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Volume 270, Number 26, Issue of June 30, 1995 pp. 15739-15746
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Expression, Domain Structure, and Enzymatic Properties of an Active Recombinant Human DNA Topoisomerase II (*)

(Received for publication, September 19, 1994; and in revised form, February 27, 1995)

Caroline A. Austin (1), (§),  Katherine L. Marsh (§), ,  Robin Ann Wasserman (2),  Elaine Willmore (1),  Penelope J. Sayer (1),  (3),  James C. Wang (2),  L. Mark Fisher (3)

From the  (1)Department of Biochemistry and Genetics, The Medical School, The University, Newcastle-upon-Tyne NE2 4HH, United Kingdom, the (2)Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts 02138, and the (3)Molecular Genetics Group, Department of Cellular and Molecular Sciences, St. George's Hospital Medical School, University of London, Cranmer Terrace, London SW17 0RE, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Human cells express two genetically distinct isoforms of DNA topoisomerase II, alpha and beta, which catalyze ATP-dependent DNA strand passage and are an important antitumor drug target. Here we report for the first time the successful overexpression of human topoisomerase IIbeta in yeast by cloning a topoisomerase IIbeta cDNA in a yeast shuttle vector under the control of a galactose-inducible promoter. Recombinant human topoisomerase IIbeta (residues 46-1621 fused to the first 5 residues of yeast topoisomerase II) was purified to homogeneity, yielding an enzymatically active polypeptide in sufficient quantity to allow analysis of its domain structure and comparison with that of recombinant human topoisomerase IIalpha. Partial digestion of beta with either trypsin or protease SV8 generated fragments of approximately 130, 90, 62, and 45-50 kDa, arising from cleavage at three limited and discrete regions of the protein (A, B, and C) indicating the presence of at least four structural domains. Recombinant human topoisomerase IIalpha and beta induced DNA breakage which was promoted by a variety of agents. Isoform differences in drug-induced DNA breakage were observed. These studies of human topoisomerase IIbeta in concert with alpha should aid the determination of their individual roles in cancer chemotherapy and should facilitate the design, targeting, and testing of cytotoxic antitumor agents.

INTRODUCTION

Topological transformations of DNA rings are carried out by DNA topoisomerases, a group of enzymes that function by introducing transient breaks in DNA strands. Eukaryotic type II topoisomerases are dimeric proteins that act by passing one DNA segment through a transient enzyme-bridged double strand break; they have been found in many organisms and cell types(1, 2, 3) . Genetic studies in yeast have revealed that topoisomerase II is essential for cell growth and plays a key role in chromosome segregation(4, 5, 6) . Yeasts and Drosophila have been reported to express only a single type II DNA topoisomerase(7, 8) . In contrast, two isoforms of murine topoisomerase II were discovered by Drake et al.(9) , these forms differ in their antigenic, biochemical, and pharmacological properties(10) . Type II DNA topoisomerases are the target for a wide range of antitumor agents(3) . The discovery of two isoenzymes of mammalian topoisomerase II has raised important questions regarding their biological roles, their potential individual roles as chemotherapeutic targets, and their implication in drug resistance(9, 10) .

Human topoisomerase IIalpha was first cloned and sequenced in 1988(11) . Two partial cDNA clones encoding different portions of human topoisomerase IIbeta were isolated independently(12, 13) , and the full coding sequence has recently been determined(14, 15) . The genes for topoisomerase IIalpha and beta have been mapped to chromosomes 17q21-22 and 3p24, respectively(11, 16) , confirming that alpha and bet a are genetically distinct isoforms. A variety of human cell lines express both the alpha and beta isoforms which have molecular masses of 170 and 180 kDa, respectively(17, 18) . Cross-species Southern blot analysis suggests that neither yeast nor Drosophila possess a beta type topoisomerase but that topoisomerase IIbeta is found in avian and mammalian species(16) .

The two isoforms are differentially expressed during the cell cycle. Topoisomerase IIalpha levels rise in late S, peak in G(2)M, and decrease as cells complete mitosis, while beta is present at a relatively constant level throughout the cell cycle(17) . They have different tissue distributions shown by studies of expression of murine topoisomerase II. Whereas alpha is associated with tissues containing proliferating cells, beta is found in a wider range of tissue types (19). Furthermore, topoisomerase IIalpha and beta appear to be differentially localized in the nucleus, with topoisomerase IIbeta reported to be nucleolar(20, 21) . Why higher eukaryotes possess two distinct isoforms of the type II enzyme while other organisms survive with one is not clear, and isoform-specific catalytic activities and cellular functions remain to be clarified, particularly for the more recently isolated topoisomerase IIbeta. Progress in this area has been hindered by the small amounts of homogeneous type II enzyme that can be purified from human cells(22) , particularly for the recently characterized beta isoform(10) . One approach in circumventing this problem is to overexpress the enzymes in yeast using the available alpha and beta cDNA clones. Overexpression of human topoisomerase IIalpha has been reported by us previously(23) . In this paper, we describe for the first time a system for the overexpression of human topoisomerase IIbeta. We have characterized the structure and activity of this isoenzyme in vitro and compared its structural and enzymatic properties to those of human topoisomerase IIalpha.

EXPERIMENTAL PROCEDURES

Materials

Amsacrine (m-AMSA) and its ortho analogue (o-AMSA) were obtained from the Drug Synthesis and Chemistry Branch, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute. Teniposide and etoposide were kindly provided by Bristol-Myers Co. Sources of quercetin, quercetagetin, and myricetin have been described previously(24) . Drugs were dissolved in dimethyl sulfoxide (Me(2)SO) or ethanol at 1 mg/ml, stored at -20 °C, and diluted in Me(2)SO or ethanol immediately before use. All other reagents were obtained from commercial suppliers.

Construction of YEphTOP2beta, a Yeast Shuttle Plasmid for Expression of Human Topoisomerase IIbeta

Plasmids pBS-CAA5-19 and SP11 containing partial cDNAs for human topoisomerase IIbeta have been reported previously(12, 13, 14) . YEpWob6 is a yeast Escherichia coli shuttle vector containing the human TOP2alpha cDNA under the control of the yeast GAL1 promoter(23) . YEpTOP2-PGAL1, a YEp24 derivative for the expression of the yeast TOP2 gene, has been described previously(25) . YEphTOP2beta was constructed using the above plasmids. The 0.9-kb SalI to AgeI fragment containing the first five codons of the yeast TOP2 gene downstream of the GALI promoter was isolated from YEpTOP2 and fused to the 46th codon of human TOP2beta at the AgeI site through the use of a polymerase chain reaction generated fragment of human TOP2beta. This was spliced to the remaining 4.6 kb of TOP2beta cDNA assembled from several partial cDNA clones(13) . The resulting GALI promoter-TOP2beta cDNA fragment was then inserted into a 6.8-kb XhoI to SalI segment derived from YEpWOB6, which contains the URA3 marker gene, the yeast 2-µm plasmid replication origin, and the beta-lactamase gene and replication origin of E. coli pBR322.

Purification of Recombinant Human Topoisomerase IIalpha and beta Proteins

Six liters of Saccharomyces cerevisiae strain JEL1 (alpha leu 2 trp1 ura3-52 prb1-1122 pep4-3 Deltahis3::PGAL 10 - GAL4) containing plasmid YEphTOP2beta were grown in glucose-free medium to an optical density of 0.4-1.0 at 600 nm, measured in a Cecil CE595 spectrophotometer. Expression of recombinant human topoisomerase IIbeta protein was induced from the plasmid-borne hTOP2beta gene by addition of galactose to the growth medium (2% final concentration). Induction times of 3-16 h were used. Cells were harvested by centrifugation, and each gram of cell pellet was resuspended in 1 ml of buffer I (containing 50 mM Tris-HCl pH 7.7, 1 mM EDTA, 1 mM EGTA, 10% glycerol (v/v), 1 mM phenylmethylsulfonyl fluoride, 1 mM 2-mercaptoethanol, 1 mM benzamidine, 2 µg/ml leupeptin, 2 µg/ml pepstatin). The cell suspension was either flash frozen in liquid nitrogen for storage at -70 °C, or cells were lysed immediately for enzyme purification. Topoisomerase IIbeta was purified by the method of Worland and Wang (25) with minor modifications as described previously for human DNA topoisomerase IIalpha(23) .

Isolation of S. cerevisiae Topoisomerase II

S. cerevisiae DNA topoisomerase II was isolated by the method of Worland and Wang(25) .

Topoisomerase II Assays

Enzyme activity was determined by the ATP-dependent relaxation of supercoiled plasmid pBR322. Standard relaxation buffer contained 50 mM Tris-HCl, pH 7.4, 100 mM KCl, 10 mM MgCl(2), 5 mM 2-mercaptoethanol, 0.5 mM EDTA, and 300 µg/ml of BSA and 0.4 µg of supercoiled plasmid DNA. One unit is the amount of enzyme required to relax 50% of the DNA in 30 min at 37 °C in standard relaxation buffer. Alternatively, topoisomerase II activity was assessed by decatenation of kinetoplast DNA as described previously (24). One unit of activity is the amount of enzyme that decatenates 0.5 µg of kDNA under standard conditions. Relaxation or decatenation reactions were stopped by addition of a gel electrophoresis loading buffer containing 25% glycerol, 5% SDS, and 0.25 mg/ml bromphenol blue, and analyzed by gel electrophoresis in 0.8% agarose gels in TBE buffer containing (89 mM Tris, 89 mM boric acid, and 2.5 mM EDTA). Gels were stained with ethidium bromide and photographed under UV transillumination using Polaroid type 665 film. Enzyme activities were estimated by inspection of band intensities on the photographic negatives.

Detection of Proteins

Proteins were electrophoretically separated on 5% SDS-polyacrylamide gels and were visualized with Coomassie Blue or detected immunologically. For immunological detection, proteins were transferred to nitrocellulose membranes either using a Bio-Rad wet blotter or an Atto semi-dry blotter, according to the manufacturers' instructions. Antibody probing of membrane blots was carried out as follows. The membrane was blocked with 3% BSA in Tris-buffered saline (TBS, 20 mM Tris-HCl pH 7.5, 500 mM NaCl) for 30 min, then incubated for 2 h at 37 °C with a topoisomerase IIbeta-specific antiserum, in Tris-buffered saline containing 0.05% Tween 20 (TTBS) plus 0.1% SDS. The membrane was washed twice in TTBS and once in TBS, then incubated with alkaline phosphatase-conjugated goat (anti-rabbit) antisera in TBS containing 3% BSA for 1 h at room temperature. The membrane was washed as above prior to color generation, and then incubated in AP buffer (100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl(2)) containing 330 µg/ml nitro blue tetrazolium and 160 µg/ml 5-bromo-4-chloro-3-indolyl phosphate. Color development was stopped by addition of a buffer containing 20 mM Tris-HCl, pH 8.0, and 5 mM EDTA. Blots were also probed with antisera raised against the C-terminal quarter of topoisomerase IIbeta. Incubations were carried out in 3% BSA at 30 °C for 2 h, and the blot was then washed and developed as described above.

Protease Digestion of Topoisomerase II

Human topoisomerase IIbeta was dialyzed into 0.2 M sodium phosphate buffer, pH 7.0, containing 20% glycerol in the presence of 1 mM DTT and protease inhibitors benzamidine (1 mM), phenylmethylsulfonyl fluoride (1 mM), leupeptin (2 µg/ml), and pepstatin (2 µg/ml). Dialysis was carried out at 4 °C for 1 h, and final protein concentration was determined by the method of Bradford(26) . The topoisomerase was then digested with 20% (w/w) or 5% (w/w) Staphylococcus aureus V8 protease or bovine pancreatic trypsin at 30 °C for various lengths of time. SV8 digestion was also carried out in acetate buffer containing 50 mM Tris acetate, pH 7.35, 165 mM potassium acetate, 5 mM magnesium acetate, 5 mM 2-mercaptoethanol, and 10% (w/v) glycerol. Reactions were quenched by addition of 2 times SDS-PAGE sample buffer (27) and boiling for 2 min. Aliquots were run on SDS-polyacrylamide gels and stained with Coomassie Blue. Trypsin and SV8 digestions were also carried out in the presence of 1 mM ATP, 1 mM AMP-PNP, or 67 µg/ml pUC19 DNA.

Peptide Sequencing

Proteins were digested and run on polyacrylamide gels as described above prior to electroblotting on to Pall, Problott, or Hyperbond membrane according to the manufacturers' instructions. Membranes were stained with Coomassie Brilliant Blue, and the desired bands were excised. N-terminal sequencing of the protein fragments was carried out on a Beckmann peptide sequencer, according to the manufacturers' instructions.

Human DNA Topoisomerase IIalpha- and IIbeta-mediated DNA Cleavage of Supercoiled pBR322

Cleavage reaction mixtures contained 0.4 µg of supercoiled pBR322 in 50 mM Tris-HCl buffer, pH 7.5, 100 mM KCl, 10 mM MgCl(2), 0.1 mM DTT, 0.5 mM EDTA, 30 µg/ml BSA, and 1 unit of human topoisomerase IIalpha or IIbeta in the presence or absence of m-AMSA, o-AMSA, teniposi de, or etoposide. Reactions were carried out in the absence of ATP for 30 min at 37 °C, stopped with 1% SDS followed by incubation with 0.5 mg/ml proteinase K for 30 min at 37 °C. Samples were analyzed by electrophoresis in 0.8% agarose in TBE buffer. Gels were stained with ethidium bromide and photographed under UV transillumination.

Human DNA Topoisomerase IIalpha- and IIbeta-mediated DNA Cleavage of End-labeled pBR322 Analyzed by Agarose Gel Electrophoresis

Supercoiled plasmid pBR322 DNA was prepared, linearized, and end-labeled as described previously(24) . Briefly, pBR322 was cut with EcoRI, incubated with calf intestinal alkaline phosphatase, and radiolabeled at its 5` ends using T4 polynucleotide kinase and [gamma-P]ATP. After HindIII digestion, the 4333-bp EcoRI-HindIII fragment was separated and purified by electrophoresis in a 1% low melting agarose gel. Cleavage reaction mixtures contained 40 mM Tris-HCl buffer, pH 7.5, 100 mM KCl, 10 mM MgCl(2), 0.1 mM DTT, 0.5 mM EDTA, 5% (v/v) Me(2)SO, 30 µg/ml BSA, 10 units of human topoisomerase IIalpha or beta, a 4333-bp EcoRI-HindIII fragment of pBR322 DNA end-labeled at the EcoRI end, and m-AMSA, ellipticine, teniposide, etoposide, or flavonoid compound at 0.25-1.5 µg/ml (total volume 20 µl). Reactions were incubated at 37 °C for 30 min and then treated with 1 µl of 10% (w/v) SDS and 1 µl of 1.5 mg/ml proteinase K at 50 °C for 30 min. DNA samples were analyzed by electrophoresis in 1% agarose in TBE buffer. Gels were blotted overnight on to DE81 paper before autoradiography.

Human DNA Topoisomerase IIalpha- and IIbeta-mediated DNA Cleavage of End-labeled pBR322 Analyzed by Polyacrylamide Gel Electrophoresis

End-labeled pBR322 was prepared by cutting with HindIII, labeling with [alphaP]dCTP an d Klenow polymerase, and then cutting with EcoRI before gel purification of the 4333-bp fragment. Cleavage was carried out by incubating 4 pmol of topoisomerase II with 5500 counts/min of labeled DNA (as measured by Cherenkov counting) in 50 mM Tris-HCl buffer, pH 7.5, 100 mM KCl, 10 mM MgCl(2), 0.1 mM DTT, 0.5 mM EDTA, 30 µg/ml BSA, and 1 mM ATP (final volume 20 µl). Where present, Me(2)SO was at 2.5%, and m-AMSA or o-AMSA at 5 or 50 µg/ml. Reactions were incubated at 37 °C for 1 h, then SDS and proteinase K were added to 1% (w/v) and 0.5 mg/ml, respectively. After incubation for a further 60 min, DNA was precipitated with ethanol and ammonium acetate in the presence of 20 µg of glycogen. After centrifugation the pellet was washed in 90% ethanol and then resuspended in 4 µl of loading buffer (80% formamide, 10 mM NaOH, 1 mM EDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). Samples were heated to 80 °C before being loaded onto a 6% polyacrylamide sequencing gel which was then run at 1100 V.

RESULTS

Expression and Purification of Recombinant Human Topoisomerase IIbeta from Yeast S. cerevisiae

We have for the first time successfully overexpressed human topoisomerase IIbeta in milligram quantities in a yeast expression system. We designed a construct, YEphTOP2beta (Fig. 1), to express human topoisomerase IIbeta in S. cerevisiae, encoding residues 46-1621 fused to sequence encoding the first 5 amino acid residues of yeast topoisomerase II. Sequence alignments of human topoisomerase IIbeta protein sequence with those of other eukaryotic type II enzymes indicated that the N-terminal 41 residues are poorly conserved and thus unlikely to be essential for catalysis(14) . In YEphTOP2beta the coding region is under the control of the galactose-inducible yeast GAL1 gene promoter, similar to YEpWOB6 which was used in the overexpression of human DNA topoisomerase IIalpha(23) .

Figure 1: Structure of YEphTOP2beta, a plasmid constructed to allow expression of human DNA topoisomerase IIbeta in yeast. SalI and XhoI restriction sites are unique in YEphTOP2beta.


YEphTOP2beta was transformed into a protease-deficient and ura3 yeast strain JEL1 and grown in minimal medium lacking uracil and containing 3% (v/v) glycerol and 2% (w/v) lactic acid. Induction of the human enzyme was achieved by the addition of galactose to the growth medium, to a final concentration of 2% and growth was continued for 3-16 h. Purification of the recombinant human topoisomerase IIbeta was carried out as described previously for human topoisomerase IIalpha(23) . Briefly, the procedure involved cell lysis, polymin P, and ammonium sulfate fractionation followed by phosphocellulose column chromatography. SDS-PAGE analysis identified a Coomassie-stained band of approximately 175 kDa in extracts of galactose-induced JEL1 containing YEphTOP2beta (Fig. 2A, lane c) which was absent from extracts of uninduced cells (lane b). This size is consistent for a 1621 amino acid protein missing the first 45 amino acids. Topoisomerase IIbeta purified to the phosphocellulose stage was >90% homogeneous and migrated with a lower mobility than recombinant topoisomerase IIalpha (Fig. 2A, lanes d and e), as expected from their predicted molecular masses. Immunoblot analysis with a topoisomerase IIbeta-specific antisera positively identified the topoisomerase IIbeta as a 175 kDa band in both the crude lysate and the phosphocellulose fraction (Fig. 2B, lanes c and d). Yields of between 300 and 700 µg/liter were obtained following purification, allowing production of milligram quantities of recombinant human topoisomerase IIbeta.

Figure 2: A, SDS-polyacrylamide gel 7.5% of fractions from the purification of recombinant human DNA topoisomerase IIbeta from yeast S. cerevisiae transformed with plasmid YEphTOP2beta. Cleared lysate from uninduced yeast cells (b), cleared lysate from galactose-induced yeast cells (c), human topoisomerase IIbeta, pooled phosphocellulose fractions (d), human recombinant topoisomerase IIalpha similarly purified to the phosphocellulose stage (e). Molecular weight markers are in lanes a and f. B, Western blot analysis of topoisomerase IIbeta. An SDS-polyacrylamide gel identical to that in A was run and blotted onto nitrocellulose. This blot was probed with an affinity-purified antipeptide antiserum against human topoisomerase IIbeta. The antiserum detects the 175 kDa beta band in both the induced extract (c) and pooled phosphocellulose fraction (d). There was no cross-reactivity to human topoisomerase IIalpha (e) or yeast topoisomerase II in extracts (b).


Catalytic Properties of Recombinant Topoisomerase IIbeta

Recombinant topoisomerase IIbeta was enzymatically active as assayed by its relaxation of supercoiled pBR322 in the presence of ATP. The specific activity of the purified beta protein ranged between 3 times 10^4 and 1 times 10^5 units/mg, similar to that reported for the native human topoisomerase IIalpha and Drosophila topoisomerase II(22, 28) . Relaxation of supercoiled pBR322 was dependent on Mg(II) and ATP. Concentrations of 6-30 mM MgCl(2) and 10-190 mM KCl were shown to support relaxation. These cation ranges are in agreement with those reported for human topoisomerase IIbeta purified from cell lines(10) .

Earlier enzymatic studies suggested that topoisomerase IIbeta was unstable and became fragmented if boiled prior to SDS-PAGE analysis (18). Stability of purified recombinant human DNA topoisomerase IIbeta was assessed by incubation for 2 min at 50, 68, 80, and 100 °C in SDS-PAGE loading buffer prior to gel electrophoresis. No differences were seen between alpha and beta treated in this way, and neither exhibited substantial protein degradation (data not shown). Topoisomerase II alpha and beta activities as measured by the DNA relaxation assay were stable for at least 24 h at 4 and 25 °C. At 37 °C, activity began to decrease after an 8-h incubation. Topoisomerase IIbeta is thus not as unstable as reported for the native enzyme from human cells(10, 18) , suggesting that the instability problem in the earlier work may have been due to the presence of a contaminating protease. However, we cannot formally eliminate the possibility that our recombinant protein is more stable due to the presence of the first five amino acids from yeast topoisomerase II or due to the lack of the first 45 amino acids of the human topoisomerase IIbeta.

Determination of the Domain Structure of Human Type II Topoisomerases by Proteolytic Cleavage and N-terminal Sequencing

Previous proteolysis studies on E. coli DNA gyrase and yeast topoisomerase II have suggested that type II topoisomerases are folded into discrete domains(29, 30, 31) . The hinge regions between these domains are more accessible and therefore less resistant to proteolytic cleavage. S. cerevisiae topoisomerase II is preferentially cleaved in three discrete protease sensitive regions, A-C. Sites A and B for S.aureus V8 protease have been mapped to the C-terminal side of residues 411 and 680, respectively, while site C is around amino acid 1200. The precise cleavage site depends on the specificity of the protease. To characterize the domain structure of topoisomerase IIbeta, we carried out proteolytic digests with trypsin and S.aureus V8 protease. To identify the resulting fragments, immunoblots were probed with two antisera: one specific for a 13-residue peptide, amino acid residues 928-940 in topoisomerase IIbeta (10) (Fig. 4), and an antibody raised against the C-terminal quarter of topoisomerase IIbeta. The exact sites of proteolytic cleavage were determined by N-terminal sequence analysis of the most prominent digestion products, (Table I).

Figure 4: Diagram showing the major proteolytic cleavage sites in topoisomerase IIbeta. Cleavage by either trypsin or SV8 protease at regions A-C generates discrete proteolytic fragments of 130, 90, 62-66, and 45-50 kDa in size. Regions A (near amino acid 470) and B (near amino acid 722) were located by N-terminal sequencing of SV8 protease and trypsin digestion products, respectively (Table I). Antibody 1 = antipeptide antiserum; antibody 2 = anti-C-terminal domain antiserum.


A time course for digestion of human topoisomerase IIbeta by trypsin is shown in Fig. 3. The C-terminal tail of topoisomerase IIbeta was most sensitive to digestion: it was cleaved at several sites to give the major digestion product of approximately 130 kDa (Fig. 3). This fragment was stable to further digestion for up to 24 h. Fragments of approximately 90 and 50 kDa became visible as the period of digestion was increased. The 90 kDa band was visualized by Western blotting and probing with the anti-peptide antibody, which indicated that it spans the centre of the protein (Fig. 4). N-terminal sequencing showed that the 50-kDa fragment starts at Val, which is 8 amino acid residues from the N terminus of the recombinant protein; this fragment therefore corresponds to the ATPase domain identified in E. coli DNA gyrase. The anti-peptide antibody also detected an approximately 62-kDa digestion product which was formed later than the other fragments and was stained less intensely by Coomassie Blue, consistent with this fragment being less abundant. N-terminal sequencing of this fragment showed that cleavage had occurred C-terminal to Phe. Fragments of a similar size to those produced with trypsin were observed with SV8 protease and their N-terminal sequences determined and listed in Table I. From the order of appearance and the intensities of the various products, it was deduced that the preferred order of cleavage in human type II topoisomerase IIbeta is region C, then region A, and lastly region B ( Fig. 4and Fig. 5, Table I).

Figure 3: A, time course for digestion of topoisomerase IIbeta by trypsin. 10 µg of the beta isoform was incubated with 0.1 µg of trypsin in phosphate buffer at 30 °C. Digestion was stopped by adding an equal volume of SDS loading buffer, and 20-µl aliquots were examined by electrophoresis on a 7.5% SDS-polyacrylamide gel. Lanes a-h, digestion was stopped at 0, 2, 5, 15, 30, 60, 90, and 240 min, respectively. The major proteolytic products of 130, 90, and 50 kDa can be seen in lane h. B, Western blot analysis of tryptic digests. An SDS-PAGE gel with the same samples as shown in A was run, blotted, and the filter probed with beta-specific antipeptide antiserum. Prominent bands of 130, 90, and 62 kDa are seen in lane h.


Figure 5: Comparison of the proteolytic cleavage patterns of human and S. cerevisiae type II topoisomerases digested with SV8 protease in the presence or absence of AMP-PNP. Lane j, protein markers. Lanes a-c, S. cerevisiae topoisomerase II (5.1 µg) digested with 0.46 units of SV8 protease for 0 min (lane a), 1 h (lane b), and 1 h in the presence of 1 mM AMP-PNP (lane c). Lanes d-i, human topoisomerase IIalpha (5.2 µg; lanes d-f) or beta (11.62 µg; lanes g-i) digested by 0.46 units of SV8 protease for 0 min (lanes d and g), 2 h (lanes e and h), and 2 h in the presence of 1 mM AMP-PNP (lanes f and i). A-C indicate the positions of yeast fragments of 90, 77, and 60 kDa, previously reported by Lindsley and Wang (29).


To investigate the degree of similarity in the domain structure of human topoisomerase IIalpha and beta, digests with SV8 protease and trypsin were carried out on the alpha isoform in the same manner as described above (Fig. 5). The patterns of fragments produced were very similar for both isoforms. Moreover, N-terminal sequencing confirmed that cleavage occurs at very similar sites in the two proteins (Table I). From the SV8 digests of the alpha isoform, it was also possible to identify two small fragments of 23 and 17 kDa: these start at Lys and Ile, respectively, and are derived from the C-terminal segment of the protein. These sequences confirm the loss of the C terminus from the major 130-kDa fragment.

The location of the sites of SV8 cleavages appeared to be similar in both alpha and beta isoforms, as confirmed by N-terminal sequence analysis (Table I). The presence of DNA slowed down the rate of proteolysis but did not alter the final fragmentation pattern (data not shown). Inclusion of AMP-PNP, a non-hydrolyzable ATP analogue did not affect the pattern of SV8 cleavage of human alpha or beta (Fig. 5, lanes e and f, h, and i). In contrast, AMP-PNP did alter the propensity for cleavage at sites in yeast topoisomerase II (Fig. 5, lanes b and c), a band of 90 kDa (A on Fig. 5) decreased while an additional two bands appeared at 77 and 60 kDa (B and C on Fig. 5), in agreement with previous work(29) . Comparison of the SV8 protease cleavage patterns of yeast topoisomerase II with that of human alpha and beta isoforms is illustrated in Fig. 5. There are similarities between the proteolysis patterns observed for recombinant human topoisomerase IIalpha and beta with those of their distant relative yeast topoisomerase II, suggesting some structural conservation at the tertiary level. However, the AMP-PNP result highlights the distinct differences in response between the human isoforms and yeast, perhaps not surprising for enzymes that differ by greater than 50% at the sequence level. This suggests caution should be used when extrapolating results from lower to higher eukaryotes.

DNA Breakage by DNA Topoisomerase IIalpha and beta: Cleavable Complex Formation Induced by Anticancer Drugs

Human DNA topoisomerase IIalpha and beta are the target for several anticancer drugs. These drugs fall into two main categories, those that inhibit type II topoisomerases by stabilizing the ``cleavable complex'' producing DNA strand breaks upon detergent treatment, and those that inhibit topoisomerase II without stabilizing this complex(3) . The in vitro effect of the cleavable complex forming drugs on recombinant topoisomerase IIalpha and beta has been investigated using DNA cleavage assays. Fig. 6illustrates cleavage of supercoiled DNA in the absence of ATP(32) . Assays with 0.4 µg of supercoiled pBR322 were incubated with 1 unit of DNA topoisomerase IIalpha or beta. In the presence of etoposide or teniposide both topoisomerase IIalpha and beta cleaved supercoiled pBR322 DNA under these conditions. Teniposide promoted similar levels of cleavage with either topoisomerase IIalpha or beta across a range of concentrations from 6 to 60 µM. Etoposide was a weaker cleavage agent than teniposide only inducing cleavage at concentrations greater than 15 µM, but again comparable levels of cleavage were seen with the two isoforms (Fig. 6A). Using this assay system, promotion of cleavage by AMSA was also investigated. Topoisomerase IIbeta produced higher levels of cleavage than alpha in the presence of m-AMSA. However, the most striking observation was that o-AMSA, the ``inactive'' ortho-isomer, promotes DNA cleavage in the presence of beta but not alpha (Fig. 6B ). No cleavage was visible at any of the concentrations of o-AMSA used with DNA topoisomerase IIalpha, while DNA topoisomerase IIbeta and o-AMSA produced cleavage.

Figure 6: A, cleavage of supercoiled pBR322 by topoisomerase IIbeta (a-j) or topoisomerase IIalpha (k-t), in the presence of teniposide (a- d and k-n), at concentrations of 60 µM (a and k), 30 µM (b and l), 15 µM (c and m), and 6 µM (d and n) or etoposide (e-h and o-r), at concentrations of 60 µM (e and o), 30 µM (f and p), 15 µM (g and q), and 6 µM (h and r) and no drug controls with ethanol (i and s) or water (j and t). B, cleavage of supercoiled pBR322 by topoisomerase IIbeta (a-j) or topoisomerase IIalpha (k-t) in the presence of m-AMSA (a-d and k-n) or o-AMSA (e-h and o-r), at concentrations of 325 µM (a, e, k, and o), 32.5 µM (b, f, l, and p), 16.3 µM (c, g, m, and q), or 8.1 µM (d, h, n, and r) and no drug controls with Me(2)SO (i and s) or water (j and t).

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Cleavage of end-labeled pBR322 DNA in the presence of topoisomerase IIbeta and drugs is shown in Fig. 7. In Fig. 7A a 4333-bp EcoRI-HindIII fragment from pBR322, uniquely end-labeled with [gamma-P]ATP, was incubated with recombinant human DNA topoisomerase IIbeta and m-AMSA, teniposide, or ellipticine. DNA cleavage was induced by the addition of detergent followed by proteinase K, and DNA products were separated according to size by agarose gel electrophoresis. Autoradiography of the gel revealed the extent of DNA cleavage and allowed mapping of cleavage sites on pBR322 DNA. With no drug, minimal cleavage was observed (lanes c and j) while m-AMSA (lanes d, g, and k), teniposide (lanes e, h, and l), or ellipticine (lanes f, i, and m) promoted cleavage, each with a different subset of cleavage sites. Addition of ATP made no difference to the sites of cleavage; however, DNA breakage was more efficient in the presence of ATP (lanes c-i). Comparison of the cleavage of end-labeled pBR322 DNA by either topoisomerase IIalpha or beta in the presence of m-AMSA or o-AMSA, after resolution on a polyacrylamide sequencing gel, is shown in Fig. 7B. Strong cleavage was seen in the presence of m-AMSA with both topoisomerase IIalpha (lanes d and h) and beta (lanes f and j), and the pattern of m-AMSA cleavage showed distinct isoform specific differences. Under these conditions cleavage was also observed with topoisomerase IIbeta and o-AMSA as can be seen in lanes g and k.

Figure 7: A, site-specific DNA cleavage by DNA topoisomerase IIbeta in the presence of m-AMSA, teniposide, or ellipticine. A 4333-bp EcoRI-HindIII fragment from plasmid pBR322 P-labeled at its 5`-EcoRI end (lane b) was incubated with human topoisomerase IIbeta in the absence (c and j), or in the presence of m-AMSA (d, g, and k), teniposide (e, h, and l), or ellipticine (f, i, and m), at concentrations of 0.25 µg/ml (d, f, k, and m), 0.5 µg/ml (e and l), 0.75 µg/ml (g and i), and 1.5 µg/ml (h). Lanes c-i also contained 1 mM ATP. DNA cleavage was induced by the addition of SDS and proteinase K and the DNA analyzed by agarose gel electrophoresis and autoradiography. Radiolabeled DNA markers were run in lane a. B, site-specific cleavage by DNA topoisomerase IIalpha and beta in the presence of m-AMSA and o-AMSA. A 4333-bp EcoRI-HindIII fragment from PBR322 labeled at the 3`-HindIII end was incubated with topoisomerase II alpha (lanes b, d, e, h, and i) or beta (lanes c, f, g, j, and k) in the presence of Me(2)SO (2.5% v/v) alone (lanes b and c), m-AMSA (lanes d, f, h, and j), or o-AMSA (lanes e, g, i, and k) at concentrations of 5 µg/ml (lanes d, e, f, and g) or 50 µg/ml (lanes h, i, j, and k). ATP was present at 1 mM. Lane a contains DNA alone. Cleavage was induced with SDS and proteinase K, and the DNA concentrated by ethanol precipitation. Samples were electrophoresed on a 6% sequencing gel before visualization by autoradiography. The positions of size markers run alongside are indicated.


Inhibition and Stabilization of the DNA-cleavable Complex by Flavonoid Compounds: Comparison of DNA Topoisomerase IIalpha and beta Isoforms

We have previously shown that the three reverse transcriptase inhibitors, the flavones quercetin (F1), quercetagetin (F2), and myricetin (F3) are able to promote DNA cleavage by calf thymus DNA topoisomerase II on end-labeled pBR322(24) . In this paper we have compared the cleavage produced by recombinant human DNA topoisomerase IIalpha and beta with quercetin (F1), quercetagetin (F2), and myricetin (F3). This work demonstrated that the site specificity of cleavage differed between topoisomerase IIalpha and beta in the presence of these plant-derived flavonoids (Fig. 8). For both isoforms the strongest cleavage pattern was observed with quercetagetin. Thus, similar to the AMSA results, human alpha and beta respond differently to flavonoid inhibitors.

Figure 8: Site-specific DNA cleavage by DNA topoisomerase IIalpha and beta in the presence of three cytotoxic flavonoid compounds at 50 µg/ml. Lane a pBR322 markers; lane b, linear end-labeled 4333-bp pBR322 fragment. Lanes c-f, DNA was incubated with topoisomerase IIalpha in the absence (c) or presence of quercetin, queretagetin, or myricetin, F1-3, respectively (d-f). Lanes g-j were incubated with topoisomerase IIbeta alone (g) or in the presence of F1-F3 (h-j).


DISCUSSION

The biological role of human DNA topoisomerase IIalpha and beta and their roles in cancer chemotherapy are two important questions that remain to be answered. Since only agonizingly small amounts of either isoform can be purified from cell lines, we have overexpressed human topoisomerase IIbeta in quantity in yeast providing protein to facilitate resolution of these questions. In the recombinant beta protein, the first 45 of the 1621 amino acids of the wild type enzyme are replaced by the first pentapeptide of yeast topoisomerase II. An inducible promoter was used to circumvent the problem that a high cellular level of topoisomerase II is detrimental to yeast cells. The yield of this expression system of between 0.3 and 0.7 mg of purified enzyme/liter of yeast culture, providing sufficient quantities of the human enzyme for biochemical, structural, and pharmacological studies. The catalytic properties of the recombinant human topoisomerase IIbeta are similar to those reported for human DNA topoisomerase IIbeta purified from cell lines except the highly purified enzyme is more stable under normal reaction conditions.

Sequence homologies between type II topoisomerases have been reported in a number of studies(14, 33, 34) . These have shown five motifs conserved between all type II topoisomerases and eight conserved between all eukaryotic type II topoisomerases(14, 34) . Their sequence conservation together with their similar catalytic activities suggested the type II topoisomerases might share common structural and mechanistic features. Evidence for structural similarities has been provided by the existence of common structural domains within the type II topoisomerases with protease-sensitive sites coinciding with junctions between these domains. Proteolysis of E. coli DNA gyrase and type II topoisomerases from S. cerevisiae, Schizosaccharomyces pombe, and Drosophila melanogaster all produced discrete protease-resistant fragments representing four discrete structural domains(29, 30, 31, 35) . The topoisomerase II sequence homology between these species is less than 50% and is not evenly distributed through the protein. The highest sequence conservation between species is seen within the N-terminal domain NH(2)-A, with the exception of the first few amino acids, which are not well conserved (Fig. 4). This region in DNA gyrase has been shown to contain the ATPase function. The three-dimensional structure of this domain has been solved for E. coli DNA gyrase(36) . The second domain, A- B contains three conserved motifs EDGSA, PLRGK, and IMTDQ(34) . The third domain, B-C, encompasses the active site tyrosine. The fourth domain C-COOH is the least well conserved at the sequence level but contains an abundance of charged amino acids in most species.

Unlike yeast and Drosophila, which have a single topoisomerase II, Homo sapiens have two isoenzyme forms of type II topoisomerase, alpha and beta. At the primary sequence level these two isoforms share 68% identity(14) . No information has previously been reported on the structural organization of either isoform. To determine the domain structure of the human isoforms, we analyzed both isoforms by proteolytic digestion and N-terminal sequencing (Fig. 3-5 and Table I). SV8 digestion of topoisomerase IIbeta produced at least five fragments, the two largest fragments both had N-terminal peptide sequences commencing at amino acid 48, the 5 yeast amino acids having been clipped off by the protease. The approximately 90-kDa fragment had an N terminus at position 471, analogous to site A at 411 in S. cerevisiae, at 406 in Drosophila, and at 394 in E. coli DNA gyrase B protein. Attempts at N-terminal sequence analysis for the two smaller fragments were unsuccessful. Trypsin digestion of topoisomerase IIbeta also produced at least five proteolytic products of similar sizes to those from SV8 digestion; N-terminal sequence could be obtained for three of these fragments. Two fragments of approximately 130 and 50 kDa had an N-terminal sequence starting at amino acid 49, the third fragment of approximately 62 kDa commenced at amino acid 723, corresponding to site B at position 680 in S. cerevisiae. The order of appearance and the N-terminal sequence data indicated that site C was cut first, then site A, and lastly site B. Human topoisomerase IIalpha exhibited cleavage with SV8 protease in equivalent positions. The preference for site C to be cleaved first in mammalian topoisomerases is consistent with information obtained for a proteolytic fragment of calf thymus topoisomerase II(37) . Our data show that both isoforms of human topoisomerase II contain four discrete structural domains. Mapping of these domains at the amino acid level provides the necessary sequence information to enable the expression of the individual domains for further structural and functional work. The conserved domain arrangement between yeast and human type II topoisomerases suggests they may share a similar tertiary structure, despite their primary sequences differing by more than 50%.

Previously, a protein conformation change was observed upon binding the non-hydrolyzable ATP analogue AMP-PNP to S. cerevisiae topoisomerase II. This change was detected by an alteration in SV8 proteolysis presumably due to altered SV8 protease accessibility. AMP-PNP is thought to act by stabilizing a conformational state that normally occurs transiently upon binding of ATP during the catalytic cycle. Yeast topoisomerase II is thought to interact with DNA as a protein clamp with the conformational change occurring as the jaws of the clamp close(38) . Human topoisomerase II most likely undergoes a similar conformational change during its catalytic cycle. However, an alteration in the protease cleavage pattern upon binding AMP-PNP was not detected with either human topoisomerase IIalpha or beta. Presumably, any conformation change caused by AMP-PNP binding to human topoisomerase IIalpha or beta does not affect the accessibility of SV8 to the proteolytic cleavage sites in these proteins (Fig. 5).

Human topoisomerase II is an important anticancer drug target(3) , but the role of the individual alpha and beta isoforms is not known. We have utilized recombinant DNA topoisomerase IIalpha and beta to determine the effects of several antitumor agents in vitro (Fig.& nbsp;6-8). Both isoforms were able to promote cleavage of supercoiled pBR322 DNA in the presence of amsacrine, teniposide, etoposide, and ellipticine. A striking difference was that topoisomerase IIbeta was able to promote cleavage with both m-AMSA and o-AMSA. It has previously been reported that o-AMSA was able to promote cleavage and to inhibit strand passage with calf thymus topoisomerase II, albeit at higher concentrations than m-AMSA(39) . However, it is not possible to determine whether these preparations of calf thymus topoisomerase II contained both alpha and beta. If they contained a mixture and only beta is sensitive to o-AMSA, this might explain the lower level of cleavage and inhibition. o-AMSA is much less cytotoxic to cells than m-AMSA, and it has been suggested that o-AMSA is unable to reach the DNA target in cells(40) ; topoisomerase IIbeta therefore may not be an in vivo target for o-AMSA. Cleavage of end-labeled pBR322 was obtained with both isoforms in the presence of three flavonoid compounds, quercetin, quercetagetin, or myricetin (Fig. 8). Human DNA topoisomerase IIbeta differed from alpha in its response to these agents; the cleavage sites were isoform-specific. The in vitro isoform-specific drug effects indicate that despite their overall structural similarities by protease studies, human alpha and beta isoforms can be distinguished by cleavable complex-forming compounds. Whether specific targeting of human topoisomerase II isoforms is of benefit in cancer chemotherapy remains to be determined. Availability of recombinant human topoisomerase IIbeta should facilitate further structural, biochemical, and pharmacological studies of this hitherto poorly characterized human isoform.

  
Table: N-terminal sequence analysis of domain boundaries

N-terminal peptide sequences of fragments produced by trypsin and SV8 protease digestion of human topoisomerase IIalpha and beta are shown in bold type. Below each peptide sequence is the closest corresponding amino acid sequence from the relevant topoisomerase, and the amino acid immediately N-terminal to the deduced site of cleavage (indicated by an arrow) is also shown.


FOOTNOTES

*
This work was supported by grants from the Science and Engineering Research Council, Cancer Research Campaign Grant SP1621-0901, The Nuffield Foundation, European Commission Concerted Action Grant PL931318 on Anticancer drug action on topoisomerase II, and 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 by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 44-191-222-8864; Fax: 44-191-222-7424.

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