Communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha.

The DNA strand passage activity of eukaryotic topoisomerase II relies on a cascade of conformational changes triggered by ATP binding to the N-terminal domain of the enzyme. To investigate the interdomain communication between the ATPase and cleavage/religation domains of human topoisomerase IIalpha, we characterized a mutant enzyme that contains a deletion at the interface between the two domains, covering amino acids 350-407. The ATPase domain retained full activity with a rate of ATP hydrolysis that was severalfold higher than normal, but the ATPase activity was unaffected by DNA. The cleavage and religation activities of the enzyme were comparable with those of the wild-type enzyme both in the absence and presence of cancer chemotherapeutic agents. However, neither ATP nor a nonhydrolyzable ATP analog stimulated cleavage complex formation. Although both conserved domains retained full activity, the mutant enzyme was unable to coordinate these activities into strand passage. Our findings suggest that the normal conformational transitions occurring in the enzyme upon ATP binding are hampered or lacking in the mutant enzyme. Consistent with this hypothesis, the enzyme displayed an abnormal clamp closing activity. In summary, the region covering amino acids 350-407 in human topoisomerase IIalpha seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.

Human DNA topoisomerase II is a multifunctional and highly complex enzyme that is able to change the topological conformation of DNA in response to different physiological alterations (1)(2)(3). Topological changes mediated by the dimeric topoisomerase II enzyme require a strict control of the passage of duplex DNA through the whole subunit interface (4) and through another duplex coordinately cleaved by the enzyme, where a correct interdomain as well as intersubunit communication is fundamental.
Topoisomerase II consists of three distinct domains. The N-terminal and central domains are highly conserved among enzymes from different eukaryotic organisms and also share homology to the gyrase B and A subunits, respectively (5)(6)(7)(8)(9). The C-terminal domain is dispensable for in vitro catalytic activity and shows no sequence conservation (9 -11). Central to the activity of the enzyme is its ability to bind and hydrolyze ATP as well as to cleave and religate DNA. The active site for ATP hydrolysis is encompassed in the N-terminal domain (12)(13)(14), while that for DNA cleavage/religation is located in the central domain (15).
Structural and biochemical data have suggested a rational model for the catalytic mechanism of eukaryotic topoisomerase II (16 -19). According to this model, the two subunits of the enzyme form a heart-shaped ring structure, where the N-terminal domains protrude as a set of jaws functioning as an ATP operated clamp. In the absence of ATP, the enzyme assumes an open conformation with a gate in the N-terminal part of the enzyme. The open state can permit a DNA segment (the socalled G-segment) to enter the enzyme through the N-terminal face and bind to the cleavage/religation domain. Binding of the DNA segment will induce the first conformational change in the enzyme, which enables the active site tyrosines to move toward each other into a position whereby cleavage can occur. Upon binding of ATP and hydrolysis of the first ATP molecule, other conformational changes occur, facilitating dimerization of the N-terminal domains (20). In this process, the other duplex segment (the so-called T-segment) that has to be transported through the broken DNA is captured by the enzyme. Besides DNA trapping, the conformational changes also ensure the creation of a gate in the G-segment by separation of the two active site tyrosines covalently linked to the broken DNA ends. The process is continued by the transport of the trapped Tsegment through the entire interfacial channel in between the two subunits and thereby through the gate in the cleaved G-segment. The movements of the cleavage/religation domain during DNA religation restrict the cavity enclosing the transported DNA, thereby releasing this duplex through a second gate formed by a disruption of the dimerization region located in the C-terminal part of the central domain (4,18,21). Finally, ATP hydrolysis of the second ATP molecule results in enzyme turnover and reopening of the N-terminal gate (20).
In a recent study by Maxwell and co-workers (22), a human topoisomerase II␣ fragment covering the N-terminal domain from amino acid 1 to 439 was shown to have an intrinsic ATPase activity, which could be further stimulated by the presence of DNA. In another study, a core domain of Drosophila topoisomerase II covering amino acids 406 -1207 was demonstrated to have wild-type levels of cleavage/religation activity (23). The results from the two studies nicely illustrate that the individual domains in topoisomerase II still preserve their intrinsic activities even when separated from the rest of the enzyme, demonstrating that they fold up as independent cata-lytic domains. However, strand passage activity requires a tightly coordinated communication between the individual domains. This is indicated by a study of the ATP consumption by topoisomerase II performed by Lindsley and Wang (24), where it was shown that a tight coupling exists between ATP usage and the DNA strand transport event under unsaturated ATP concentrations. Furthermore, several studies have revealed a stimulatory effect of ATP on topoisomerase II-mediated cleavage, also illustrating the communication between enzyme subdomains (25,26).
In order to investigate the communication between the ATPase and cleavage/religation domains of human topoisomerase II␣, we characterized a mutant enzyme having a deletion at the interface between the two domains. The enzyme contained both ATPase and cleavage/religation activities, but no strand passage occurred. Furthermore, the DNA cleavage activity was independent of ATP, and rates of ATP hydrolysis were unaffected by the presence of DNA. Finally, the mutant enzyme lacked a normal clamp closing activity. In summary, the deleted region seems to be essential for correct interdomain communication and probably is involved in signaling ATP binding to the rest of the enzyme.

EXPERIMENTAL PROCEDURES
Yeast Strains and Plasmids-The Saccharomyces cerevisiae strains BJ201 (Mat␣ ura3 trp1 pep4::HIS3 prb1 can1 top2::TRP1) and JEL1⌬Top1 (kindly provided by J. C. Wang) were used for complementation and overexpression of topoisomerase II constructs, respectively. Plasmid pBY105 contains the yeast TPI promoter inserted into the polylinker region of the LEU2/ARS-CEN plasmid pRS315, which was used as the backbone for pHT212 and pHT⌬350 -407, carrying the wild-type human TOP2␣ cDNA and the human TOP2␣ cDNA with a deletion spanning amino acids 350 -407, respectively. Both pHT212 and pHT⌬350 -407 contain a bicomposite tag at the C-terminal end consisting of the c-Myc epitope and a hexahistidine tail (9). Modified versions of YEpWOB6 were used for overexpression of the hexahistidine-tagged human topoisomerase II␣ and h⌬350 -407 enzymes.
Construction of Plasmids-The construction of pHT212 and pHT⌬350 -407 was described by Jensen et al. (9). For overexpression of the hexahistidine versions of human topoisomerase II␣ and h⌬350 -407 enzymes, the topoisomerase II␣ cDNA of YEpWOB6 was first modified with a hexahistidine tail at the C terminus. For this purpose, a Cterminal fragment of topoisomerase II␣ fused to a hexahistidine tail was generated via polymerase chain reaction using pHT212 as template. The 3Ј primer was designed with a stretch of 54 overhanging nucleotides containing the hexahistidine tail and three restriction sites for cloning. The annealing segment of the primer was the reverse complementary sequence of the human c-Myc epitope (5Ј-TCC CCC CGG GGC GGC CGC CTC GAG CTA ATG ATG GTG GTG ATG GTG GCT CCC ACG GTT CAA GTC TTC TTC AGA GAT CAA C-3Ј). The 5Ј primer sequence was identical to nucleotides 2973-2991 of the topoisomerase II␣ cDNA sequence (5Ј-GAG AGA GTT GGA CTA CAC-3Ј). The generated polymerase chain reaction fragment was used to replace the corresponding fragment of human topoisomerase II␣ in YEpWOB6 employing BlnI and XmaI as 5Ј and 3Ј cloning sites, respectively. For overexpression of the h⌬350 -407 enzyme in the YEpWOB6 system, the BsrGI/BlnI fragment in YEpWOB6 was substituted with the corresponding fragment from pHT⌬350 -407.
Yeast Transformation and Complementation-Yeast cells were transformed by using a modified version of the LiAc method of Ito et al. (27). To test the ability of the h⌬350 -407 enzyme to complement the lack of endogenous topoisomerase II in BJ201, the LEU2-based construct pHT⌬350 -407 was transformed into BJ201, and cells were transferred to media plates containing 5Ј-fluoro-orotic acid (1 mg/ml) to select against the URA3 plasmid carrying the Schizosaccharomyces pombe TOP2 gene (9). pHT212 was used as the positive control.
Human Topoisomerase II␣ Induction, Overexpression, and Purification-The recombinant human topoisomerase II enzymes were overexpressed in yeast strain JEL1⌬Top1 by the addition of galactose to glucose-free medium (12). Yeast cells were extracted with 2 volumes of extraction buffer (50 mM Tris-HCl, pH 7.8, 1 M NaCl, 1 mM phenylmethylsulfonyl fluoride) and 1 volume of acid-washed glass beads (425-600 m; Sigma). Further preparation of yeast extracts was done according to the procedure of Jensen et al. (9). The initial purification step using a 6-ml Ni 2ϩ -nitrilotriacetic acid-agarose column was as described previously by Biersack et al. (28). For further purification of the recombinant enzymes to near homogeneity, the fractions pooled from the Ni 2ϩ column were loaded onto a 5-ml heparin-Sepharose column (Amersham Pharmacia Biotech), and elution was performed by a 75-ml linear gradient having a NaCl concentration ranging from 200 mM to 1 M. Fractions containing topoisomerase II were further applied to a phosphocellulose column (P11 cellulose phosphate; Whatman) for concentration of the enzyme. Elution was performed in a buffer containing 750 mM KCl, 0.5 mM dithiothreitol, 0.1 mM EDTA, 40% glycerol, 50 mM Tris-HCl, pH 7.7. Fractions containing topoisomerase II enzyme were pooled and stored in liquid nitrogen for later use. Homogeneity of the topoisomerase II preparations was determined based on the analysis in 8.5% SDS-polyacrylamide gels stained with Coomassie Blue dye.
Topoisomerase II-mediated DNA Relaxation-DNA relaxation was performed by incubating 0.05 g (5 nM) of topoisomerase II and 0.3 g of supercoiled pBR322 DNA in assay buffer (50 mM Tris-HCl, pH 7.9, 100 mM KCl, 0.1 mM EDTA, 5 mM MgCl 2 , and 2.5% glycerol) supplemented with 1 mM ATP. Reactions were incubated at 37°C and stopped at different times by the addition of 3 l of 0.77% SDS and 77 mM EDTA. Samples were subjected to electrophoresis in 1% agarose gels in TBE (100 mM Tris borate, pH 8.3, 2 mM EDTA). Gels were stained with 1 g/ml ethidium bromide, visualized by UV light, and photographed using Polaroid type 665 positive/negative films.
Topoisomerase II-mediated pBR322 Cleavage-Topoisomerase IImediated cleavage was performed by incubating 1.5 g (150 nM) of topoisomerase II and 0.3 g of negatively supercoiled pBR322 DNA in a total volume of 20 l in assay buffer. Samples were incubated at 37°C for 6 min, and cleavage products were trapped by the addition of SDS to 1%. Samples were treated with 2 l of 0.8 mg/ml Proteinase K before subjection to electrophoresis in 1% agarose gels in TAE (40 mM Tris acetate, pH 8.3, 2 mM EDTA) containing 1 g/ml ethidium bromide. When topoisomerase II-mediated DNA cleavage was carried out in the presence of ATP or the ATP analog AMP-PNP 1 (Roche Molecular Biochemicals), the concentration of these nucleotides was 1 mM. When amsacrine or VM26 (teniposide) was included in the cleavage reaction, the final concentration of the drug was 100 M. Levels of DNA cleavage were quantified by scanning the cleavage bands in photographic negatives with an EC apparatus model EC910 scanning densitometer in conjunction with Hoefer GS-370 Data System software Oligonucleotides-DNA oligonucleotides were synthesized on a DNA synthesizer model 394 by DNA Technology Corp. and purified by preparative polyacrylamide gel electrophoresis as described by Andersen et al. (29). The 28-mer used as the bottom strand in the suicide substrate was modified at the 3Ј-end by the amino link -O-PO 2 -O-CH 2 -CHOH-CH 2 -NH 2 to inhibit ligation to this end.
Topoisomerase II-mediated Cleavage of Suicide Substrates-Hybridization and labeling of the synthetic oligonucleotides were done according to the procedures described by Andersen et al. (29). For topoisomerase II-mediated cleavage, 75 nM topoisomerase II was incubated with 0.1 pmol of labeled substrate in 50 l of 10 mM Tris-HCl, pH 7.0, 2.5 mM MgCl 2 , 2.5 mM CaCl 2 , 30 mM NaCl, 15 g/ml bovine serum albumin, and 0.1 mM EDTA (cleavage buffer) at 37°C, and reactions were stopped at different times by the addition of SDS to 1%. Covalent topoisomerase II-DNA cleavage complexes were recovered from a phenol/water interphase according to Gocke et al. (30). Complexes were subsequently ethanol-precipitated and treated with proteinase K (500 g/ml) for 2 h at 45°C. One volume of gel loading buffer (50% formamide, 0.05% bromphenol blue, 0.03% xylene cyanole, 5 mM EDTA) was added, and the material was subjected to electrophoresis in a 12% denaturing polyacrylamide gel. The level of cleavage was quantified using a Phos-phorImager (Molecular Dynamics, Inc., Sunnyvale, CA).
Topoisomerase II-mediated DNA Ligation-A topoisomerase II-mediated suicide cleavage reaction was performed as described above. After incubation at 37°C for 60 min, the cleavage reaction was stopped by the addition of NaCl to 0.4 M, thereby preventing further cleavage during the ligation reaction. Ligation was initiated by the addition of a 45-mer ligation substrate in a 200-fold molar excess relative to the cleavage substrate. After further incubation (with incubation times as indicated in the figure legends), the reaction was stopped by the addition of SDS to 1%. Samples were ethanol-precipitated, proteinase Kdigested, and analyzed by electrophoresis in a 12% denaturing polyacrylamide gel.
Hydrolysis of ATP by Topoisomerase II-The ATPase assay was based on the method of Osheroff et al. (31). Reactions contained 15 nM topoisomerase II and, when indicated, 1 g of negatively supercoiled pBR322. Reactions were carried out in 20 l of assay buffer containing a final concentration of 1 mM cold ATP and 1 mM [␥-32 P]ATP (3000 Ci/mmol; Amersham Pharmacia Biotech). Mixtures were incubated at 37°C, and 2.5-l aliquots were removed at various times and spotted onto thin layer cellulose plates impregnated with poly(ethylenimine) (Baker-flex precoated flexible TLC sheets). Chromatography was performed using freshly made 0.4 M NH 4 HCO 3 . Levels of free PO 4 were quantified using a PhosphorImager.
Clamp Closing Assay-For clamp closing experiments, 0.06 pmol of topoisomerase II was preincubated with 6 ng of supercoiled pBR322 and 6 ng of linearized pBR322 at 37°C for 5 min in a total volume of 20 l of assay buffer. After preincubation, AMP-PNP or ATP was added to a final concentration of 1 mM, and the reactions were incubated for an additional 5 min at 37°C. The reactions were next stopped by the addition of either NaCl or SDS to final concentrations of 800 mM and 1%, respectively. The sample volume was increased to 70 l by adding 50 l of an 800 mM NaCl solution. To trap enzyme-DNA catenanes, phenol extraction was performed by adding 1 volume of phenol. The samples were vortexed and centrifuged at 13,000 rpm in an Eppendorf centrifuge for 15 min. The water phase was removed, and 35 l of it was ethanol-precipitated and dissolved in 10 l of TE buffer for gel analysis. The combined phenol and phenol interphase was washed three times with 500 l of 800 mM NaCl after increasing the sample volume to approximately 100 l with phenol. Upon removal of the water phase after the last wash, the remaining material was ethanol-precipitated and dissolved in 10 l of Proteinase K buffer containing 1 mg/ml Proteinase K and 0.5% SDS. The samples were next subjected to electrophoresis in 1% agarose gels in TBE. Southern blotting was performed using Zeta-Probe GT membranes (Bio-Rad), and random primed plasmids were used for hybridization (Roche Molecular Biochemicals). A PhosphorImager (Molecular Dynamics) was used for gel scanning.

Purification and Characterization of a Human Topoisomerase II␣ Enzyme Lacking the Interface between the ATPase and the Cleavage/Religation
Domains-ATP binding and hydrolysis by topoisomerase II is known to mediate sequential conformational changes in the enzyme reaching from the N-terminal clamp to the C-terminal dimerization region (16 -20). In order to investigate the communication between the ATPase and the cleavage/religation domains of human topoisomerase II␣, we characterized a mutant enzyme (h⌬350 -407) that contains a deletion of amino acids 350 -407 at the very C-terminal end of the N-terminal ATPase domain (Fig. 1A). The deletion mutant has been presented earlier as one in a series of human topoisomerase II␣ mutants all having deletions in highly conserved subdomains (9). In a complementation assay using an S. cerevisiae top2 deletion strain, h⌬350 -407 failed to sustain mitotic growth in contrast to a wild-type human topoisomerase II␣ enzyme, suggesting that the mutant enzyme has lost its in vivo activity (Fig. 1B). For studies of the in vitro capabilities of h⌬350 -407, the mutant enzyme fused to a hexahistidine tail at the C-terminal end was overexpressed in a yeast top1 null strain and purified to homogeneity as seen from the Coomassiestained gel in Fig. 1C.
To investigate the DNA strand passage activity of h⌬350 -407, a DNA relaxation assay was performed, where the catalytic activity of the mutant enzyme was compared with that of the wild-type enzyme (Fig. 2). While the wild-type enzyme relaxed all the supercoiled DNA within 10 min, the deletion mutant showed no sign of relaxation up to 15 min. Thus, consistent with the lack of in vivo complementation, h⌬350 -407 appears to have lost its in vitro relaxation activity, or it is diminished to under detectable levels. A similar lack of DNA strand passage was observed in a DNA decatenation assay (data not shown). These results demonstrate that deletion of the C-terminal 57 amino acids of the ATPase region in human topoisomerase II␣ is detrimental to the enzyme. Either it abrogates the DNA strand passage activity of the enzyme, or it disrupts correct folding of the protein.
Characterization of the N-terminal ATPase Domain of h⌬350 -407-In order to determine whether the individual domains of h⌬350 -407 still retain their residual activities although the enzyme is unable to convert these activities to full catalysis, we analyzed the capability of the N-terminal domain of the deletion mutant to hydrolyze ATP. According to the model presented by Lindsley, ATP hydrolysis is responsible for A low copy ARS/CEN plasmid carrying either the h⌬350 -407 (h⌬350 -407) or the wild-type human topoisomerase II␣ (h␣-wt) cDNA behind a TPI promoter was transformed into the yeast strain BJ201. As a control, the cells were transformed with plasmid DNA lacking the TOP2 cDNA (control). In BJ201, the chromosomal TOP2 gene has been disrupted by insertion of the structural TRP1 gene, while the essential topoisomerase II activity is provided by the S. pombe TOP2 gene carried on a low copy URA3-based plasmid. After transformation, cells were grown on media containing 5Ј-fluoro-orotic acid to counterselect against the URA3 plasmid. C, purification of the h⌬350 -407 and wild-type enzymes. The enzymes were purified after overexpression in yeast through three different steps involving Ni 2ϩ -nitrilotriacetic acid-agarose, heparin-Sepharose, and phosphocellulose column chromatography. The homogeneity of the topoisomerase II preparations was determined after analysis of the proteins in an 8.5% SDS-polyacrylamide gel stained with Coomassie Blue. Protein size markers are indicated to the right of the gel. the conformational changes leading to DNA transport and final enzyme turnover (20). ATP hydrolysis was investigated using thin layer chromatography, and hydrolysis rates were assigned using a linear regression least squares analysis (Fig. 3). Strikingly, the ATPase activity of h⌬350 -407 was ϳ6-fold higher than that of the wild-type enzyme, demonstrating that the N-terminal domain of the mutant enzyme indeed still folds into a catalytic active domain able to bind and hydrolyze ATP.
Earlier studies on topoisomerase II-mediated ATP hydrolysis have shown that this activity is stimulated in the presence of DNA, both when the ATPase region is embodied in a fulllength enzyme or in an N-terminal topoisomerase II fragment (22,24,(31)(32)(33)(34). In the present study, the ATPase activity of the wild-type human topoisomerase II␣ enzyme was stimulated 2-3-fold in the presence of supercoiled plasmid DNA (Fig. 3,  inset). However, no stimulation was observed with h⌬350 -407, indicating that the ATPase activity of the mutant enzyme is unaffected by DNA. The significant rise in the ATPase activity and its DNA independence combined with the lack of strand passage activity indicate an uncoupling of the N-terminal domain from the rest of the enzyme. The uncoupling might be caused either by an inability of the mutant enzyme to transmit essential conformational changes as a result of ATP binding and hydrolysis or an inability to interact properly with DNA.
Characterization of the Central Cleavage/Religation Domain of h⌬350 -407-In order to test if the central domain of the h⌬350 -407 enzyme still retained its intrinsic DNA binding and cleavage activities, cleavage experiments were performed using either oligonucleotides or supercoiled circular DNA as substrate.
The suicide substrate, which is schematically illustrated in Fig. 4A (upper panel) consists of a 16-base-long 5Ј-recessed top strand with only three nucleotides 5Ј to the cleavage position and a 28-base-long bottom strand. Use of the suicide substrate has been demonstrated to cause an uncoupling of the cleavage and ligation half-reactions due to the release of the trinucleotide 5Ј to the cleavage position on the top strand (29). The substrate was labeled at the 3Ј-end of the recessed top strand and incubated with either h⌬350 -407 or the wild-type enzyme. Samples were withdrawn at different times, and after termination of the cleavage reaction by SDS, the protein-linked cleavage complexes were isolated from a phenol/water interphase. Samples were analyzed in a 12% denaturing polyacrylamide gel after proteinase K treatment (Fig. 4A, middle panel). As seen from a schematic presentation of the obtained cleavage levels (Fig. 4A, lower panel), the mutant enzyme cleaved the suicide substrate to a level similar to the wild-type enzyme, indicating that the cleavage domain retained normal DNA binding and cleavage activities.
The topoisomerase II cleavage complex generated upon cleavage of a suicide substrate is kinetically competent. As demonstrated previously, such a complex is able to perform ligation if a suitable ligation substrate is added to the cleavage mixture as schematically illustrated in Fig. 4B (upper panel) (29). To investigate if the central domain of h⌬350 -407 also withholds ligation activity, topoisomerase II-DNA cleavage complexes were prepared for the ligation assay as described under "Experimental Procedures." After an increase in the salt concentration to inhibit further cleavage, ligation was initiated by the addition of a 45-mer DNA oligonucleotide able to hybridize to the bottom strand of the cleaved substrate. At different times, aliquots were taken and treated with SDS and proteinase K before analysis in a 12% polyacrylamide gel. The results are depicted graphically in Fig. 4B (lower panel), where the levels of ligation at different times are given relative to the amount of initial cleaved material to take into account differences in the cleavage level at the start of ligation. The relative rates of ligation as visualized from the slope of the curves are comparable for the two enzymes, further substantiating that the central domain is folded into an entity retaining normal properties.
The optimal conditions for topoisomerase II-mediated cleavage of small oligonucleotides vary to some extent from those giving maximum cleavage of longer duplexes including circular DNA (29). Also, whereas oligonucleotides only require contacts to a very restricted area of the cleavage domain, circular DNA might contact the enzyme in place of both the T-and G-segments (33). Cleavage of circular DNA or long duplex DNA substrates might therefore require a higher extent of correct interdomain communication. This is also indicated from the stimulatory effect of ATP on topoisomerase II-mediated cleavage of such substrates (25,26) as compared with the negligible effect of ATP on cleavage of small oligonucleotides (35). Therefore, in order to investigate if h⌬350 -407, although operating normally on oligonucleotides, has an altered behavior toward longer substrates, we performed cleavage experiments using supercoiled plasmid DNA as substrate. As shown in Fig. 5A and schematically presented in Fig. 5B, h⌬350 -407 is able to cleave supercoiled DNA to almost the same level as the wildtype enzyme (compare lanes 4 and 5 with lanes 2 and 3). Therefore, the mutant enzyme also appeared to display normal DNA binding and cleavage activity with supercoiled plasmid substrates. This conclusion is further supported by the similar response of the h⌬350 -407 and the wild-type enzymes to the two anti-tumor agents VM26 and amsacrine with respect to cleavage complex formation (Fig. 5C).
To further test if cleavage mediated by h⌬350 -407 is still influenced by ATP binding to the ATPase domain, topoisomerase II-mediated cleavage of the supercoiled substrate was performed in the presence of ATP or the ATP analog, AMP-PNP. In contrast to the 2-3-fold stimulation of cleavage obtained with the wild-type enzyme, cleavage complex formation by h⌬350 -407 was not enhanced by the presence of ATP or the ATP analog (Fig. 5A, compare lanes 6, 7, 10, and 11 with lanes  8, 9, 12, and 13). In conclusion, our data suggest that the central domain operates normally with respect to DNA binding, cleavage, and religation and therefore constitutes a full functional domain in the mutant enzyme. However, the inability of ATP to stimulate the cleavage reaction of the enzyme favors the hypothesis of an uncoupling of the ATPase and cleavage/religation domains.
Characterization of the N-terminal Clamp Closing Activity of h⌬350 -407-The existence of two functional domains in h⌬350 -407, combined with the lack of strand passage activity, strongly suggests that the two domains of the enzyme are unable to communicate. Several studies suggest that the mode of communication in topoisomerase II is through a cascade of conformational changes taking place in the enzyme upon ATP binding and hydrolysis, starting with the trapping of a Tsegment by N-terminal clamp closure (16, 19, 20, 36, 37). The changes are transmitted to the rest of the enzyme, leading to a coordinated separation of the two active site tyrosines. The newly created gate in the G-segment allows the T-segment to pass through and leave the enzyme after opening of the primary C-terminal dimerization region. To investigate if the initial conformational changes including N-terminal clamp closure occur in h⌬350 -407 upon ATP binding, we performed a clamp closing assay taking advantage of phenol extraction for collection of enzyme-DNA complexes that have become interlinked due to enzyme clamp closure. The h⌬350 -407 or wildtype enzyme was incubated with a DNA mixture containing equal amounts of circular and linear DNA, where most of the circular DNA was in a supercoiled form. Following preincubation, an ATP analog was added to close the N-terminal clamp, and after stopping the reaction by salt, the samples were treated with phenol. While the phenol water phase, containing free DNA, was loaded directly on an agarose gel after alcohol precipitation, the material in the phenol interphase was washed several times with 0.8 M salt to remove free DNA before the samples were treated with proteinase K, alcohol-precipitated, and loaded on the gel. In the case of the wild-type enzyme, the presence of an ATP analog resulted in almost all of for the suicide cleavage reaction the wild-type or h⌬350 -407 enzyme was incubated with the 3Ј-end labeled substrate, and cleavage was stopped at different times by the addition of SDS to 1%. Covalent topoisomerase II-DNA complexes were recovered from a phenol/water interphase. Complexes were subsequently ethanol-precipitated and treated with proteinase K, before they were subjected to electrophoresis in a 12% polyacrylamide gel. Lane 1, DNA size marker increasing in steps of two bases; lane 2, labeled DNA substrate; lanes 3-8, time course of the cleavage reaction performed with the wild-type enzyme; lanes 9 -14, time course of the cleavage reaction performed with the h⌬350 -407 enzyme. S, the cleavage substrate remaining in the interphase after phenol extraction. Cl, the cleavage product, for which migration was retarded with ϳ1 base due to residual undigested protein. *, cleavage products with a longer protein fragment covalently linked, due to partial proteinase K digestion. Lower panel, schematic representation of the time course experiment shown in the middle panel. Cleavage levels were measured by Phospho-rImager scanning and are presented in arbitrary units relative to the cleavage level obtained with the wild-type enzyme after 90 min. B, investigation of the suicide ligation reaction performed with the h⌬350 -407 and wild-type enzymes. Upper panel, schematic illustration of the topoisomerase II-mediated ligation reaction on the 5Ј-recessed substrate performed by topoisomerase II covalently linked to the cleaved suicide substrate. The 45-mer added to the reaction is the incoming ligation substrate, which in the 3Ј end is complementary to the single-stranded region of the bottom strand. The arrowheads indicate the position of topoisomerase II-mediated cleavage. The asterisk represents radioactive labeling. Lower panel, graphic illustration of a topoisomerase II-mediated time course ligation experiment using the wild-type or h⌬350 -407 enzyme. A suicide cleavage reaction was performed as described in A. After 60 min, the cleavage reaction was stopped by the addition of salt, thereby preventing further cleavage during the ligation reaction. Ligation was initiated by the addition of a 45-mer ligation substrate in a 200-fold molar excess relative to the cleavage substrate. Samples were withdrawn at the indicated times, treated with SDS, and subjected to electrophoresis in a 12% denaturing polyacrylamide gel. Levels of ligation were measured by PhosphorImager scanning of the gel and are expressed in arbitrary units relative to initial cleaved material. the supercoiled DNA and none of the linear DNA being found in a protein-linked form in the interphase, in accordance with the principles of DNA trapping due to enzyme clamp closure (Fig.  6, upper panel). In contrast, only a trace amount of the supercoiled DNA was found in the interphase for h⌬350 -407, and enzyme-mediated trapping of DNA occurred independent of the ATP analog. The efficiency of the clamp closure event for the mutant enzyme relative to the wild-type enzyme is depicted graphically in the histogram presented in Fig. 6 (lower panel). The results strongly indicate that the mutant enzyme is disturbed in its clamp closing activity, being unable to stably close the clamp in the presence of high salt, although it still binds and hydrolyzes ATP. The increased ATPase activity of the mutant enzyme might be caused by an elimination of a time lag normally existing due to a strict coordination of the ATPase activity with the transitions occurring in the rest of the enzyme during strand passage. The negligible amount of supercoiled DNA trapped by the mutant enzyme independent of the ATP analog might be caused by a slight change in the affinity of the enzyme for the G-segment in the central domain, as also suggested from the inability to totally reverse enzyme-DNA binding in the presence of 1 M salt (data not shown).
FIG. 5. Investigation of the DNA cleavage activity of h⌬350 -407 using supercoiled plasmid DNA. A, the wild-type or the h⌬350 -407 enzyme was incubated with pBR322 plasmid DNA at 37°C in the absence or presence of ATP or the ATP analog, AMP-PNP. The reactions were stopped after 6 min by the addition of SDS to 1%, and following proteinase K treatment, they were subjected to electrophoresis in 1% agarose gels containing excess ethidium bromide in the electrophoresis buffer. Each experiment was carried out in duplicate. Lane 1, DNA standard; lanes 2 and 3 and lanes 4 and 5 represent cleavage by the wild-type enzyme and the h⌬350 -407 enzyme, respectively, in the absence of nucleotide. Lanes 6 and 7 and lanes 8 and 9 represent cleavage by the wild-type enzyme and the h⌬350 -407 enzyme, respectively, in the presence of ATP. For the clamp closing experiment, the enzymes were incubated with equal amounts of circular and linear DNA. After preincubation, ATP or the ATP analog AMP-PNP was added to a final concentration of 1 mM. The reactions were further incubated before they were stopped by the addition of either NaCl to a final concentration of 800 mM or SDS to a final concentration of 1%. The samples were next phenol-extracted before the water phase was removed and ethanol-precipitated. The remaining phenol interphase was carefully washed three times in high salt before the material was ethanol-precipitated, proteinase K-digested, and subjected to electrophoresis in a 1% agarose gel together with the water phase samples. Southern blotting was performed with random primed plasmids as probes. The enzyme used in the experiment is indicated above the lanes. The presence of ATP or AMP-PNP in the reaction mixture and whether the samples were treated with SDS or salt is indicated above the lanes (ϩ). w and i indicate phenol water phase or phenol interphase, respectively. RC, L, and SC indicate the positions of relaxed, linear, and supercoiled plasmid DNA, respectively. Lower panel, graphic illustration of the clamp closing experiment shown in the upper panel. Levels of supercoiled plasmid DNA trapped in the interphase were quantified using a PhosphorImager. The amount of supercoiled plasmid DNA trapped by the wild-type enzyme in the presence of AMP-PNP and 0.8 M salt relative to the total amount of supercoiled plasmid DNA was set as 1.

DISCUSSION
The ability of eukaryotic topoisomerase II to change the topological conformation of DNA is based on a highly controlled communication between the individual subdomains in the dimeric enzyme, allowing transport of a DNA duplex through the whole intersubunit channel and through a second duplex hold and cleaved by the enzyme (19). In the present study, we investigated the interdomain communication between the Nterminal ATPase and the central cleavage/religation domains by studying a human topoisomerase II␣ enzyme that contained a deletion at the interface between the two domains, spanning amino acids 350 -407. In accordance with a lack of strand passage activity of the mutant enzyme, h⌬350 -407 was unable to sustain mitotic growth of a yeast top2 null strain. The mutant enzyme displayed a high ATPase activity and cleaved either suicide substrates or supercoiled plasmid DNA to wildtype levels. These results indicate that the enzyme consists of two functional domains that have lost their ability to coordinate their activities into strand passage. Further supporting an uncoupling of the N-terminal and central domain activities is the observation that the N-terminal ATPase activity was unaffected by DNA and that the cleavage/religation activity of the central domain was not stimulated by ATP. An examination of the clamp closing activity of the N-terminal domain revealed an inability of the mutant enzyme to close the clamp properly, strongly favoring the hypothesis that the mutant enzyme has lost its interdomain communication due to a failure in the generation and/or transmission of the correct conformational changes upon ATP binding and hydrolysis.
The region deleted in h⌬350 -407 constitutes a highly conserved domain of human topoisomerase II␣, implying that this region is very important for overall enzyme activity (9). This is further supported by results obtained from a linker insertion analysis, which showed that even a 2-amino acid linker inserted at position 350 or 407 was detrimental for enzyme activity, whereas similar insertions in regions flanking other highly conserved domains were tolerated by the enzyme (9). Based on homology to DNA gyrase, the deleted region constitutes the wall of a cavity existing in the dimeric form of the N-terminal region of Gyr B, as visualized after crystallization of this fragment of the gyrase enzyme (13). Assuming that the N-terminal region of Gyr B and human topoisomerase II␣ fold into similar structures, the outer skeleton of the enzyme would probably be left undisturbed after the deletion. The overall frame structure of the N-terminal region in the mutant enzyme would thus be kept intact, as also indicated from the ability of the enzyme to still efficiently hydrolyze ATP and cleave DNA. The size of the cavity in Gyr B is 20 Å, large enough to accommodate a DNA duplex, and it has been suggested that the cavity is a DNA binding pocket that binds the T-segment to be transported during the strand passage reaction (13). In light of this, h⌬350 -407 might suffer from an inability to interact properly with the T-segment, which eventually would disturb the whole communication pathway in the enzyme.
As revealed by biochemical and structural analyses of both eukaryotic topoisomerase II and the prokayrotic DNA gyrase, ATP binding and subsequent hydrolysis of one of the bound ATP molecules trigger a series of conformational changes resulting in N-terminal clamp closure and T-segment transport through the intersubunit channel (17,19,20). For the gyrase enzyme, the crystal structure of the N-terminal fragment of Gyr B has revealed that ATP upon binding contacts amino acids located in the wall facing the cavity encompassing the T-segment (13). These contacts were suggested to provide a mechanism for signaling ATP binding to the rest of the enzyme. Since this contact region is lacking in our deletion mutant, ATP binding and hydrolysis might not be sensed properly by the enzyme and transmitted further to allow correct T-segment binding and/or movements as well as further conformational changes in the enzyme.
Several studies performed either with fragments of DNA gyrase or eukaryotic topoisomerase II or with the full-length enzymes have shown a stimulatory effect of DNA on topoisomerase II-catalyzed ATP hydrolysis (22,24,(31)(32)(33)38). Although it is still unclear whether this stimulation is caused by T-and/or G-segment binding, an N-terminal fragment of human topoisomerase II covering amino acids 1-439 (and therefore lacking the ability to interact with the G-segment) displayed a DNA-stimulated ATPase activity. This result favors the involvement of the T-segment in stimulating ATP hydrolysis (22) and supports the hypothesis of a hampered T-segment interaction in h⌬350 -407, which displayed a DNA independent ATP hydrolysis although its interaction to the G-segment was normal. In a study of a gyrase mutant having a point mutation at Arg 286 located in the wall of the cavity in the N-terminal part of Gyr B, the enzyme was also suggested to have a disturbed T-segment interaction (38). The gyrase mutant showed several similarities to h⌬350 -407, in that it had a DNA-independent ATPase activity and cleaved DNA, but did not perform ATP-dependent DNA strand passage. Wang and co-workers (34) have recently shown that the stimulatory effect of DNA on the ATPase activity of topoisomerase II fragments is increased if the enzyme fragment besides the N-terminal ATPase domain also holds the BЈ region of the enzyme. Since this region normally is involved in G-segment binding (19), the result strongly indicates that the G-segment influences topoisomerase II-catalyzed ATP hydrolysis. To this end, Lindsley and co-workers (39) have recently suggested, based on results obtained with yeast topoisomerase II, that binding of the Gsegment per se primarily stimulates ATP binding to the enzyme, whereas binding of the T-segment stimulates ATP hydrolysis, so that both DNA segments affect the activities of the ATPase domain. h⌬350 -407 efficiently binds and cleaves the G-segment. A potential stimulatory effect of the G-segment on topoisomerase II-catalyzed ATP hydrolysis might in the mutant enzyme be prevented due to loss of communication between the N-terminal and central domains.
Although the ATPase activity of h⌬350 -407 is independent of DNA, the mutant enzyme had a very high rate of ATP hydrolysis compared with the wild-type enzyme. However, our study of the N-terminal clamp closing activity of h⌬350 -407 revealed that, in contrast to the wild-type enzyme, the mutant enzyme was unable to keep the clamp tightly closed in high salt, since only very limited amounts of DNA were trapped by the enzyme in the presence of an ATP analog. The dimerization of the N-terminal arms, which occurs upon ATP binding, has been suggested to be a prerequisite for ATP hydrolysis, since dimerization might bring together key residues in both halves of the enzyme, forming a pocket essential for ATP hydrolysis (13). However, our data on h⌬350 -407 demonstrate that ATP hydrolysis can occur efficiently in the absence of correct Nterminal dimerization.
The model recently presented by Lindsley on topoisomerase II action suggests that ATP hydrolysis occurs sequentially, where hydrolysis of the first ATP precedes transport of the T-segment and hydrolysis of the second ATP is responsible for enzyme turn over (40,41). The very high level of ATP hydrolysis observed in h⌬350 -407 can be interpreted in light of the lack of DNA strand passage activity in the mutant enzyme. Thus, due to the inability of the enzyme to perform DNA strand passage, it avoids the time lag generated as a consequence of the whole cascade of conformational changes it has to undergo from the time of ATP binding to ATP hydrolysis of the second ATP molecule. Our observations to some extent correlate with observations obtained previously by Lindsley and Wang (24) using yeast topoisomerase II, where they found that uncoupling of ATP hydrolysis and DNA strand passage due to high ATP concentrations correlated with an increased rate of ATP hydrolysis.
Besides the stimulatory effect of DNA on topoisomerase IIcatalyzed ATP hydrolysis, the communication between the Nterminal and central domains is also manifested during topoisomerase II-mediated DNA cleavage, where ATP shifts the DNA cleavage/religation equilibrium toward cleavage (25,42). In h⌬350 -407, however, the stimulatory effect of ATP on cleavage was lacking. Studies of topoisomerase II-mediated cleavage have revealed that two different cleavage/religation equilibriums exist (43). One is the pre-strand passage equilibrium, which occurs in the absence of ATP and therefore in the absence of strand passage. The other is the post-strand passage equilibrium existing in the presence of ATP or an ATP analog, which involves T-segment transport through a gate in the G-segment formed by a separation of the two active site tyrosines (43). The conformation of the enzyme is presumed to be different in the two situations. In the pre-strand passage equilibrium, the N-terminal arms most likely are in an open conformation and the tyrosines close together. However, in the post-strand passage equilibrium, the N-terminal arms might be closed and the tyrosines allowed to move back and forth between a closed and a widely separated conformation, thereby shifting the equilibrium toward cleavage. Our data on h⌬350 -407 are consistent with the mutant enzyme being unable to switch to the post-strand passage equilibrium in the presence of ATP. Rather, the mutant enzyme stays in the pre-strand passage equilibrium under all conditions.
In conclusion, the highly conserved region deleted in h⌬350 -407 is essential for correct two-way communication, which normally exists between the N-terminal and central cleavage/ religation domains in eukaryotic topoisomerase II, resulting in a loss of DNA strand passage activity. Thus, although the two domains retain their normal intrinsic activities, the DNA cleavage activity of the enzyme becomes independent of ATP, and the ATPase activity does not respond to DNA due to the lack of interdomain communication. The mutant enzyme is unable to perform the correct conformational changes upon ATP binding and hydrolysis as evident from an inability to perform a correct clamp closure. The enzyme might also suffer from a disturbed T-segment trapping, which could by itself hamper clamp closure and inhibit DNA strand passage.