Rapid Purification and Characterization of DNA Topoisomerase I from Cultured Mouse Mammary Carcinoma FMSA Cells*

We have previously shown that a DNA topoisomerase I from mouse mammary carcinoma cells is inhibited by heparin. Taking advantage of this enzyme-heparin in-teraction, we developed a rapid and efficient method of purification of this enzyme to near homogeneity by extraction of chromatin with 0.15 M phosphate buffer followed by two-step column chromatography on hep- arin-Sepharose and phenyl-Sepharose. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gels re- vealed that the final preparation is composed of two polypeptides with apparent M, - 98,000 (p98) and 102,000 (p102), p98 comprising 70% and p102 30%. Extraction and renaturation of the polypeptides from the gel shows that both p98 and p102 seem to possess topoisomerase activity. Partial proteolytic digestion of p98 and p102 with Staphylococcus aureus V8 and chymotrypsin yielded a series of identical peptides, indicating that the two polypeptides are structurally related. The enzyme sedimented through sucrose density gradient with s20,w of 4.0 s, and thus is monomeric in solution. polyacryl-amide). Various amounts of the protease were added to each well and electrophoresis was carried out. Proteins were partially digested during electrophoresis, and resulting peptides were separated on the gel. Other Procedures-Protein concentration was determined by the Bio-Rad protein assay kit. Silver staining of proteins on polyacryla- mide gels was performed using the Bio-Rad silver stain kit. Sucrose gradient sedimentation of topoisomerase was performed as follows. Two-tenths ml of the phenyl-Sepharose fraction of topoisomerase (50 pg of protein) was overlaid on 5 ml of a 10 to 25% linear sucrose density gradient containing 40 mM Tris-HC1, pH 8.0, 0.25 M NaCI, and 0.2 mM PMSF, and centrifuged for 27 h at 19,500 X g at 4 "C in a Beckman SW 50.1 rotor.

topoisomerase, as is termed according to the recommendations of Wang and Liu (1979), have been the subject of speculation (Champoux, 1978;Wang and Liu, 1979;Cozzarelli, 1980;Gellert, 1981). Although they were originally assumed to form a swivel which would facilitate replication and/ or transcription, recent discoveries have suggested additional roles: the finding that the bacteriophage X int protein is a type I topoisomerase (Kikuchi and Nash, 1979) and recent isolation and characterization of Escherichia coli mutants lacking topoisomerase I (Sternglanz et al., 1981) suggested that one of the possible roles of this enzyme may be in the recombinational processes. Pruss et al. (1982) and DiNardo et al. (1982) recently reported results clearly demonstrating that all of the previously isolated topoisomerase I-deficient mutants of Escherichia coli possessed a second additional mutation in either of the two gyrase subunit loci gyr A and gyr B, thus compensating for the lack of topoisomerase I in maintaining the proper degree of superhelicity of chromosomal DNA. Weisbrod (1982) and Javaherian and Liu (1983) * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom reprint requests should be addressed. have shown in Xenopus laeuis erythrocytes and in HeLa cells, respectively, that topoisomerase is bound in active nucleosomes presumably in association with high mobility group proteins. These results strongly suggested that topoisomerase I is an essential enzyme involved in various genetic processes and hence indispensable for survival.
We describe in this paper a rapid and efficient method of purification of the topoisomerase I from cultured mouse mammary carcinoma cells for various studies using purified enzyme. The method involves extraction of purified chromatin with low salt concentration followed by successive chromatography on heparin-Sepharose and phenyl-Sepharose. After the two chromatographic steps, the enzyme was purified to near homogeneity. From 10 g of wet pellet of the mouse mammary carcinoma cells, we typically obtain about 100 pg of topoisomerase within 2 days. The final preparation contained two polypeptides with M , = 98,000 (p98) and 102,000 (p102), p98 comprising about -70% and p102 "30%. Both polypeptides seem to possess topoisomerase activity. Furthermore, the two polypeptides were shown to be structurally related by peptide mapping after partial proteolysis by Stuphylococcus aureus V8 and chymotrypsin.

EXPERIMENTAL PROCEDURES
Materials-Heparin-Sepharose 6B and phenyl-Sepharose 4B were purchased from Pharmacia; Seakem agarose (ME) was from Marine Colloids, Inc.; acrylamide and methylenebisacrylamide were from Seikagaku Kogyo, Inc.; tetramethylenediamine was from Wako Pure Chemicals, Inc.; BSA' was from Bethesda Research Laboratories; S. aureus V8 protease was from Miles; a-chymotrypsin type IA was from Sigma. Col E l DNA was prepared from E. coli A745met-thy-(col E l ) by the method of Sakakibara and Tomizawa (1974).
Topoisomerase Assay-The principle of the assay is the decreased mobility in an agarose gel of supercoiled DNA after treatment with topoisomerase (Keller, 1975). Assays contained 10 mM Tris-HC1, pH 7.5, 0.5 mM EDTA, 1 mM dithiothreitol, 1 mM spermidine, 0.2 mM PMSF, 150 mM sodium chloride, 100 rg/ml of BSA, 10% glycerol, and 1 pg of supercoiled col E l DNA in a final volume of 50 11. Reactions were started by addition of 5 pl of the sample to be assayed and were allowed to proceed for 15 min at 37 "C. Reactions were terminated by addition of an equal volume of double strength sample buffer (40 mM Tris-HC1, pH 7.5, 0.4 mM EDTA, 20% sucrose, 2% SDS, and 0.01% bromphenol blue) and incubation at 45 "C for 15 min. The samples were loaded on a 1.2% agarose gel cast in a horizontal slab apparatus. The electrophoresis buffer contained 36 mM Tris-HC1, pH 7.7, 30 mM sodium phosphate, 1 mM EDTA. For discrimination of relaxed closed circular DNA from nicked circular form I1 DNA, samples were electrophoresed in agarose gels containing chloroquine as described by Shure et al. (1977).
Purification of Topoisomerase-All steps in the purification were carried out at 0-4 "C. Centrifugations were for 15 min at 7000 X g. Chromatin extract containing topoisomerase activity was prepared from cultured mouse mammary carcinoma FM3A cells (Nakano, Mouse Mammary Carcinoma DNA Topoisomerase I 1966) by the method of Germond et al. (1975). About 10 g of wet cells ("5 X los cells) from a 4-liter culture was usually processed in one batch. The cell pellet washed with phosphate-buffered saline (0.14 M NaCl. 0.03 M KC1 in 0.01 M phosphate buffer) was suspended in 10 volumes of hypotonic buffer A (0.2 mM sodium phosphate, pH 7.5, 0.1 M sucrose, and 0.2 mM PMSF) and lysed by dropwise addition of an equal volume of NP40 solution B (0.5% NP40,0.2 mM EDTA, 0.2 mM PMSF, pH 7.5). Crude chromatin obtained by centrifugation of the lysate was purified by suspending in 5 to 10 volumes of NP40 solution B and centrifuging through the layer of hypotonic buffer A adjusted at pH 8.5. The same washing procedure was repeated two more times except that the hypotonic buffer A used was at pH 7.5. Purified chromatin was extracted by suspension in an equal volume of 0.3 M phosphate buffer, pH 7.5, containing 1 mM dithiothreitol, 0.5 mM EDTA, 0.2 mM PMSF, 20% glycerol followed by centrifugation.
Chromatin extract usually containing 150-200 pg of protein/ml was applied at a flow rate of 50 ml/h to a column (0.7 X 1.0 cm) of heparin-Sepharose equilibrated with elution buffer (10 mM Tris-HC1, pH 7.5, 1 mM dithiothreitol, 0.5 mM EDTA, 0.2 mM PMSF and 10% glycerol) containing 0.15 M NaCI. Unless otherwise mentioned, solutions used hereafter for heparin-and phenyl-Sepharose chromatography contained the elution buffer. After application, the column was washed with 5 ml of 0.4 M NaCl, and eluted with a total of 20 ml of a linear gradient of 0.4-0.9 M NaCl. One-half-ml fractions were collected and 5 pl of appropriately diluted samples 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 an equal volume of 2 M ammonium sulfate. This sample, typically 10 ml, was applied to a column (1.0 X 2.0 cm) of phenyl-Sepharose equilibrated with 1 M ammonium sulfate, and the column was washed with 10 ml of 1 M ammonium sulfate. Topoisomerase was eluted from the column with a total of 25 ml of a linear decreasing gradient of ammonium sulfate from 1.0 to 0.2 M. One-half-ml fractions were collected, and 5 pl of appropriately diluted samples were assayed for topoisomerase activity. The fractions containing the bulk of the activity were pooled and stored frozen a t -80 "C. The activity was maintained practically unaltered for at least 1 month.
Renaturation of Topoisomerase after Electrophoresis-SDS-polyacrylamide gel electrophoresis was carried out with the discontinuous buffer system of Laemmli (1970) and with prescription of acrylamide and methylenebisacrylamide according to Blattler et al. (1972). The procedure used for elution of proteins from the gel, removal of SDS, and renaturation of enzymatic activity was as described by Hager and Burgess (1980) and Dynan et al. (1981).
Peptide Mopping Analysis of Topoisomerase-Partial proteolytic digestion with S. aureus V8 and chymotrypsin was done according to Cleavland et al. (1977). A sample of about, 2 pg of the phenyl-Sepharose fraction was subjected to SDS-polyacrylamide gel electrophoresis. The gel was stained with Coomassie blue, and regions corresponding to protein bands were cut out. Each gel slice was placed in a well of another SDS-polyacrylamide slab gel consisting of a stacking gel (3% polyacrylamide) and separating gel (15% polyacrylamide). Various amounts of the protease were added to each well and electrophoresis was carried out. Proteins were partially digested during electrophoresis, and resulting peptides were separated on the gel. Other Procedures-Protein concentration was determined by the Bio-Rad protein assay kit. Silver staining of proteins on polyacrylamide gels was performed using the Bio-Rad silver stain kit. Sucrose gradient sedimentation of topoisomerase was performed as follows. Two-tenths ml of the phenyl-Sepharose fraction of topoisomerase (50 pg of protein) was overlaid on 5 ml of a 10 to 25% linear sucrose density gradient containing 40 mM Tris-HC1, pH 8.0, 0.25 M NaCI, and 0.2 mM PMSF, and centrifuged for 27 h at 19,500 X g at 4 "C in a Beckman SW 50.1 rotor.

RESULTS
Comments on the Purification-Topoisomerase activity can be detected in whole cell extract, but total activity calculated tends to be lower than that of chromatin extract, suggesting the interference with some materials in the extract. Chromatin extract containing virtually all of the enzymatic activity was prepared according to Germond et al. (1975). In order to recover most of the activity in this fraction, extraction buffer in the preparation and washing of chromatin should contain 0.2 mM PMSF. Briefly, cells were lysed with 0.25% NP40 in hypotonic buffer and resulting chromatin in the lysate was pelleted by centrifugation. The crude chromatin was washed three times by suspending the pellet and centrifuging through the 0.1 M sucrose layer. The purified chromatin was extracted by suspending in an equal volume of 0.3 M sodium phosphate followed by centrifugation. This fraction contained virtually all of the enzymatic activity and approximately 2.4% of the protein of the cell, and thus nearly IO-fold purification, is attained by this step. Since we have previously shown that the topoisomerase is strongly inhibited by heparin (Ishii et al., 1982), we took advantage of this enzyme-heparin interaction and used heparin-Sepharose for the purification of this enzyme. Thus, the chromatin extract was applied to a heparin-Sepharose column.
A typical heparin-Sepharose column profile is shown in Fig.  1. There is no topoisomerase activity in the column flowthrough or wash fractions. Since the peak of activity, which elutes between 0.6 and 0.8 M NaCl, contains only about 1.6% of the protein applied to the column, almost 50-fold purification is obtained at this step. As shown in Fig. lB, a prominent band with apparent M, = 98,000 (p98) indicated by an arrow seemed to be intimately associated with enzymatic activity. To determine which polypeptide on the gel the topoisomerase activity was associated with, we ran a sample of the heparin-Sepharose fraction on an SDS-polyacrylamide gel, cut the region from the top of the gel to the marker dye into 12 sections, and subjected each section to the protein elution and renaturation procedure described under "Experimental Procedures" (Fig. 2). We then correlated activity with the positions of polypeptides. As shown in the lower figure, we found that most of the topoisomerase activity was in region number 5 which contained doublet bands. Fig. 3 shows further that the enzymatic activity seems to be associated with both of the two polypeptides in regions a and b. The possibility of the enzymatic activity associated with the minor band a being a result of the contamination of the major band b, however, cannot be excluded. The molecular weight of the major enzyme was determined to be approximately 98,000 by comparison with marker proteins on SDS-polyacrylamide gels.
The concentration of ammonium sulfate in the pooled heparin-Sepharose fractions was increased to 1 M by addition of 2 M ammonium sulfate solution in the elution buffer, and then applied to a phenyl-Sepharose column. The peak of activity eluted between 0.6 and 0.4 M ammonium sulfate (Fig.  4A). Results shown in the lower figure (Fig. 4B)  onstrated that in the final preparation of active fraction the major topoisomerase band was still accompanied by a minor band with apparent M, = 102,000 (~102). The major 98,000-Da polypeptide in the final preparation makes up about 70% of the mass of proteins in the peak fractions shown in Fig.   4A. Table I summarizes the results of this purification. About 180-fold purification with an overall yield of 54% was obtained starting from chromatin extract. The purified topoisomerase was shown to be free of contaminating protease by incubating aliquots of the enzyme either at room temperature for 48 h or at 4 "C for 72 h, and then analyzing by SDS-polyacrylamide gel electrophoresis. Nuclease was not detected in the prepa-  ration by incubation of supercoiled col E l DNA with the enzyme at 20 "C for 4 h and analysis of the product by agarose gel electrophoresis containing chloroquine.

Peptide Composition of the Two Proteins p98 and plO2-To
test the structural similarity of the two proteins p98 and p102, we compared the patterns of their partial proteolytic digests by S. aureus V8 and chymotrypsin. As shown in Fig. 5, the electrophoretic patterns of these peptides on SDS-polyacrylamide gels changed with the concentrations of the protease used, but with fixed concentrations the patterns of the products of the two proteins were almost indistinguishable: both p98 and p102 gave rise to several discernible and indistinguishable peptide bands when digested either with V8 protease or chymotrypsin, strongly suggesting that p98 and p102 are structurally similar and related each other.
Sucrose Density Gradient Centrifugation of Topoisomerase I-As analyzed by sucrose density gradient centrifugation, phenyl-Sepharose-purified topoisomerase sedimented as a single symmetrical peak on the lighter shoulder of BSA (Fig.  6A). The sedimentation coefficient (sz0J of topoisomerase was calculated to be 4.0 S. SDS-polyacrylamide gel electrophoresis of aliquots of fractions demonstrated that topoisomerase is still M, = 98,000 and 102,000 after centrifugation.
These results suggest that the topoisomerase assumes rather rod-like conformation and is monomeric in solution.

Mouse Mammary
Carcinoma DNA Topoisomerase I

DISCUSSION
In the present work, we described a rapid and convenient method of purification of topoisomerase I from a cultured line of mouse mammary carcinoma FMSA cells. The method consisted of low salt extraction of chromatin followed by two steps of column chromatography on heparin-Sepharose and phenyl-Sepharose. Table I summarizes the result of the purification. We obtained about 180-fold purification with an overall yield of 54% starting from the chromatin extract. Since almost all enzymatic activities are recovered in chromatin extract, nearly 7000-fold purification was attained starting from whole cell extract. Completion of this purification procedure takes about 48 h in succession. This time can be reduced to one-third with similar yield and purity by introduction of the following modifications. Enzyme is adsorbed to heparin-Sepharose by mixing with chromatin extract. After packing the beads onto a column and washing with 0.53 M NaCl in elution buffer, enzyme is eluted in one step with 1.0 M NaCI. This eluate is adjusted to 1.0 M ammonium sulfate by addition of an equal volume of 2.0 M ammonium sulfate and applied to a column of phenyl-Sepharose. After washing with 0.8 M ammonium sulfate, enzyme is eluted with 0.4 M ammonium sulfate.
The finding that partial proteolytic digestion of the two proteins p98 and p102 in the purified preparation gave rise to a series of identical peptides clearly demonstrated that they are structurally similar and related. One could be a modified form of the other. The modification could be either phosphorylation, glycosylation, or poly(ADP)-ribosylation. This is under investigation. Since the mass ratio of these two polypeptides does not change with or without the addition of a protease inhibitor PMSF throughout the purification procedure, the possibility of p98 being derived from p102 by partial proteolysis during purification would be negligible. However, if it occurs it would be an in vivo phenomenon. It is relevant to refer to Liu and Miller (1981) who showed that the 67-kDa species of topoisomerase was formed by proteolysis of the 100-kDa species in HeLa cells. We isolated here for the first time the structurally related two polypeptides of topoisomerase I with very close M , -100,000. We could not detect any smaller species with M , = 60,000-70,000 as often reported in various organisms such as human KB cells (Keller, 1975), rat liver (Champoux and McConaughy, 19761, mouse L cells (Tang, 1978), and human HeLa cells (Liu and Miller, 1981).
The establishment of the rapid and efficient method of purification enables us to obtain the enzyme in large quantity for various studies including further structural analyses.