Hindering the Strand Passage Reaction of Human Topoisomerase IIα without Disturbing DNA Cleavage, ATP Hydrolysis, or the Operation of the N-terminal Clamp*

DNA topoisomerase II is an essential enzyme that releases a topological strain in DNA by introduction of transient breaks in one DNA helix through which another helix is passed. While changing DNA topology, ATP is required to drive the enzyme through a series of conformational changes dependent on interdomain communication. We have characterized a human topoisomerase IIα enzyme with a two-amino acid insertion at position 351 in the transducer domain. The mutation specifically abolishes the DNA strand passage event of the enzyme, probably because of a sterical hindrance of T-segment transport. Thus, the enzyme fails to decatenate and relax DNA, even though it is fully capable of ATP hydrolysis, closure of the N-terminal clamp, and DNA cleavage. The cleavage activity is increased, suggesting that the transducer domain has a role in regulating DNA cleavage. Furthermore, the enzyme has retained a tendency to increase DNA cleavage upon nucleotide binding and also responds to DNA with elevated ATP hydrolysis. However, the DNA-mediated increase in ATP hydrolysis is lower than that obtained with the wild-type enzyme but similar to that of a cleavage-deficient topoisomerase IIα enzyme. Our results strongly suggest that the strand passage event is required for efficient DNA stimulation of topoisomerase II-mediated ATP hydrolysis, whereas the stimulation occurs independent of the DNA cleavage reaction per se. A comparison of the strand passage deficient-enzyme described here and the cleavage-deficient enzyme may have applications in other studies where a clear distinction between strand passage and topoisomerase II-mediated DNA cleavage is desirable.

Type II DNA topoisomerases are highly complex enzymes that are able to change the topological conformation of DNA by introducing a transient double strand break in one DNA helix, the G-segment, whereas another intact helix, the T-segment, is transported coordinately through the gated DNA (1). The process depends on ATP, which drives the enzyme through a series of conformational changes (2)(3)(4)(5).
The homodimeric eukaryotic topoisomerase II enzyme contains two highly conserved catalytic entities, which also share homology with the bacterial DNA topoisomerase II counterpart, DNA gyrase. The ATPase activity is found in the Nterminal region, whereas the DNA cleavage and ligation activities are held by the central region (6). The extreme C termini of the type II topoisomerases are divergent and dispensable for catalytic activity in vitro (7)(8)(9).
The structural data reveal that the N-terminal region of topoisomerase II forms an ATP-operated clamp, which closes upon ATP binding (10,11). The dimeric N-terminal clamp contains two domains in each protomer (10). The most Nterminal domain is responsible for dimer interactions during clamp closure and also holds the ATP-binding site. The second domain, called the transducer domain, forms the walls of the clamp and connects it to the enzyme core. Furthermore, communication between the ATP-binding domain and the central domain of the enzyme, responsible for DNA cleavage/ligation, has been suggested to go through the transducer domain (4,(12)(13)(14)(15). Upon clamp closure, the transducer domain of each protomer approaches one another, creating a very tight cavity that is actually too small to hold a T-segment (10). The surprisingly small hole of the eukaryotic topoisomerase II has been suggested to enable the enzyme to couple T-segment interactions to G-segment cleavage and opening (10).
The core region of topoisomerase II that is responsible for DNA cleavage and ligation is comprised of two domains, AЈ and BЈ, showing homology to the subunits of DNA gyrase, gyrase A and gyrase B, respectively (16). The AЈ domain contains both the active site tyrosine covalently attached to the 5Ј-end of the DNA during cleavage and the primary dimerization region (6,16). The BЈ domain constitutes the interface between the transducer domain and the AЈ domain (16) and is essential for DNA cleavage (16,17).
At low ATP concentrations, the ATP consumption and the DNA strand passage activity of DNA topoisomerase II are coupled tightly (18). The catalytic actions of both the cleavage/ ligation site and the ATPase site are governed by extensive interdomain communication, which probably facilitates an appropriate temporal order of conformational changes in the complex catalytic cycle of topoisomerase II and thereby reduces the energy usage. Thus, the binding of ATP to the N-terminal domain stimulates the cleavage of the G-segment bound by the core region (19), and the presence of DNA increases the turnover rate of ATP hydrolysis (2). Studies of a cleavage-deficient topoisomerase II enzyme have shown that an inability to cleave DNA severely reduces the stimulatory effect of DNA on ATP hydrolysis (20). In this enzyme, both DNA cleavage and strand passage were abolished. Therefore, it was suggested that the normal increase in ATP turnover either depends on the ability to cleave the G-segment or is mediated through T-segment binding/transfer (20). Thus, in contrast to ATP-mediated stimulation of DNA cleavage, which only requires ATP binding, a full increase in ATP hydrolysis seems to depend on a catalytic action on DNA rather than just DNA binding.
We have characterized a human topoisomerase II␣ enzyme with two amino acids inserted at position 351 in the transducer domain (351i). The insertion abolishes the strand passage reaction of the enzyme but leaves both catalytic domains active. Based on the newly published structure of the N-terminal fragment of yeast topoisomerase II (10), we find it very likely that the insertion impairs the strand passage reaction via sterical hindrance. Our results show that the insertion at position 351 slightly increases topoisomerase II-mediated DNA cleavage complex formation, consistent with a role of the transducer domain in controlling DNA cleavage (15). The biochemical characterization of 351i has revealed further that the enzyme retains DNA-stimulated ATP hydrolysis, although at a lower level compared with that of the wild-type enzyme. The stimulatory effect of DNA in 351i, which lacks strand passage activity, is equal to that obtained with a cleavage-deficient topoisomerase II␣ enzyme. Our results demonstrate that DNA-mediated stimulation of ATP hydrolysis is independent of DNA cleavage and rather requires the strand passage reaction per se.

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 analysis and overexpression of topoisomerase II enzymes, respectively. Plasmid pBY105 contains the yeast TPI promotor inserted into the polylinker region of the LEU2/ARS-CEN plasmid pRS315. pRS315 was used as the backbone for pHT300 and pHT351i (pHT350-2), carrying the wild-type human TOP2␣ cDNA and the human TOP2␣ cDNA with an insertion of 6 nucleotides encoding two amino acids (Val-Asp) at position 351, respectively. Modified versions of YEpWOB6 were used for overexpression of the hexahistidine-tagged human topoisomerase II␣ and 351i enzymes.
Construction of Plasmids-The construction of pHT300 and pHT351i (pHT350-2) was described by Jensen et al. (8). For the construction of the hexahistidine version of the human topoisomerase II␣ enzyme, see Bjergback et al. (4).
Yeast Transformation and Complementation-Yeast cells were transformed by using a modified version of the LiAc method of Ito et al. (21). To test the ability of 351i to complement the lack of endogenous topoisomerase II in BJ201, the LEU2-based construct pHT351i was transformed into BJ201 and cells were transferred to medium plates containing 5Ј-fluoro-orotic acid (1 mg/ml) to select against the URA3 plasmid carrying the Schizosaccharomyces pombe top2 ϩ gene (8). pHT300 was used as a positive control.
Topoisomerase II-mediated k-DNA Decatenation-k-DNA decatenation was performed by incubating the indicated amount of topoisomerase II with 150 ng k-DNA (TopoGEN) in a total volume of 20 l of buffer (50 mM Tris-HCl, pH 8, 140 mM KCl, 1 mM EDTA, 8 mM MgCl 2 , 1 mM ATP). Reactions were incubated at 37°C and stopped after 15 min by the addition of SDS to 1%. Samples were subjected to electrophoresis in a 1% agarose gel containing 1 g/ml ethidium bromide. Electrophoresis was performed in TBE (100 mM Tris borate, pH 8.3, 2 mM EDTA), and DNA was visualized by UV light.
Topoisomerase II-mediated DNA Relaxation-DNA relaxation was performed by incubating 1 or 10 nM topoisomerase II and 6.5 nM of supercoiled pUC19 DNA in a total volume of 20 l of buffer (50 mM Tris-HCl, pH 8, 140 mM KCl, 1 mM EDTA, 8 mM MgCl 2 supplemented with 1 mM ATP). The reactions were incubated at 37°C and stopped after 10 min by the addition of SDS and EDTA to a final concentration of 0.1% and 10 mM, respectively. The samples were subjected to electrophoresis in 1% agarose gels in TBE buffer. DNA was stained with 1 g/ml ethidium bromide and visualized by UV light.
Topoisomerase II-mediated Cleavage of Circular DNA-Topoisomerase II-mediated DNA cleavage was performed by incubating 100 nM topoisomerase II and 6.5 nM of negatively supercoiled pUC19 DNA in a total volume of 20 l of buffer (50 mM Tris-HCl, pH 8, 140 mM KCl, 1 mM EDTA, 8 mM MgCl 2 ). Samples were incubated at 37°C for 7 min, and cleavage products were trapped by the addition of SDS to 1%. After proteinase K digestion (0.8 mg/ml), samples were subjected to electrophoresis in 1% agarose gels in TAE buffer (40 nM Tris acetate, pH 8.3, 2 mM EDTA). DNA was visualized by ethidium bromide staining (1 g/ml). When topoisomerase II-mediated cleavage was carried out in the presence of ATP or AMP-PNP 1 (Roche Applied Science), the concentration of the nucleotide was 1 mM.
Oligonucleotides-DNA oligonucleotides were obtained from DNA Technology Corporation and purified by preparative polyacrylamide gel electrophoresis as described by Andersen et al. (22). The oligonucleotides used for suicide DNA cleavage and ligation were as described previously (15).
Topoisomerase II-mediated DNA Ligation-Hybridization and labeling of the synthetic oligonucleotides were done according to the procedures described by Andersen et al. (22). To study DNA ligation, kinetically competent topoisomerase II DNA cleavage complexes were generated by incubating 20 nM topoisomerase II with 0.02 pmol of labeled suicide substrate in 20 l of buffer (10 mM Tris-HCl, pH 7.0, 2.5 mM MgCl 2 , 2.5 mM CaCl 2 , 20 mM NaCl, 0.1 mM EDTA) at 37°C. After incubation for 60 min, the cleavage reactions were 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 4 pmol of a 45-mer ligation substrate. After further incubation (with incubation times as indicated in the figure legends), the reactions were stopped by the addition of SDS to 1% and subjected subsequently to SDS-polyacrylamide gel analysis. Covalent cleavage complexes were revealed by the transfer of the radiolabeled oligonucleotide to the topoisomerase polypeptide, and ligation was measured as the reduction in the amount of cleavage complexes with time. Reaction products were visualized and quantified using a Molecular Imager (Bio-Rad) Clamp-closing Assay-For clamp-closing experiments, 10 nM topoisomerase II was preincubated with 6.5 nM of negatively supercoiled pUC19 DNA for 5 min in a total volume of 20 l of buffer (50 mM Tris-HCl, pH 8, 140 mM KCl, 1 mM EDTA, 8 mM MgCl 2 ). After preincubation, AMP-PNP was added to a final concentration of 1 mM and the reactions were incubated further for 5 min. The reactions were stopped next 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 10 min. The waterphase was removed, ethanol-precipitated, and dissolved in 10 l of TE buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA) for gel analysis. The combined phenol phase and phenol/water interphase were washed once in high salt washing buffer (2 M NaCl, 50 mM Tris-HCl, pH 7.9, 5 mM MgCl 2 ) and once in 0.6 M LiCl. Upon the removal of the waterphase after the last wash, the remaining material was ethanol-precipitated and dissolved in 10 l of TE buffer containing 1 mg/ml proteinase K. The samples were subjected next to electrophoresis in TBE in a 1% agarose gel containing 1 g/ml ethidium bromide.
Clamp Closing Analyzed by Analytical CsCl Density Gradient Ultracentrifugation-The technique used is essentially that described previously by Morris et al. (20). Reaction mixtures of 30 l containing 150 nM topoisomerase II, 250 nM relaxed pBR322 (topoGEN), or linearized pUC19 plasmid DNA in sample buffer (50 mM Tris-HCl, pH 8, 60 mM NaCl, 1 mM EDTA, 8 mM MgCl 2 ) were incubated for 5 min at 30°C. AMP-PNP was added to a final concentration of 1 mM, and incubation was continued for 10 min. To each sample, 130 l of sample buffer and 334 l of saturated CsCl were added. The samples were run at 20°C and at 40,000 rpm in a Beckman XL-A analytical ultracentrifuge (Beckman Instruments). Scans were taken at 260 and 280 nm until equilibrium was reached (ϳ40 h).
Topoisomerase II-mediated Hydrolysis of ATP-The ATPase assays were performed using an ADP-sensitive-linked enzyme assay (23,24). Reactions contained 30 -200 nM topoisomerase II and, when indicated, 20 nM of negatively supercoiled pUC19 plasmid DNA. Reactions were carried out in volumes of 60 l containing 1.25 mM ATP, 50 mM Tris-HCl, pH 8, 110 mM KCl, 1 mM EDTA, 8 mM MgCl 2 , 0.4 mM phosphoenolpyrovate, and 0.2 mM NADH, 1.5 l of pyruvate kinase/lactate dehydrogenase (Sigma). Reactions were initiated by the addition of enzyme and monitored spectrophotometrically for 10 min at 37°C (Uvicon 923 spectrophotometer). The rate of ATP hydrolysis was monitored as the rate of oxidation of NADH and determined by the absorbance decrease at 340 nm.

Insertion of Two Amino Acids at Position 351 in Human
Topoisomerase II␣ Abolishes the Strand Passage Activity of the Enzyme-In a previous study performed by Jensen et al. (8), it was observed that insertion of two amino acids at position 351 in human topoisomerase II␣ disrupted the ability of the enzyme to complement growth of a yeast strain in which the endogenous topoisomerase II gene was deleted. Fig. 1A verifies the complementation inability of the mutant enzyme (351i). The alignment with the crystallized S. cerevisiae topoisomerase II fragment suggests that the insertion in 351i is situated at the inner wall of the transducer domain in the DNA transport path of the enzyme as shown in Fig. 1B. To determine the underlying causes of the complementation inability, 351i was purified to near homogeneity as seen from the Coomassie Bluestained gel in Fig. 1C and subjected to biochemical analysis. To investigate the DNA strand passage activity of the mutant enzyme, a decatenation assay was performed first (Fig. 1D,  upper panel). Whereas wild-type topoisomerase II␣ under the given reaction conditions decatenated k-DNA, no release of mini-circles occurred with 351i even at high enzyme concentrations. A similar lack of DNA strand passage activity was observed in a DNA relaxation assay (Fig. 1D, lower panel) and in a DNA cleavage assay performed in the presence of ATP ( Fig.  2A, lane 6). In the latter experiment, the enzyme was present in an ϳ15-fold excess, which excludes that 351i can perform even a single round of strand passage. Thus, consistent with the lack of in vivo complementation, 351i has lost its overall catalytic activity.
351i Has Retained DNA Cleavage Activity-Topoisomerase II is a multidomain enzyme, which is capable of both ATP hydrolysis and of DNA cleavage/ligation. The two activities reside in different regions of the enzyme and can work independent of each other; however, a strict coordination is required to obtain topoisomerase activity. Coordination of the two catalytic activities of topoisomerase II is obtained through domain communication. When investigating topoisomerase II-FIG. 1. Human topoisomerase II␣ containing an insertion of two amino acids at position 351 is catalytic-inactive and fails to complement growth in a yeast top2 deletion strain. A, a single copy ARS/CEN plasmid carrying either the 351i (pHT351i) or the wild-type human topoisomerase II␣ (pHT300) cDNA behind a TPI promotor was transformed into the yeast strain BJ201. As a control, the cells were transformed with the empty vector (pRS315). In BJ201, the chromosomal TOP2 gene has been disrupted by the insertion of the structural TRP1 gene, whereas the essential topoisomerase II activity is provided by the S. pombe top2 ϩ gene carried on a single copy URA3-based plasmid. After transformation, cells were grown on medium containing 5Ј-fluoro-orotic acid to counterselect against the URA3 plasmid. B, the structure of the N-terminal fragment of yeast topoisomerase II (10) showing the location of position 340 (spheres, indicated with arrows), corresponding to position 351 in human topoisomerase II␣. The figure is prepared with PYMOL (26). C, purification of 351i and wild-type human topoisomerase II␣. The enzymes were overexpressed in yeast and purified through three different steps involving Ni 2ϩ -nitriloacetic acid-agarose, heparin-Sepharose, and phosphocellulose column chromatography. The homogeneity of the enzyme preparations was determined in a 10% SDS-polyacrylamide gel stained with Coomassie Blue. Protein size markers are indicated to the left of the gel. Bovine serum albumin (BSA) has been loaded as a marker for protein concentration. D, upper panel, to investigate DNA strand passage activity, k-DNA and topoisomerase II were incubated in the presence of ATP at 37°C for 15 min. The reactions were stopped by adding SDS to 1% and analyzed on an agarose gel. The enzyme and enzyme concentration used in the reactions are denoted above the lanes. The positions of k-DNA (kDNA) and the mini-circles are indicated. Lower panel, DNA relaxation was investigated by incubating DNA and topoisomerase II at 37°C for 10 min in the presence of ATP. The reactions were stopped by adding SDS and EDTA to 0.1% and 10 mM, respectively, and analyzed on an agarose gel. The enzyme and enzyme concentration used in the reactions are indicated above the lanes. The positions of supercoiled (SC), relaxed (R), and catenated (Cat) plasmid DNA are indicated. wt, wild-type. mediated DNA cleavage, domain communication can be revealed by the addition of a nucleotide that binds to the N terminus of the enzyme and thereby increases the DNA cleavage activity of the cleavage/ligation domain (19,25).
To test whether the lack of topoisomerase II activity seen with 351i is caused by an inability of the enzyme to perform DNA cleavage, 351i and the wild-type enzyme were incubated first with supercoiled plasmid DNA in the absence of nucleotide. The reactions were stopped by SDS, which immediately denatures the enzyme. After proteinase K treatment, cleavage products were separated from uncleaved plasmid DNA by agarose gel electrophoresis ( Fig. 2A, upper panel, compare lanes 2  and 5). In the absence of the nucleotide, 351i actually displays a DNA cleavage level higher than that of the wild-type enzyme as also shown in the histogram in Fig. 2A, lower panel. The result shows that the mutant enzyme indeed has retained its cleavage activity.
The addition of the ATP analog, AMP-PNP, to the cleavage reaction increases the DNA cleavage level obtained by 351i ( Fig. 2A). However, the stimulatory effect of the nucleotide is low compared with the effect obtained with the wild-type enzyme. In the presence of ATP, the wild-type enzyme relaxes the DNA. Even with these high enzyme concentrations, ATP only allows 351i to cleave DNA in support of its lack of overall catalytic activity.
In the assay described above, the DNA cleavage level reflects an equilibrium between topoisomerase II-mediated DNA cleavage and ligation. To investigate the DNA ligation ability of 351i, an advantage was taken of the topoisomerase II suicide system, which allows a separation of the DNA cleavage and ligation half-reactions (Fig. 2B, upper panel) (22). Since the topoisomerase II cleavage complex generated upon cleavage of a suicide substrate is kinetically competent (22), it is able to perform ligation if a suitable ligation substrate is added to the cleavage mixture.
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. Ligation reactions were stopped with SDS at different time points and subjected to electrophoresis in an 8% SDS gel. The cleavage complexes were identified because of the covalent linkage of labeled DNA, whereas ligation was revealed as a disappearance of cleavage products upon the addition of ligation substrate (Fig. 2B,  middle panel).
The results obtained in the ligation assay shows that 351i and the wild-type enzyme have very similar rates of ligation (Fig. 2B, lower panel). Thus, the slightly increased DNA cleavage level obtained by 351i is probably not the result of a lower ligation rate of this enzyme.
The results obtained in the DNA cleavage and ligation assays revealed that the insertion of two amino acids at position 351, although detrimental to overall enzyme catalysis, does not stopped by the addition of NaCl to 0.4 M, and ligation was initiated by the addition of a 45-mer ligation substrate. Samples were withdrawn at the indicated time points and subjected subsequently to electrophoresis in an 8% SDS gel and visualized on a Molecular Imager (Bio-Rad). DNA ligation is revealed as a disappearance of cleavage complexes due to release of topoisomerase II from the labeled oligonucleotide upon ligation to the 45-mer ligation substrate.  topoisomerase II suicide system. Middle panel, topoisomerase II-mediated DNA ligation was tested using the topoisomerase II suicide system. In this system, kinetically competent topoisomerase II-DNA cleavage complexes were formed by incubating topoisomerase II and a substrate designed to prevent ligation due to the release of a trinucleotide 5Ј to the cleavage position on the top strand upon DNA cleavage. Cleavage reactions were disrupt the DNA cleavage/ligation domain of topoisomerase II␣ and does not abolish interdomain communication as measured by nucleotide-stimulated DNA cleavage. Thus, the lack of strand transfer in 351i is not because of an inability of the enzyme to mediate DNA cleavage/ligation.
The N-terminal Clamp of 351i Is Functional-One of the essential steps in the catalytic cycle of topoisomerase II involves the closure of the ATP-operated N-terminal clamp, whereby a Tsegment may be trapped for subsequent transport through the G-segment. To test whether 351i has retained this essential function, a clamp-closing assay was performed (modified from Bjergbaek et al. (4)). In the assay, topoisomerase II was preincubated with supercoiled plasmid DNA followed by the addition of the non-hydrolyzable ATP analog, AMP-PNP. When the wildtype enzyme was used in the assay, this led to the formation of a salt-stable enzyme-DNA-interlinked complex, which was collected readily from a phenol-water interphase (Fig. 3A). A similar result was obtained with 351i, showing that it is able to keep circular DNA trapped in high concentrations of salt. Thus, the insertion of two amino acids at position 351 does not disturb the ability of human topoisomerase II␣ to undergo stable clamp closure. None of the enzymes allowed the trapping of DNA upon the addition of SDS, which disrupts the interlink between topoisomerase II and DNA, or in the absence of AMP-PNP where stable clamp closure does not occur.
To confirm that 351i retains a normal clamp function, clamp closure was analyzed using analytical CsCl density gradient centrifugation as described under "Experimental Procedures." The assay takes advantage of the sedimentation differences of clamp complexes and free DNA in CsCl because of the different densities of protein and DNA. Free DNA will migrate to the bottom of a CsCl gradient, whereas the lighter species of DNA complexed with protein will sediment in the upper part of the gradient. In the presence of AMP-PNP, a peak was revealed in the upper part of the CsCl gradient both with 351i and the wild-type enzyme, demonstrating that both enzymes under these conditions form a stable complex with circular DNA (Fig.  3B, upper panel). Two control reactions also were performed. In one reaction, the circular DNA was substituted with linear DNA, which cannot be linked topologically to topoisomerase II (Fig. 3B, middle panel), and in another, AMP-PNP was excluded, preventing stable closure of the topoisomerase II clamp (Fig. 3B, lower panel). The assay revealed that the wild-type enzyme and 351i are able to generate similar amounts of the salt-stable closed clamp complex in the presence of the nonhydrolyzable ATP analog, AMP-PNP, and therefore confirmed that 351i retains a functional N-terminal clamp. Thus, the lack of strand transfer observed in 351i is not the result of a malfunction of the N-terminal clamp.
351i Has Retained Intrinsic as Well as DNA-stimulated ATPase Activity-The catalytic activity of topoisomerase II is dependent on ATP hydrolysis. Thus, even though a non-hydrolyzable ATP analog can lead to clamp closure, hydrolysis is required for consecutive strand passage events. To test whether the strand passage defect of 351i is attributed to an inability of the enzyme to hydrolyze ATP, 351i and wild-type FIG. 3. 351i retains a functional N-terminal clamp. A, 351i or wild-type human topoisomerase II␣ was preincubated for 5 min with plasmid DNA before the addition of AMP-PNP. After 5 min of further incubation, the reactions were stopped by the addition of either NaCl or SDS to 800 mM and 1%, respectively. The samples then were phenolextracted. The waterphase was removed and ethanol-precipitated, whereas the remaining phenol/water interphase was washed in high salt before the material was ethanol-precipitated, proteinase K-digested, and subjected to electrophoresis in a 1% agarose gel containing ethidium bromide next to the waterphase samples. Enzyme, nucleotide, and stop solutions used in the samples are indicated above the gel. i and w denote interphase and waterphase, respectively. SC and Cat indicate the position of supercoiled and catenated plasmid DNA, respectively. Catenated DNA also becomes topologically trapped by topoisomerase II because of its circular form. B, for the analysis of clamp closure by analytical ultracentrifugation, enzyme, DNA, and AMP-PNP were incubated as described under "Experimental Procedures." The samples were loaded on a CsCl gradient and spun for 40 h. Absorbance traces at 260 nm of the CsCl density gradients are shown for each reaction. The major peak at the bottom of the gradient corresponds to DNA without any protein bound, whereas the peak in the upper part of the gradient represents enzyme bound to one plasmid. The enzyme bound to catenated plasmids sediments as a minor peak in the middle of the gradient. The small peak observed in the control experiment with linear DNA is due to a contaminating fraction of non-linearized plasmid DNA in the linear DNA preparation. wt, wild-type. human topoisomerase II␣ were subjected to an NADH-coupled ATPase assay (Fig. 4A, upper panel) (23,24). The intrinsic ATPase activities of the enzymes were measured first in the absence of DNA. The results, which are shown in Fig. 4A, lower  panel (columns 1 and 3), demonstrate that this activity is not influenced by the two-amino acid insertion at position 351.
Optimal coordination of the catalytic steps in DNA topoisomerase II requires a two-way communication between the N-terminal and core domains. Besides the ATP-stimulated DNA cleavage, normal topoisomerase II operation also requires a DNA-stimulated ATPase activity. To test whether DNA still stimulates the ATPase activity of 351i, the ATPase activities of 351i and the wild-type enzyme were also probed in the presence of supercoiled plasmid DNA. Both enzymes displayed a DNAmediated stimulation of the ATPase activity. In the present study, the addition of DNA stimulated the ATPase activity of the wild-type topoisomerase II␣ ϳ14-fold, whereas 351i displayed a ϳ4-fold stimulation (Fig. 4A).
The results show that 351i has retained intrinsic ATPase activity and is also able to respond to the presence of DNA with increased ATP hydrolysis. Therefore, the lack of topoisomerase II activity in 351i is not due to a defective ATPase activity.
Taken together, the insertion at position 351i specifically hinders the DNA strand passage step of human topoisomerase II␣ without disturbing DNA cleavage, operation of the N-terminal clamp, and ATP hydrolysis.
DNA Strand Passage and Not DNA Cleavage Is Important for the DNA-mediated Stimulation of ATP Hydrolysis in Topoisomerase II-The exact events leading to DNA-mediated stimulation of the ATPase activity of topoisomerase II are not known. However, the ATPase activity of a cleavage-deficient yeast topoisomerase II has been shown to display a reduced stimulation upon the addition of DNA compared with that of the wild-type enzyme (20). This finding suggests that either DNA cleavage or the DNA strand passage reaction is required to obtain efficient stimulation.
To test whether DNA cleavage or DNA strand passage plays a role in the stimulatory mechanism, the ATPase activity of the strand passage-deficient but cleavage-competent 351i enzyme was compared with that of a cleavage-deficient human topoisomerase II␣ enzyme, Y805S. In the latter enzyme, the active site tyrosine has been substituted with a serine and the enzyme can neither perform DNA relaxation nor DNA cleavage to a detectable level (29). The extent of stimulation obtained with Y805S is very similar to that obtained with 351i, showing that the DNA cleavage reaction per se does not contribute significantly to the stimulation of topoisomerase II-mediated ATP hydrolysis (Fig. 4B). Taken together with the results obtained with the wild-type enzyme (Fig. 4A), which on top of the activities held by 351i can perform strand passage, efficient DNAmediated stimulation of ATP hydrolysis in topoisomerase II requires the DNA strand passage reaction per se.

DISCUSSION
Topoisomerase II is a multidomain enzyme, which is capable of both ATP hydrolysis and DNA cleavage/ligation. The two activities reside in different regions of the enzyme and can work independent of each other. However, a coordination is required to obtain topoisomerase activity (4). Communication between the ATPase domain and the central domain of the enzyme responsible for DNA cleavage/ligation seems to go through the transducer domain (4,(13)(14)(15). The conformational changes caused by ATP binding to the N-terminal region thus reach all the way to the core domain and also change the catalytic features of the DNA cleavage/ligation reaction of the enzyme. Likewise, DNA interactions stimulate the ATPase activity of topoisomerase II, but in this case, the scenario seems more complex because DNA binding per se is not sufficient to efficiently increase the ATPase activity (20). The shift in ATPase turnover rather requires an enzymatic action on DNA.
In this study, we have characterized a human topoisomerase II␣ enzyme bearing a two-amino acid insertion at position 351 in the transducer domain. The insertion is situated in the core proximal part of the inner wall suggested to interact with the T-segment prior to its passage through the G-segment (10,27). The characterization has revealed that this enzyme, although capable of N-terminal clamp closure, ATP hydrolysis, and DNA cleavage/ligation, still lacks strand passage activity. 351i has retained a certain extent of interdomain communication. First, AMP-PNP stimulates 351i-mediated DNA cleavage slightly, demonstrating that the enzyme has preserved a tendency to increase the cleavage level upon nucleotide binding, either by stabilizing the cleaved conformation or by increasing the forward cleavage rate. Second, DNA stimulates the ATPase activity of 351i.
We previously characterized a human topoisomerase II␣ enzyme bearing an insertion at position 408 in the transducer domain (15), which based on alignment with the crystallized N-terminal fragment from yeast topoisomerase II is located close to position 351 in the tertiary structure. However, the two insertions result in enzymes with very different phenotypes where only 351i is able to perform clamp closure. This finding suggests that the defects observed in the two mutant enzymes do not result from a misfolding of the entire transducer domain. Rather, the two insertions cause a specific local disturbance resulting in distinct enzymatic defects. Whereas the insertion at position 408 interferes with the clamp function of topoisomerase II, the insertion at position 351 abolishes strand passage activity and leaves the clamp unaffected. Together with the proposed localization of 351 at the inner part of the clamp arms, our results suggest that the insertion disturbs the strand passage reaction per se. This can be attributed to a lack of T-segment interaction of the enzyme in the open clamp conformation, but for two reasons, we find it more likely that it is the result of a direct impair of the transport event. First, according to alignment with the structure of the N-terminal yeast fragment, position 351 in human topoisomerase II␣ is located at the bottom of the cavity formed upon clamp closure in close proximity to the same position in the other subunit.
The T-segment normally can be transported through this narrow passage, but insertion of two amino acids is likely to cause a sterical hindrance of this event. Second, the N-terminal part of topoisomerase II only has very weak DNA interactions that are difficult to detect by biochemical analyses (23). Thus, it seems that the T-segment is presented to the clamp mainly by coincidence, and therefore, a disrupted T-segment interaction would not be expected to result in the complete inhibition of strand passage as is the case in 351i.
351i is able to cleave plasmid DNA, showing that the G-segment can easily access the DNA binding site in the core of the enzyme. Thus, the insertion apparently only introduces a hindrance to the transport of the T-segment, probably because this event occurs right upon clamp closure where the transducer domain of each subunit is oriented differently from what it is during G-segment entering. The DNA cleavage level displayed by 351i in the absence of nucleotide slightly exceeds that of the wild-type enzyme. This catalytic trait is shared with the enzyme bearing an insertion at position 408 in the transducer domain (15) and indicates that an important function of the transducer domain is to control the cleavage level of the core domain.
DNA only stimulates the ATPase activity of 351i ϳ4-fold as compared with the ϳ14-fold stimulation seen with wild-type topoisomerase II. However, the level of DNA-mediated stimulation is not significantly different from that obtained with a cleavage-deficient topoisomerase II. With the latter enzyme a ϳ3-fold stimulation was obtained, which is similar to the stimulation seen with a cleavage-deficient yeast topoisomerase II as reported by Morris et al. (20). Because these cleavage-deficient enzymes can neither perform DNA cleavage nor strand passage, the remaining stimulatory effect of DNA on the ATPase activity in these enzymes probably reflects the stimulatory effect of DNA binding per se. Our observations that 351i and Y805S have similar ATPase activities lead us to conclude that the ATPase activity is not stimulated through the DNA cleavage reaction. This is in agreement with studies of a DNA gyrase enzyme in which the DNA gate was locked by cysteine crosslinking. The gyrase enzyme was able to cleave DNA, but still no DNA stimulation of the ATPase activity of the enzyme occurred (28). Thus, the step(s) leading to the pronounced increase in ATP hydrolysis is limited to the events that are very tightly coupled to DNA strand passage, most probably T-segment interactions that occur during the transport event. Alternatively, G-segment opening may be essential for the stimulatory effect of DNA, because we cannot completely rule out that 351i is unable to enter this conformation.
In conclusion, we have identified a human topoisomerase II␣ mutant enzyme, where the DNA strand passage reaction is inhibited, neither due to a lack of clamp closure, a defect in the ATPase domain or due to an active site mutation that inhibits DNA cleavage. Rather, the strand passage reaction per se has been abolished by a two-amino acid insertion at position 351, which seems to block the normal DNA transport path in topoisomerase II by a sterical hindrance. The mutant enzyme thus may have further applications in studies where a clear distinction between DNA cleavage and strand passage is desired. Our results with 351i have suggested strongly that the strand passage reaction of topoisomerase II is required for efficient DNAmediated stimulation of the ATPase activity of topoisomerase II, whereas this stimulation is independent of topoisomerase II-mediated DNA cleavage. Furthermore, the studies have confirmed that changes in the transducer domain are likely to influence the DNA cleavage level of the enzyme.