose separates macromolecules on the basis of their hydrophobicity, a relatively new method of protein fractionation which has not previously been used for topo- isomerase purification. After the two chromatographic steps, our enzymes is approximately 60% pure and is free of protease and endodeoxyribonuclease activity. From 500 g of wheat germ we typically obtain 250 pg of topoisomerase, which is an amount sufficient to prepare gram quantities of relaxed DNA for use in physicochemical or transcription studies.

EXPERIMENTAL PROCEDURES

Materials and Buffers-The buffer used throughout the purifica- tion contained 50 mM Tris-HCl, pH 7.9, at 25 “C, 1 m~ EDTA, and 1 mM dithiothreitol, plus ammonium sulfate as indicated (TED buffer). Agarose (standard low -Mr), acrylamide, methylenebisacryl- amide, ammonium persulfate, tetramethylethylenediamine, and Coo- massie blue were obtained from Bio-Rad. Bio-Rex 70, from Bio-Rad, was prepared as described by Lowe et al. (1979). Phenyl-Sepharose was from Pharmacia. Before pouring into a column, it was washed in TED buffer containing 0.05 M ammonium sulfate, degassed, and equilibrated with 1 M ammonium sulfate in TED buffer. Wheat germ was generously provided by General Mills Corporation. (Vallejo, CA). Polymin P was diluted and neutralized before use as previously described (Jendrisak and Burgess, 1975).

Renaturation of Topoisomerase after Electrophoresis-SDS’- polyacrylamide gel electrophoresis was carried out according to La- emmli (1970) with the modifications previously described (Burgess and Jendrisak, 1975). The procedure used for elution of protein from SDS-polyacrylamide gels, removal of SDS, and renaturation of en- zymatic activity will be described in detail elsewhere (Hager and Burgess, 1980). Briefly, segments of the gel were crushed, protein was allowed to elute by diffusion, the suspension was centrifuged to remove polyacrylamide, and protein was precipitated with acetone to concentrate it and to remove SDS. The pellets were washed with 80% acetone/20% TED buffer, and were resuspended in buffer containing 6 M guanidine hydrochloride. The samples were then diluted 50-fold into a buffer consisting of 50 r n ~ Tris-HC1, 1 mM EDTA, 1 mM dithiothreitol, 50 mM NaC1, 5 m~ MgC12, 20% glycerol, 100 pg/ml of bovine serum albumin. Topoisomerase activity was assayed as de- scribed below.

Topoisomerase Assay-The principle of the assay is the decreased mobility in an agarose gel of supercoiled DNA after treatment with

’ The abbreviation used is: SDS, sodium dodecyl sulfate.

5860

Wheat Germ DNA Topoisomerase I 5861

topoisomerase (Keller, 19758). Assays contained 50 mM Tris-HCI, pH 7.9, 1 mM EDTA, 1 mM dithiothreitol, 208 glycerol. 0.05 M NaCI, and 200 ng of supercoiled pBR322 DNA in a final volume of 50 pl. Reactions were started by addition of 5 pl of the sample to be assayed and were allowed to proceed for 5 min a t 37 "C. Reactions were terminated by addition of SDS to 0.5% and bromphenol blue to 0.0058. The samples were loaded on a 0.8% agarose gel cast in a horizontal slab apparatus (McDonnell et al., 1977). The electropho- resis buffer contained 40 mM Tris-HCI, pH 7.9, 1 mM EDTA, 5 mM sodium acetate. After electrophoresis a t 2 V/cm for 4 h, gels were stained for 15-30 min in 0.5 pg/ml of ethidium bromide and the UV fluorescent material was photographed.

I t was usually necessary to dilute enzyme samples 100- to 1000-fold before assaying. The dilution buffer was the same as the reaction buffer except that it contained 100 pg/ml of bovine serum albumin and did not contain DNA. Topoisomerase is labile at low concentra- tion, so dilutions were made at 0 "C, and assays were performed as rapidly as possible after dilution. When necessary, two rows of slots were cast one above the other, so that as many as 28 samples could be run on a single slab gel (15 X 15 cm).

The DNA used as substrate for topoisomerase was the 4.3-kilobase plasmid pBR322, propagated in Escherichia coli strain HB101. After overnight amplification with chloramphenicol (Clewell, 1972), cells were harvested and a cleared lysate prepared (Katz et al., 1973). The DNA was banded twice in CsCl/ethidium bromide density gradients. After extensive dialysis, the DNA was precipitated with ethanol, resuspended in buffer containing 10 mM Tris-HCI. pH 7.9, 1 mM EDTA, 100 mM NaCI, and stored frozen a t -20 "C or -55 "C. We estimate that DNA prepared by this method is 90-958 supercoiled. Recombinant DNA containing SV40 in pBR322 was prepared by the same method, in accordance with National Institutes of Health guide- lines (P2, EK1, certified vector).

Purification of Topoisomerase-All steps in the purification were carried out a t 0-4 "C. Centrifugations were for 15 min at 10.000 X g.

The initial steps in the purification procedure were the same as for purification of wheat germ RNA polymerase I1 (Jendrisak and Bur- gess, 1975). Grinding buffer was TED containing 0.075 M ammonium sulfate, 23 pg/ml of phenylmethylsulfonyl fluoride, 10 Komberg In- ternational Units/ml of Trasylol. Wheat germ (500 g) was ground in 2 liters of this buffer for 1 min a t full speed in a Waring Blendor. An additional 500 ml of 0.075 M ammonium sulfate in T E D buffer were added, the extract was centrifuged, and the pellet was discarded.

The supernatant was filtered through Miracloth (Calbiochem), its volume was measured, and 0.075 volume of 10% Polymin P solution was added slowly while stirring a t low speed in a Waring Blendor. Stirring was continued for 5 min, a t which time the suspension was centrifuged. The pellet was either used for preparation of RNA polymerase I1 or discarded. The volume of the supernatant was measured, and 23.1 g of solid ammonium sulfate/100 ml were added. After stirring for 30 min, the solution was centrifuged and the pellet was discarded. A further 6.3 g of ammonium sulfate/100 ml of Polymin P supernatant were added, and the solution was stirred for 30 min. This solution was centrifuged and the supernatant discarded. The pellet was redissolved in TED buffer slowly and without mechanical homogenization. The redissolved material was diluted with T E D buffer until its conductivity was equal to 0.15 M ammonium sulfate in T E D buffer. The volume at this stage was typically 560 ml.

This material was applied to a 50-ml Bio-Rex 70 column at a flow rate of 50 ml/hr. The column was washed with 5 volumes of 0.15 M ammonium sulfate in T E D buffer and eluted with a &column volume gradient of 0.15-0.3 M ammonium sulfate in T E D buffer. Fractions were collected, and 5 pl of a 1:lOO dilution of each fraction were assayed for topoisomerase activity. The fractions containing the bulk of the activity were pooled.

These pooled fractions were adjusted to 1 M ammonium sulfate by addition of 2 M ammonium sulfate in T E D buffer. This sample, typically 120 ml, was applied to a 10-ml bed volume phenyl-Sepharose column at 10 ml/hr, and the column was washed with 50 ml of 1 M ammonium sulfate in TED buffer. Topoisomerase was eluted from the column by decreasing the ammonium sulfate concentration in the buffer; a 9-column volume linear gradient from 1-0.5 M ammonium sulfate in T E D buffer was used. Fractions were collected, and 5 1-11 of a 1:500 dilution of each fraction were assayed for topoisomerase activity. The fractions containing the bulk of the activity were pooled and dialyzed against storage buffer containing 50 mM Tris-HCI, pH 7.9, 1 mM EDTA, 1 mM dithiothreitol, 50% glycerol, 0.5 M NaCI. Aliquots were stored a t -20 "C or frozen at -55 "C.

This storage buffer was chosen as a result of tests to see how stable

the enzyme activity was to thermal inactivation in various buffers. In this buffer only about one-half of the activity was lost when the enzyme was heated to 60 "C for 5 min. Similar inactivation occurred at 50 "C with buffers containing 5% glycerol or 0.1 M NaCI, and a t 40 "C with a buffer containing both 5 8 glycerol and 0.1 M NaCI.

RESULTS AND DISCUSSION

Comments on the Purification-Topoisomerase activity can be detected in the wheat germ extract even before Poly- min P fractionation. However, the level of activity is low, and fractions must be extensively diluted before assaying, appar- ently because of interfering material in the extract. The first centrifugation removes the bulk of the chromatin, as well as bran, starch, and other debris. The remaining nucleic acids and some acidic proteins are precipitated by Polymin P and are removed in the second centrifugation. Topoisomerase activity appears to remain in the supernatant during these two centrifugations. Our preliminary experiments detected no activity in chromatin or in the protein fraction eluted from the Polymin P pellet by 0.2 M ammonium sulfate.

T o determine the optimum concentration of ammonium sulfate for precipitation of topoisomerase, a series of very narrow ammonium sulfate cuts were made and assayed. To- poisomerase activity was recovered in fractions precipitating between 33-36%, 36-39%, and 39-42% of saturation. Accord- ingly, for large scale purification, material precipitating below 33% of saturation was removed by centrifugation and dis- carded. The concentration of ammonium sulfate was then increased to 42% of saturation, and the precipitate was col- lected by centrifugation. This precipitate was resuspended in TED buffer and applied to a Bio-Rex 70 column.

A Bio-Rex column profile is shown in Fig. 1. There is almost no topoisomerase activity in the column flow-through or wash fractions. Since the peak of activity, which elutes between 0.19 and 0.23 M ammonium sulfate, contains only about 0.1% of the protein applied to the column, there is a large apparent purification at this step. In our experience the topoisomerase

BIOREX 70 PROFILE

/ FQOLFD

FRACTION NUMBER

FIG. 1. Profile of Bio-Rex 70 column. An extract from 500 g of wheat germ was fractionated with Polymin P and ammonium sulfate and run on a Bio-Rex column as described under "Experimental Procedures." Absorbance a t 280 nm was monitored continuously during gradient elution (-) and 12.5-1111 fractions were collected. The absorbance profile of the flow-through and wash is not shown. Ammonium sulfate concentration was determined by measurement of conductivity (- - -), and topoisomerase activity was determined as described under "Experimental Procedures." For all fractions, 5 pI of a 1:100 dilution were assayed. The inset shows the assay results. Column on-put, flow-through, and numbered fractions are as marked. The positions of supercoiled plasmid starting material ( S ) and relaxed plasmid product ( R ) are indicated at the right. Fractions 10-14, containing the peak of topoisomerase activity, were pooled and sub- jected to chromatography on phenyl-Sepharose.

5862 Wheat Germ DNA

activity in the peak fractions from this column is stable for several days or more at 4 “C.

The concentration of ammonium sulfate in the pooled Bio- Rex 70 fractions must be increased to promote binding to phenyl-Sepharose. This can be accomplished by addition of solid salt or by addition of a concentrated ammonium sulfate solution. Although both methods will work, we prefer the latter because it avoids the foaming produced by gases ad- sorbed on the solid salt crystals. We find that a 1 M ammonium sulfate concentration is sufficient to retain all the topoisomer- ase activity on the column without being so high a concentra- tion as to risk precipitating the enzyme.

Topoisomerase is among the first proteins to elute from the column as the ammonium sulfate concentration is decreased. Fig. 2 shows topoisomerase assays and SDS-polyacrylamide gels of fractions from a phenyl-Sepharose column. The peak of activity eluted between 0.85 and 0.75 M ammonium sulfate (Fractions 23-26).

The pooled phenyl-Sepharose fractions were dialyzed against storage buffer containing 50% glycerol. A 3-fold con- centration of the enzyme occurs during dialysis. All further characterization of wheat germ DNA topoisomerase was done using this pooled, concentrated material from the 2nd column.

Identification of the Actioe Polypeptide-The molecular weights of the major and minor polypeptides in the final fraction from the topoisomerase purification were determined by comparison with marker proteins in SDS-polyacrylamide gels (Fig. 3). The most prominent band (labeled topo) has a

ON FT 14 1822242628X)?i23640C - - TOP0

t

FIG. 2. Phenyl-Sepharose column fractions. The pooled frac- tions from the Bio-Rex 70 column in Fig. 1 were adjusted to 1 M ammonium sulfate by addition of the solid salt. This 73-ml sample was applied to a 10-ml bed volume phenyl-Sepharose column, and the column was washed with 5 volumes of 1 M ammonium sulfate in T E D buffer. Elution was with a 9-column volume gradient of 1-0.5 M ammonium sulfate in TED buffer. Fractions of 1.65 ml were collected. Ammonium sulfate concentration was determined by mea- surement of conductivity, and topoisomerase activity was assayed as described under “Experimental Procedures.” For the column on-put and flow-through samples, 5 p1 of a 1:100 dilution were assayed; for other fractions, 5 pl of a 1:500 dilution were assayed. Assay results are shown at the top of the figure. The positions of supercoiled plasmid starting material (S) and relaxed plasmid product ( R ) are marked. The slot labeled C is a control to which no topoisomerase was added. The bottom of the figure shows SDS-10% polyacrylamide gels. T o each gel 50 pl of sample were applied. Topoisomerase activity is associated with the 11 1,000-dalton polypeptide labeled topo (see text). The polypeptide labeled with an arrow is a 51,000-dalton contaminant that co-elutes with topoisomerase. Fractions 23-26 were pooled and dialyzed against storage buffer.

Topoisomerase I

A B C D

E

- 1 F L : 1 2 3 4 5

F 1 2 3 4 5

FIG. 3. Molecular weight of the active polypeptide. Samples were analyzed on SDS-8.759, polyacrylamide gels. Track A contains molecular weight markers. The proteins and their respective molec- ular weights are as follows, from top to bottom: E . coli RNA polvm- erase p’ subunit, 155,000, E . coli RNA polymerase /i subunit, 145.000; 15. coli /i-galactosidase. 116,250; rabbit muscle phosphorvlase h. 97,400, E . coli RNA polymerase D subunit, 9O,ooO, bovine serum albumin, 66,300; beef liver catalase, 58,000, ovalbumin. 43,000; and E . coli RNA polymerase a subunit, 36,500. Sources and references are given in Lowe et al. (1979). Track B is a mixture of molecular weight markers and topoisomerase, and track C contains topoisomerase alone. The major enzyme band at 11 1,OOO and the major contaminant a t 51,000 are marked. Track D shows topoisomerase run twice as far as usual. This gel was stained for 2 h and destained for 8 h and was cut into five sections as indicated. Each section was soaked for 15 min in 2 M Tris-HCI to neutralize the destaining solution, and elution of protein and renaturation of activity were carried out as described under “Experimental Procedures.” Assays were performed using 10 pI ( E ) or 3.3 pl ( F ) of renatured enzyme from each section. The positions of supercoiled starting material and relaxed product are marked at right.

M , = 11 1,000; below this area are two minor bands of M , = 104,000 and 94,000, respectively. The amounts of these two bands were variable. This enzyme preparation, purified in the absence of protease inhibitors, had more of these polypeptides than the preparation in Fig. 2. There are also two smaller peptides M , = 51,000 (arrow) and 48,000.

T o determine which polypeptide was topoisomerase, we ran a sample of our preparation on an SDS-polyacrylamide gel, cut the region from the top of the gel to the marker dye into sections, and subjected each section to the protein elution and renaturation procedure described under “Experimental Pro- cedures.’’ We then correlated activity with the positions of polypeptides in a gel run in parallel and stained with Coo- massie blue. We found that most of the topoisomerase activity was in the region of the gel containing the 111,000-dalton species and the other large polypeptides. Little or no activity was recovered from the regions encompassing the 51,000- dalton and smaller polypeptides.

In order to determine precisely which large polypeptide was topoisomerase, we eluted and renatured activity from a Coo- massie blue-stained gel. Although the efficiency of recovery was lower from stained gels, it was possible to correlate activity with the various polypeptides in an unequivocal way.

The gel in truck D of Fig. 3 was used for a renaturation experiment. This gel was run twice as long as those in tracks A-C in order to achieve a better separation of the three large polypeptides. After brief staining and destaining, the bands were individually excised, eluted, and renatured. Each sample

Wheat Germ DNA Topoisomerase I

was assayed at two concentrations. The assays in the upper row (Fig. 3, E ) had 3 times as much enzyme added as those in the lower row (Fig. 3, F). Both the 111,000- and 104,000- molecular weight polypeptides have activity associated with them, with the 111,000 one having at least 3 times more than the 104,000 (compare samples 2 and 3 in Fig. 3). The simplest explanations for this heterogeneity, given the variable amount of the 104,000-dalton band, is that it arises as an artifact of proteolysis.

It was believed until recently that the molecular weight of mammalian DNA topoisomerase I was in the range 60,000- 70,000 (Keller, 1975a, and McConaughy and Champoux, 1976). Recent data suggest that the molecular weight of HeLa cell enzyme is actually 110,000 (Liu, 1980). A smaller form corre- sponding to Keller's 60,000-dalton enzyme can be derived from the 110,000-dalton form by mild proteolysis (Liu, 1980). Re-examination of Keller's data shows that in his final enzyme fraction there is a minor 110,000-dalton polypeptide in addi- tion to the major 60,000-dalton species. Our value for the polypeptide molecular weight of wheat germ topoisomerase I is in close agreement with the revised value for the molecular weight of the HeLa enzyme, suggesting that the topoisomerase gene has been highly conserved during evolution.

Activity a n d Purity-The 11 1,000-dalton polypeptide makes up about 60% of the mass of protein in the peak fractions shown in Fig. 2, as determined by scanning of the stained gels. Based on the intensity of staining as compared with known amounts of bovine serum albumin, approximately 250 pg of the 11 1,000-dalton polypeptide are obtained in a typical purification starting with 500 g of wheat germ.

The major contaminating polypeptide of M, = 51,000 is not required for topoisomerase activity, but may be associated with the native topoisomerase enzyme molecule, since it co- purifies with the 11 1,000-dalton polypeptide on Sephacryl S- 200. Preliminary experiments show that both polypeptides bind to and can be eluted with a salt step from hydroxylapatite (Hypatite C, Clarkson Chemical Co., Williamsport, PA), Ci- bacron blue-agarose (Bio-Rad, Affigel blue), and DEAE-Seph- arose CLGB (Pharmacia). A gradient elution from DEAE- Sepharose CLGB gives a light resolution of the two polypep- tides, with the smaller one eluting a t higher ammonium sulfate concentration. From the renaturation experiments we con- clude that the amount of renaturable activity, if any, associ- ated with the 51,000-dalton polypeptide is below the limit of sensitivity of our assay. However, this finding does not pre- clude the possibility that this polypeptide is related to the topoisomerase.

Fig. 4A shows a titration of topoisomerase activity by serial dilution. The concentration of the 11 1,000-dalton polypeptide was approximately 0.1 mg/ml in this sample. The 25 nl which gave full relaxation represents an enzyme to DNA molar ratio of approximately 1:3, demonstrating that each enzyme mole- cule isomerizes more than one DNA molecule in the course of even a 5-min reaction.

The results of an assay for contaminating endonuclease activity are shown in Fig. 4B. This gel contained 0.02 pg/ml of ethidium bromide. Plasmid DNA molecules relaxed by topo- isomerase, because they remain covalently closed, take on a positively supercoiled conformation in the presence of ethid- ium bromide and become the fastest migrating species. Nicked DNA molecules, which would be produced by contaminating endonuclease, do not become superhelical in the presence of ethidium bromide and so are the most slowly migrating spe- cies. Negatively supercoiled starting material loses some of i c s superhelicity in the presence of ethidium bromide and mi- grates at an intermediate position. The positions of the three major DNA species are marked on the right of Fig. 4R.

A. 50

B.

-R

- S

5863

-N - S

-R

FIG. 4. Dilution series and endonuclease activity. A, dilution series. Topoisomerase was serially diluted and assayed as described under "Experimental Procedures." Assays were for 5 min at 37 "C, and contained 200 ng of pRR322 DNA. Each track is labeled according to the number of nanoliters of undiluted enzyme contained in the assay. Positions of supercoiled starting material (S) and relaxed produce ( R ) are indicated. B, endonuclease activity. Topoisomerase was serially diluted and assayed by incubation with a supercoiled 9.5- kilobase plasmid consisting of sv40 cloned in pRR322. Assays were for 30 min at 37 "C, and the reaction buffer was supplemented with 5 mM MgC12. Reactions were terminated by pheno1:chloroform (1:l) extraction. The products were analyzed on an 0.58 agarose gel con- taining 0.02 yg/ml of ethidium bromide. The presence of the ethidium bromide changed the relative mobilities of the different forms of DNA, as described in the text. The positions of supercoiled starting material (S) . relaxed product ( R ) , and contaminating nicked circles ( N ) are marked. Each track is labeled according to the number of nanoliters of undiluted enzyme/200 ng of DNA in the assay.

The sensitivity of this assay has been increased by using a recombinant plasmid twice the size of pBR322 and by increas- ing the incubation time from 5 to 30 min. Magnesium ion was included in the reaction buffer to activate possible divalent cation-requiring nucleases. No increase in the amount of ma- terial migrating at the nicked position was detected, indicating that this topoisomerase preparation is essentially free of en- donuclease activity.

Topoisomerase was also assayed for contaminating protease activity by incubating aliquots of the enzyme overnight at 37 "C in the presence or absence of 0.1% SDS, then analyzing by SDS-polyacrylamide gel electrophoresis. No proteolysis was apparent (results not shown).

Contaminating ribonuclease was assayed by incubating to- poisomerase for 30 min a t 37 "C with ,'"P-labeled in vitro transcripts of phage h DNA prepared using E. coli RNA polymerase (Calva and Burgess, 1980). The mobility of the RNA on 3.5% polyacrylamide gels containing 7 M urea was compared for samples incubated with different amounts of topoisomerase. This assay, which is very sensitive, did reveal trace contamination with ribonuclease, which varied from one topoisomerase preparation to another (results not shown). We are uncertain whether this ribonuclease actually co-purifies with topoisomerase or whether it is an incidental contaminant acquired during the later stages of purification. The contam- inating ribonucleases (as well as topoisomerase itself) can be inactivated by treatment with 0.1% diethylpyrocarbonate (Eh- renberg et al., 1976).

Mono- a n d Divalent Cation Requirements-The effect of NaCl concentration on topoisomerase activity is illustrated by the experiment shown in Fig. 5. The optimal salt concentration is in the range 0.05-.15 M, which is somewhat lower than the 0.15-0.2 M NaCl optimum that has been reported for other eukaryotic DNA topoisomerases (Champoux, 1976, Baase and Wang, 1974, Keller, 1975a. Mattoccia et al., 1976). A pattern of intermediately relaxed DNA isomers is seen when the reaction is carried out a t 0.2 M salt, suggesting that the enzyme is assocating and dissociating with its substrate several times during the course of the reaction. These intermediately re-

5864 Wheat Germ DNA Topoisomerase I

FIG. 5. NaCl optimum. Topoisomerase reactions were carried out as described under “Experimental Procedures.” Assays were for 5 min at 37 OC, and the concentration of NaCl in the assay was varied between 0.02 M and 0.30 M as indicated. The positions of supercoiled starting material ( S ) and relaxed product ( R ) are marked.

laxed species are not seen when reactions are carried out a t 0.05 or 0.02 M NaCl but with insufficient enzyme to allow the reaction to proceed to completion (Fig. 4A and 5).

The presence of magnesium decreases the NaCl optimum, as would be expected for an enzyme whose interaction with DNA is primarily electrostatic (Record et aL, 1976). Aside from this, magnesium and ATP appear to have no effect on the activity of topoisomerase.

Characterization of DNA Product-When supercoiled DNA is treated with DNA topoisomerase, a distribution of topological isomers is generated, with topological linking num- bers differing in increments of 1 (Keller, 1975, a and b, and Pulleyblank et al., 1975). When a single band is excised from the distribution, purified, and re-treated with type I DNA topoisomerase, the full distribution of isomers is regenerated (Pullyblank et al., 1975).

Recently, however, it has been discovered that there is a second class of DNA topoisomerases than changes the topol- ogical linking number in increments of f 2 (Brown and Cozzarelli, 1979, Liu, 1980, and Mizuuchi et al., 1980). These type I1 DNA topoisomerases, of which E. coli DNA gyrase and T4 DNA topoisomerase are examples, do not interconvert molecules with odd and even topological-linking numbers. If a single band from the distribution is excised and re-treated with a type I1 enzyme, every other band in the full distribution is generated. Type I1 topoisomerases work by introducing transient double strand breaks in DNA in contrast to the type I topoisomerases, which appear to work by introducing a transient single strand nick. All known type I1 enzymes either require ATP or have activities that are very much affected by the presence of ATP.

Wheat germ DNA topoisomerase is a type I, or nicking- closing type topoisomerase. This is shown by the experiment in Fig. 6A. In this experiment supercoiled pBR322 was treated with topoisomerase at 20 “C, with 50 mM NH&l substituted for NaCl in the reaction buffer. The DNA was subjected to preparative scale electrophoresis in an agarose gel. When electrophoresis begins, the sodium ion in the electrophoresis buffer exchanges with the aNA-bound ammonium ion, induc- ing a slight positive superhelicity in the DNA (Anderson and Bauer, 1978). The degree of superhelicity is sufficient to resolve topological isomers with different winding numbers. Individual bands were excised and purified as described in the figure legend, then re-treated with wheat germ DNA topo- isomerase. The full distribution of topological isomers was regenerated. This result, together with our previous observa- tion that magnesium and ATP do not affect the activity of the enzyme, clearly identifies wheat germ topoisomerase as a type I enzyme.

When a single relaxed DNA isomer is re-treated with to- poisomerase and converted to a form migrating more slowly in the gel system in Fig. 6, there is an algebraic decrease in its topological winding number, that is, a change opposite in direction from the removal of the negative supercoils in the original starting material. Since more slowly migrating forms are generated, it is clear from the experiment in Fig. 6A that wheat germ DNA topoisomerase is capable of catalyzing the swiveling of DNA in both directions.

For certain kinds of experiments it is desirable to prepare circular DNA with an equilibrium distribution of winding numbers, that is, DNA that is equivalent to a sample which has been nicked, allowed to swivel freely at the point of nicking, then ligated. Fig. 6B shows that the distribution of relaxed DNA isomers produced by wheat germ DNA topo- isomerase is equivalent to that produced by successive treat- ment with DNase I and E. coli DNA ligase. In this experiment,

FIG. 6. Characterization of the reaction products. A, regen- eration of a distribution of topological isomers. A relaxation reaction was carried out as described under “Experimental Procedures,” except that 0.05 M NH.,CI was substituted for NaCI, and the temperature was 20 “C instead of 37 “C. After 75 min, the reaction was stopped by addition of SDS, and the reaction products were subjected to electro- phoresis for 8 h at 2 V/cm on a preparative 0.8% agarose slab gel. The gel was stained for 15 min with 1.0 pg/ml of ethidium bromide, the DNA was visualized with long wave ultraviolet light, and the bands were individually excised from the gel. The excised section was placed in 3 ml of buffer containing 0.2 M sodium acetate, 0.05 M Tris-HCI, 0.001 M EDTA, 0.1% SDS, 3.3 pg/ml of tRNA, and was broken into small pieces. Elution was allowed to proceed for 16 h, a t which time the suspension was filtered through glass wool to remove the agarose particles. The DNA was precipitated with ethanol, resuspended, and twice extracted with pheno1:chloroform (l:l), then reprecipitated with ethanol. The DNA was resuspended and treated with topoisomerase, again in the standard reaction buffer with NHnCl substituted for NaCI. The reaction was allowed to proceed for 75 min a t 20 “C, and the starting material and products were analyzed by agarose gel electrophoresis. Slots 2 and 4 show the bands eluted from the fvst gel prior to retreatment with topoisomerase. Tracks 1 and 3 show the distribution of topological isomers generated from the DNA in tracks 2 and 4, respectively. Track 6 shows untreated supercoiled plasmid DNA, and track 5 shows the distribution of topological isomers generated from it by topoisomerase. B, comparison of nicked and ligated DNA with topoisomerase-treated DNA. Supercoiled pBR322 DNA was subjected to limited treatment with DNase I. Approxi- mately 3 pg of DNase were incubated with 1 pg of DNA for 1 h a t 14 “C in a reaction volume of 25 pl in a buffer containing 50 mM Tris- HCI, 10 mM MgCI,, 10 pg/ml of bovine serum albumin. The reaction was terminated by addition of 0.18 diethylpyrocarbonate. After over- night incubation a t 4 “C to allow hydrolysis of the diethylpyrocarbon- ate, the reaction mixture was adjusted to contain 50 mM NH,CI, 60 p~ NAD, and an excess of MgC12 over EDTA of 7 mM. To the reaction was added 1 unit of E. coli DNA ligase (New England Biolabs, Beverly, MA). Ligation was allowed to proceed for 6 h a t 20 “C and was terminated by addition of SDS to 0.1%. A sample of supercoiled DNA was incubated under identical conditions with topoisomerase, and both samples were analyzed by agarose gel electrophoresis. Track 1, nicked-ligated sample; track 2, topoisomerase-treated sample; track 3, supercoiled starting material.

Wheat Germ DNA Topoisomerase 1

topoisomerase treatment and ligation were carried out in identical buffers, both reactions containing NH4C1, SO as to produce a distribution of isomers readily separated on the gel. There is some unrelaxed material in the DNase I/ligase- treated sample, evidently because the amount of DNase I used was insufficient.

As a method for preparing relaxed circular DNA, treatment with wheat germ DNA topoisomerase offers several advan- tages. The product is equivalent to that produced by DNase and ligase, but it is a one-step procedure; there is no need to carefully titrate the enzyme as is the case with DNase I, and the requirement for the presence of divalent cation and a nucleotide cofactor during ligation is avoided. Moreover, by varying the temperature or by adding salt or ethidium bro- mide to the topoisomerase reaction, it is possible to generate DNA species covering a broad range of supercoiling density (Keller, 1975b).2 A procedure using topoisomerase to prepare relaxed templates for in vitro transcription will be described in the accompanying paper (Dynan and Burgess, 1981).

Acknowledgment-We thank Dr. Leroy Liu for helpful discussions during the preparation of this manuscript and for making results available prior to publication.

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