Identifying Lys359 as a Critical Residue for the ATP-dependent Reactions of Drosophila DNA Topoisomerase II*

Tao Hu, Steve Chang, and Tao-shih HsiehDagger

From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Substituting Lys359 with either Gln or Glu in the highly conserved QTK-loop in the DNA gyrase B protein homologous domain of Drosophila topoisomerase II inactivates its catalytic activities. Although strand passage and DNA-dependent ATPase activities are affected in these mutant proteins, their DNA cleavage activity is comparable with the wild-type enzyme and can be stimulated to the same level by topoisomerase-targeting anticancer drugs. The sequence specificity in the DNA cleavage reaction remains unaltered for the mutant proteins. We have used both glass fiber filter binding assay and CsCl density gradient ultracentrifugation to monitor the formation of a salt-stable, protein-clamp complex. Both Gln and Glu mutant proteins can form a clamp complex in the presence of 5'-adenylyl-beta ,gamma -imidodiphosphate, albeit with a lower efficiency than the wild-type enzyme. However, the mutant proteins can form a stable complex either in the presence of ATP or in the absence of any cofactors. These results are in an interesting contrast with the wild-type enzyme, which cannot form a stable complex under similar conditions. Our data suggest that Lys359 is critical for the catalytic activity of topoisomerase II and may have an important function in the ATP signaling process.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Type II DNA topoisomerases are ubiquitous enzymes that have critical functions in many aspects of DNA metabolism, including replication, transcription, recombination, and genome stability (reviewed in Ref. 1). It is dimeric in structure and can pass DNA through a transient DNA gate generated by reversible transesterification from a pair of active site tyrosine residues. In vitro, topoisomerase II (topo II)1 catalyzes the reaction of DNA supercoiling and relaxation, catenation/decatenation, and knotting and unknotting of DNA rings. In vivo, the essential biological functions of topo II are in the segregation of replicated and topologically interlocked chromosomes (Ref. 2; also reviewed in Ref. 3). Both eukaryotic and prokaryotic topo II share very similar biochemical mechanisms and are related by strong sequence homologies (4). The N-terminal and central domains of the eukaryotic topo II subunit are homologous to the bacterial gyrB and gyrA subunits, respectively. Recent high-resolution structural data suggest that the sequence homology between eukaryotic and bacterial topo II can be translated into structural similarity (5, 6).

The biochemical mechanism of topo II in freely passing DNA helices through each other is no less complicated than those described for other macromolecular machineries. The reaction starts with the binding of a DNA segment to the enzyme surface at the active site tyrosines. This stretch of DNA will serve as a protein-mediated gate and is termed the G segment. Upon the binding of ATP, a second segment of DNA is captured by the closure of the topo II N-terminal protein clamp, known as the N-gate (7), and transported through the G segment. The binding of ATP to eukaryotic topo II also induces an allosteric change in the N-terminal domain in concert with the clamp closure (8, 9). After the passage of DNA through the reversible break in the G segment, the transported DNA helix is held and then expelled from the enzyme through the C-terminal protein clamp, known as the C-gate, formed by the gyrA homologous domain (10, 11). The hydrolysis of ATP in this reaction allows the turnover of the catalytic cycle (12-14). Although ATP binding can trigger the closure of N-gate, its role in other steps of the catalytic cycle, such as the strand passage and exit of DNA from the C-gate, remains to be delineated. Furthermore, the topoisomer distribution in the topo II-catalyzed reactions including supercoil relaxation, catenation, and knotting is apparently below the equilibrium position (15). It is likely that the free energy from the hydrolysis of ATP concomitant with topo II-mediated strand passage reactions is the driving force to bias the chemical equilibrium. How this is achieved in biochemical and mechanistic terms is yet to be determined.

We are interested in identifying important residues in the ATP-dependent reactions of topo II and analyzing the biochemical consequences of mutations at these residues. Such information provides insight into the mechanism of topo II reactions and guides the future experiments to test various hypotheses. We report here the result from site-specific mutagenesis of a conserved Lys359 that is situated in close proximity to the gamma -phosphate of bound ATP. Although the double strand DNA cleavage reaction is not affected by the mutations tested, Lys359 mutants have greatly reduced catalytic activities and have altered clamp closure response with respect to ATP cofactors. Thus, Lys359 is implicated in the ATP-triggered reactions of topo II.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Site-directed Mutagenesis-- The expression of Drosophila topo II was driven by the GAL1 promoter in a YEp24-based yeast vector (16). Site-directed mutagenesis was carried out by the method described by Kunkel (17). Three 21-mer oligonucleotides, covering the top2 cDNA nucleotides 1325-1345, contain sequence variations of AGG (Arg mutant), GAG (Glu mutant), and CAG (Gln mutant) in place for the original Lys359 codon AAG. DNA sequences were determined surrounding the mutagenic site to confirm that only the desired mutation was introduced.

Yeast Complementation Assay-- Complementation of yeast top2ts alleles by Drosophila top2 was carried out as described (16). The growth of yeast cells at a nonpermissive temperature of 37 °C in medium containing galactose demonstrated the expression of a functional Drosophila top2. The color sectoring assay was performed to test the ability of wild-type and mutant Drosophila top2 to complement a yeast top2 null mutation (18). The formation of red/white sectored colony indicated that a Drosophila topo II construct was functional and could replace the only functional yeast TOP2 on the plasmid (19).

Purification of Wild-type and Mutant Topo II-- Drosophila topo II was overexpressed in yeast BCY123 (pep4-, prb1-, bar1-, lys2::GAL1-GAL4, ura3-; a generous gift from Dr. Janet Lindsley, University of Utah). The expression of Drosophila top2 constructs were induced in YEP medium containing 2% galactose. An 8-liter culture of yeast cells overproducing Drosophila topo II was harvested and lysed as described in Ref. 20. The cleared lysate was loaded onto a 100-ml of Bio-Rex 70 (Bio-Rad) column. The column was eluted by a NaCl gradient in Buffer P (15 mM NaDi, pH 7.0, 0.1 mM EDTA, and 10% Glycerol), with topo II coming off at 0.5 M NaCl. The active fractions were then pooled, diluted 1:1 with 0.1 M Tris-HCl (pH 8.0), and loaded onto a 7.8-ml Poros HQ column (PerSeptive Biosystems, Framingham, MA). The column was eluted by a NaCl gradient in 20 mM Tris-HCl (pH 8.0), and topo II was eluted at 0.8 M NaCl. The peak fractions were combined, diluted 4-fold with 50 mM NaPi (pH 8.0), and loaded onto a 1.7-ml heparin column (Poros HE, PerSeptive Biosystems). The Poros HE column was eluted by a NaCl gradient in 10 mM NaPi (pH 8.0). The peak fractions eluted at 0.3 M NaCl were then pooled and dialyzed against the storage buffer containing 15 mM NaPi, pH 7.0, 0.1 mM EDTA, 0.1 M NaCl, and 50% glycerol for 16 h. 0.1 mM of dithiotreitol and a mixture of protease inhibitors including 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, and 1 µg/ml pepstatin A were included in all solutions used in the purification procedures. Protein concentrations were determined by Coomassie dye-binding assay (21), and purification at each step was monitored by SDS-PAGE (22).

CsCl Density Gradient Ultracentrifugation-- A 40-µl reaction mixture containing 10 mM Tris-HCl, pH 8.0, 50 mM KCl, 100 mM NaCl, 10 mM MgCl2, 0.1 mM EDTA, 50 µg/ml bovine serum albumin, 25 nM of pCaSpeRhs83 DNA (circular or linearized), and 75 nM of topo II proteins was incubated at 30 °C for 15 min. Some reactions also included 0.5 mM of a nucleoside triphosphate cofactor (ATP or AMPPNP). The reaction was terminated by adding a chilled mixture of 330 µl of saturated CsCl solution and 107 µl of 10 mM Tris-HCl, pH 8.0, to make the final density of the solution 1.65 g/ml. The mixture was spun at 40,000 rpm in an analytical ultracentrifuge (XL-A ultracentrifuge, Beckman Instruments) at 20 °C for 36 h before scanning the DNA concentrations across the gradient at wavelengths of 260 and 280 nm. The density difference between protein/DNA complex and DNA was used to calculate the protein molecular weight in the salt-stable complex (23). The plot of (1/rho c-n - 1/rho d)/(1/rho p - 1/rho c-n)·MWd versus n produced a straight line with slope MWp, the molecular weight of protein moiety in the complex. In this equation, n is the number of protein molecules bound in the complexes, MWd is the DNA molecular weight, and rho p, rho d, and rho c-n are the buoyant densities of protein, DNA, and a complex of DNA bound with n protein molecules, respectively.

Other Methods-- Topo II strand passage activities were tested by the unknotting of P4 knotted DNA, supercoiled relaxation, catenation, and decatenation assays as described in (24). ATPase assay was done as described in Ref. 25 except that a 8.6-kilobase plasmid DNA, pCaSpeRhs83 (26), was used, and the radioactivities were quantified by PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Topo II-mediated double-stranded DNA cleavage was carried out using plasmid DNA substrate as described in (27). The uniquely end-labeled DNA containing the intergenic region of heat shock gene HSP70 was used to map the topo II cleavage sites as described earlier (28). Protein-mediated binding of DNA to GF/C glass fiber filters (29) was done as described by Roca and Wang (7). The radiolabeled DNA substrate used in the filter binding assays was generated by nick translation of nicked pHC 624 (30) carried out in the presence of [alpha -32P]TTP (0.5 Ci/mmol) for 1 h followed by religation with T4 DNA ligase in the presence of 5 µg/ml ethidium bromide for 3 h. The mixture was then phenol extracted and run through a Sephadex G-50 spin column (Amersham Pharmacia Biotech) to remove the unincorporated nucleotides. Half of the labeled DNA sample was linearized by the restriction enzyme EcoRI. An equal mixture of linear and circular DNA samples was used in each assay.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Lys359 Mutants of Drosophila Topoisomerase II-- The x-ray crystal structure of the 43-kDa N-terminal fragment of gyrB protein reveals a two-domain structure: the N-terminal half (residues 2-220) is the ATP-binding moiety and the C-terminal half (residues 221-392) forms a 20-Å cavity (31). Examining the cavity-forming domain shows that a loop with a sequence of QTK (residues 335-337) extends away from the rest of the domain and is in close contact with gamma -phosphate of the bound ATP analog (Ref. 31; see Fig. 1A). The N---O distance between the epsilon -amino group in Lys337 and gamma -phosphate in ATP is 2.6 Å, suggesting that either hydrogen bonding or ionic interaction could exist between them. It is also interesting to note that QTK and the sequence surrounding it are highly conserved among topo II (Fig. 1B).


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Fig. 1.   A, AMPPNP and some of its interacting residues in gyrB. The diagram was drawn with Kinemage software (51) using the coordinates from the crystal structure of a 43-kDa E. coli gyrB fragment complexed with AMPPNP (31). AMPPNP and the Lys337 side chain are shown in black. The side chains from other residues (Glu42, Lys103, Gln335, and Thr336) and backbone C-alpha s are in dark gray and light gray, respectively. The dotted line marks the 2.6-Å interatomic distance from epsilon -amino nitrogen to the gamma -phosphate oxygen in AMPPNP. Earlier works demonstrated that mutations in Glu42 (45) or Lys103 (50) inactivate DNA gyrase. B, amino acid sequence alignment surrounding Lys337 of gyrB. The highly conserved residues, either identities or homologous substitutions, are boxed for sequences of gyrB from E. coli (EcGyrB) and Bacillus subtilis (BsGyrB), gene 39 protein from phage T4 topo II, human topo IIalpha (HumA), and topo II from Saccharomyces cerevisiae (Sc), Schizosaccharomyces pombe (Sp), and Drosophila melanogaster (Dm). The Lys359 substitution mutants reported in this work are given under the sequence for Dm.

To test the importance of the lysine residue in the QTK loop in topo II reactions, we generated site-specific mutations converting lysine to arginine, glutamine, and glutamate (see "Experimental Procedures"). The homologous residue of Lys337 of gyrB is Lys359 in Drosophila enzyme (Fig. 1B). The activity of the site-specific mutants was first tested by genetic complementation assays that were based on our previous observation that a functional Drosophila top2 gene could rescue either a conditional yeast top2 mutation (16) or a null mutation (18). In these in vivo functional assays, the K359R mutant is active but K359Q and K359E are not (data not shown). These results are also confirmed by the topoisomerase assays in which the crude extracts yeast cells expressing K359R are active, whereas those expressing K359Q and K359E are inactive (data not shown). Therefore, both in vivo and in vitro assays suggest that lysine is critical for topoisomerase functions; a homologous substitution with arginine does not significantly alter topoisomerase activities, and mutations converting it into glutamine and glutamate result in a loss of its activities.

Purification of K359Q and K359E Mutant Proteins and Their Biochemical Characterizations-- To analyze the effects of substitutions of Lys359, we have purified wild-type topo II and K359Q and K359E mutant proteins from overproducing yeast cells. The cleared extracts were fractionated on a cation-exchange column (BioRex-70), followed by anion exchanger chromatography (Poros HQ), and affinity chromatography (Poros HE). Wild-type and mutant proteins all had identical elution profiles in these chromatographic steps (see "Experimental Procedures") and were purified to over 90% homogeneity (Fig. 2).


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Fig. 2.   Purification of wild-type and mutant Drosophila topo II. 2 µg each of molecular weight size markers, wild-type, and two Lys359 mutant proteins were run in a 7% Laemmli gel (22) and stained with Coomassie Blue.

The DNA strand passage activity of these proteins were determined by the P4 DNA unknotting assays (Fig. 3). Two-fold serial dilutions in the linear range of activity were assayed for each protein. With 3.2 fmol of wild-type protein, P4 DNA could be completely unknotted (Fig. 3, lane 2). This corresponds to a specific activity of about 106 units/mg, which is the same as topo II purified from Drosophila cells (25, 27). Even with the highest amount of mutant proteins tested here, about 500-fold more than the wild-type enzyme, the unknotting reaction was only about 50 and 10%, respectively, in completion (Fig. 3, lanes 7 and 11). Other strand passage assays including supercoil relaxation, catenation, and decatenation of circular DNA produced similar results (data not shown). Furthermore, the unknotting activity is ATP-dependent; no unknotting was observed for wild-type and mutant proteins at the highest concentrations used in these assays in the absence of ATP (Fig. 3, lanes 1, 6, and 10). Other strand passage assays are also ATP-dependent (data not shown). Therefore, substitution of Lys359 with a neutral residue (K359Q) has resulted in 103-fold reduction in topoisomerase activity, whereas its substitution with a negatively charged residue (K359E) further lowers the enzymatic activity.


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Fig. 3.   Unknotting assays of wild-type and mutant topo II. P4 knotted DNA rings were used as the substrate, and the reactions were carried out either without ATP (lanes 1, 6, and 10) or in the presence of 1 mM ATP (all other lanes). The amounts of topo II added to reaction mixtures were 3.2, 3.2, 1.6, and 0.8 fmol for wild-type (WT, lanes 1-5), 1.8, 1.8, 0.9, and 0.4 pmol for the K359Q mutant (lanes 6-9), and 2.2, 2.2, 1.1, and 0.5 pmol for the K359E mutant (lanes 10-14). Lane 14 shows the control, with no enzyme. The positions of nicked circle and knotted nicked circle are marked by NC and KNC, respectively.

In parallel with the greatly reduced topoisomerase activity, K359Q and K359E also lost most of their ATPase activity (Fig. 4). The DNA-dependent ATPase activity of glutamine and glutamate mutants dropped to 5.9 and 4.0%, respectively, of the wild-type enzyme activity (Fig. 4, solid columns). Most of these residual ATPase activities in the mutant proteins can be attributed to the DNA-independent activities, which are not significantly altered among the topo II proteins tested here (Fig. 4, shaded columns). This DNA-independent ATPase activity is likely intrinsic to topoisomerase proteins rather than from a contaminating ATPase. We tested the sensitivity of ATPase activity toward two topo II-targeting, antitumor drugs: ICRF159 and teniposide (VM26). ICRF159 and other drugs in the family of bisdioxopiperazine inhibit the catalytic activities of topo II (32) and can promote the clamp formation of yeast topo II (33). Bisdioxopierazines can also inhibit the ATPase activity of yeast topo II (33). In the presence of ICRF159, the DNA-independent ATPase activity was reduced by 50% (Fig. 4, hatched versus shaded columns), a level of inhibition similar to that observed for yeast topo II. The same level of inhibition by ICRF159 was also demonstrated for K359Q and K359E mutant proteins (Fig. 4, hatched versus shaded columns). A second topo II inhibitor, teniposide, can promote the formation of the DNA cleavage complex of eukaryotic topo II (reviewed in Refs. 34 and 35; see also Ref. 36), and it can inhibit the ATPase activities in the mutant protein to a similar extent as ICRF159 (data not shown).


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Fig. 4.   ATPase activities of wild-type and mutant topo II. ATPase assays contained 0.9 pmol of topo II proteins either with 2.6 pmol of a 8.6-kilobase pair plasmid DNA pCaSeRhs83 (solid) or without (shaded). ATPase assays were also carried out in the absence of DNA but with the addition of 1 mM of ICRF159 (hatched). The rates were determined from the linear regression analysis of the linear portion of the rate curve for ATP hydrolysis and were the averages of at least five independent measurements. The bar above each column shows the S.D. of these measurements. The ATPase rates were normalized against the rate of DNA-dependent ATP hydrolysis for wild-type topo II, which was determined to be 5.8 µM min-1.

DNA Cleavage Reactions for Glutamine and Glutamate Mutants-- In addition to DNA strand passage and hydrolysis of ATP, another hallmark reaction of topo II is the double strand DNA cleavage. In this reaction, the addition of a strong denaturant traps the formation of a DNA cleavage complex in which the active site tyrosine of topo II is covalently linked to the phosphoryl end at DNA cleavage site. We used a 8.6-kilobase pair plasmid DNA, pCaSpeRhs83, as the substrate, and the cleavage reaction was monitored by the appearance of linear DNA fragments (Fig. 5). In contrast to topoisomerase and ATPase activities, the double strand DNA cleavage in the absence of any cofactors is comparable for all three proteins (Fig. 5, lanes 1-3). Although ATP stimulates the DNA cleavage by wild-type and K359Q proteins, it only has a marginal effect on K359E protein (Fig. 5, lanes 10-12). The ATP-stimulatory effect appears to correlate with the ATP-dependent strand passage activity in these mutant proteins.


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Fig. 5.   Wild-type and mutant topo II have comparable double strand DNA cleavage activities. In the DNA cleavage assays, 1.5 pmol of wild-type topo II (K, lanes 1, 4, 7, 10, 13, and 16), K359Q (Q, lanes 2, 5, 8, 11, 14, and 17), or K359E (E, lanes 3, 6, 9, 12, 15, and 18) were incubated at 30 °C for 30 min with 0.05 pmol of supercoiled pCaSpeRhs83 DNA in either the presence (lanes 10-18) or absence (lanes 1-9) of 1 mM ATP. The effect of topo II drugs was tested by including 1 µM of VM26 (lanes 4-6 and 13-15) or mAMSA (lanes 7-9 and 16-18). Lane 19 shows the control, with no enzyme, and the supercoiled plasmid is marked SC. Lane 20 shows the linearized plasmid DNA (L). Note that both wild-type and K359Q topo II have higher strand passage activity, and under the low-salt DNA cleavage reaction conditions, they could promote the formation of catenated networks (CAT; see lanes 10 and 11).

A unique feature in the DNA cleavage reaction mediated by topoisomerase is that it can be stimulated by a number of antitumor agents (reviewed in Refs. 34 and 35). To examine the effects of antitumor drugs on the cleavage by mutant proteins, we have used representatives from two distinct classes of topo II-targeting agents: amsacrine (mAMSA) and teniposide of epipodophyllotoxin (VM26) (36, 37). They differ by their ability to intercalate into DNA. Although mAMSA can inhibit the strand passage step of topo II, another member of epipodophyllotoxin drugs, etoposide, does not affect this step in the topo II reaction (38). In the absence of ATP, VM26 at a concentration of 1 µM can stimulate the DNA cleavage by all three proteins to identical levels (Fig. 5, lanes 7-9). mAMSA at the same concentration also affects the DNA cleavage to a similar degree (Fig. 5, lanes 4-6). The presence of ATP greatly stimulates the drug-promoted DNA cleavage, as evidenced by the nearly quantitative conversion of plasmid DNA into full-length linear molecules and smaller fragments, by wild-type and glutamine mutant proteins (Fig. 5, lanes 13 and 16 and lanes 14 and 17, respectively). For the glutamate mutant, the ATP stimulatory effect is negligible (Fig. 5, lanes 15 and 18). Despite the greatly reduced catalytic activities for Lys359 mutants, these mutations do not affect the formation of DNA cleavable complexes and also do not interfere with the interaction of topo II-targeting drugs. However, depending on the lysine substitutions, the effects of ATP cofactor in these reactions are variable.

Sequence Specificity in the Topo II-mediated DNA Cleavage Reaction-- The DNA cleavage reaction has been used to assess the sequence preference in the topo II/DNA interactions (39-41). Previously, we have used DNA cleavage and nuclease footprinting to demonstrate the coincidence of topo II cleavage and binding sites in the intergenic region of a heat shock gene HSP70 (28). For this study, we prepared a similar end-labeled DNA substrate to map the DNA cleavage sites for wild-type and mutant proteins (Fig. 6). Without any added cofactors, identical DNA cleavage patterns were observed for all three proteins, and the relative intensities at different cleavage sites are consistent with earlier data (Fig. 6, lanes 1-3). The strongest cleavages occur in sites marked as T4-T6, which contain tracks of alternating purine/pyrimidine sequences. Addition of teniposide, VM26, greatly stimulates the cleavage by topo II and mutant proteins to a similar extent, except for site T3, where cleavage is enhanced to a lesser degree with mutant proteins (Fig. 6, lanes 4-6). We have tested the stimulation of topo II-mediated, double-strand DNA cleavage by VM26 at concentrations ranging from 0.2 to 25 µM. At higher concentrations, the drug stimulated DNA cleavage to a higher extent, and the degree of such stimulation was similar for both the wild-type and mutant proteins (data not shown). Under these conditions of DNA cleavage, most of the DNA substrate remained uncleaved, and the cleavage was correlated with the amount of topo II protein present in the reaction (data not shown). The data from DNA cleavage experiments using either the plasmid or the end-labeled DNA substrate demonstrate that Lys359 mutations do not affect DNA cleavage reactions of topo II and do not alter the sequence specificity in their interactions with DNA substrate.


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Fig. 6.   Mutant and wild-type topo II have similar DNA sequence specificity in the cleavage reaction. Plasmid DNA 6/122, a derivative of pBR322 with an insertion of 1.7-kilobase pair SalI fragment from the intergenic region of Drosophila heat shock gene HSP70, was used to prepare the uniquely end-labeled linear DNA. 6/122 was linearized by HindIII, radiolabeled at its 5'-ends, and treated with EcoRI to remove one of the end labels. 75 nM wild-type topo II (K, lanes 1 and 4), K359Q (Q, lanes 2 and 5), or K359E (E, lanes 3 and 6) were incubated at 30 °C for 30 min with 0.5 nM of 5'-end labeled 6/122 DNA (2.5 × 106 cpm/pmol) in either the presence (lanes 4-6) or absence (lanes 1-3) of 1 µM VM26. Lane 7 shows the control, with no enzyme. The strong topo II binding and cleavage sites, marked as T1-T6 in order of increasing strengths (28), and size markers (in base pairs) are shown on the left and right sides, respectively, of the autoradiogram.

Closure of N-Gate in the Protein Clamps of Topo II and Lys359 Mutants-- Recent structural and biochemical data suggest a two-gate model for DNA transport by topo II (7, 10, 11). A key element in this proposed mechanism is that the jaws of the N-gate are closed upon the binding of ATP and are open when ATP is hydrolyzed and dissociated. We have used a glass fiber filter binding assay (7, 29) to test the retention of circular versus linear DNA by topo II proteins. An equal-weight mixture of negatively supercoiled and linear DNA was used in these binding assays. The results obtained with the wild-type protein have demonstrated that the formation of a salt-stable complex with circular DNA is only observed in the presence of a nonhydrolyzable ATP analog, AMPPNP, and the linear molecules were found almost exclusively in the high-salt eluate (Fig. 7, lanes 7 and 8). In the absence of any cofactors, there was no filter-retainable, salt-stable complex (Fig. 7, lane 4). The total absence of filter-retainable complex in the presence of ATP (Fig. 7, lane 6) is presumably due to the continuing hydrolysis of ATP and opening of the protein clamp. In an interesting contrast, for K359Q and K359E mutants, clamp complex formation is detectable in the conditions of no cofactors or with ATP (Fig. 7, lanes 10 and 12 and lanes 16 and 18, respectively). The inclusion of AMPPNP stimulates the clamp complex formation for K359Q (Fig. 7, lane 14), but it has no effect for K359E (Fig. 7, lane 20). The efficiency of the clamp complex formation with the mutant proteins under the conditions tested here is always lower than that for the wild-type protein in the presence of ATP analog, suggesting that Lys359 is critical for the clamp closure reaction. The mutant protein with glutamate substitution is less efficient in the clamp complex formation than that of the glutamine mutant. Furthermore, the formation of clamp complex either without any cofactor or with ATP suggests that Lys359 may be involved in the pathway for ATP-induced closure of N-gate, and its substitutions with other residues result in an anomaly in this signaling process.


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Fig. 7.   Lys359 mutant topo II proteins can form salt-stable, filter-retainable complex with circular DNA. Wild-type topo II (lanes 3-8), K359Q (lanes 9-14), K359E (lanes 15-20), or control, with no enzyme (lanes 21 and 22), were incubated with a mixture of labeled linear and circular DNA either in the presence of 1 mM ATP (lanes 5, 6, 11, 12, 17, and 18), in the presence of 1 mM AMPPNP (lanes 7, 8, 13, 14, 19, and 20), or without any cofactors (lanes 3, 4, 9, 10, 15, and 16). The binding reactions were quenched by the addition of NaCl to 1 M and filtered through GF/C filters. The filters were further washed with 1 M NaCl before final elution with 1% SDS. The combined filtrates and 1 M NaCl eluates (odd-numbered lanes 3-21) and SDS eluates (even-numbered lanes 4-22) were analyzed by 1.6% agarose gel electrophoresis in the presence of 1 µg/ml ethidium. The gel was dried and autoradiographed. Lane 1 shows the linear DNA marker, and lane 2 shows the DNA substrates used in the filter-binding assays. The positions for supercoiled DNA (SC), relaxed circle (RC), nicked circle (NC), linear (L), and supercoiled dimer (SCD) are marked on the left. Circular DNA can form stable complex with mutant topo II in high salt even without cofactors (lanes 10 and 16) or with ATP (lanes 12 and 18).

A unique feature for the clamp complex is that the topological link between a protein ring and circular DNA is stable in high salt; the filter-retainable complex is resistant to 1 M NaCl wash (Ref. 7; also Fig. 7). Earlier data also demonstrated by gel mobility shift assays that the complex formed by topo II and circular DNA in the presence of AMPPNP is resistant to dissociation in 0.5 M NaCl (14). We used the analytical ultracentrifugation to monitor the formation of these salt-stable complex in CsCl density gradient. In the buoyant density gradient experiments, circular DNA with increasing numbers of complexed topo II molecules band at distinct positions with progressively lighter buoyant densities (Fig. 8). The wild-type topo II did not form any stable complex with circular DNA either without any cofactors or with ATP, because only a single free DNA peak appeared in the bottom of a density gradient (Fig. 8, A and B). In contrast, the presence of AMPPNP resulted in the generation of DNA species with multiply interlocked protein rings (Fig. 8C). For the K359E mutant, the formation of these complexes can be observed regardless of the cofactor present (Fig. 8, G, H, and I). The complex with K359Q can also form without any cofactors (Fig. 8D) but it can be stimulated by ATP and, more efficiently, by AMPPNP (Fig. 8, E and F). The control experiments with linear DNA suggest that the complex formed is a result of the capture of DNA by the closure of a protein clamp (Fig. 9). The protein clamps can slide off a linear DNA substrate under high salt conditions. No complex with linear DNA was observed in the absence of cofactors or in the presence of ATP for K359Q and K359E mutant proteins (Fig. 9, D, G, E, and H), and only a minor DNA/protein complex peak was observed for wild-type topo II (Fig. 9, A and B). In the presence of AMPPNP, a peak was observed for linear DNA complexed with wild-type topo II (Fig. 9C) or K359Q (Fig. 9F), but none with K359E (Fig. 9I). The salt-stable complex formed between topo II and linear DNA is most likely due to a DNA cleavage complex with one of topo II subunits covalently linked to a single strand DNA break (42-44). This was also confirmed by the following experiment. When wild-type topo II was incubated with labeled linear DNA in the presence of AMPPNP, a trace amount of DNA was retained on the glass fiber filter, which was analyzed by two-dimensional agarose gel electrophoresis and was found to comigrate with the full-length linear DNA (data not shown). This result supports the notion that the complex formed under these conditions is due to single-strand DNA cleavage, because DNA in such a complex should remain as a full-length linear molecule. In all instances, the complex formed between linear DNA and topo II is always much less than those with circular DNA under identical conditions. The results obtained from CsCl density gradient experiments are in complete accord with the data from filter binding assays. Furthermore, it shows the protein clamp due to the closure of N-gate is stable in very high concentration of salt solutions (~5 M CsCl) and for a period at least as long as ultracentrifugation time (~30 h). The buoyancy shifts between the DNA/protein complex and DNA can be used to determine the molecular weight of protein in the complexes (23) (see "Experimental Procedures"). For all three proteins, it can be shown that the multiple species of complexes correspond to a single DNA circle interlocked with multiple molecules, up to 4, of a protein with a molecular mass of 350 kDa, consistent with it being a dimer of the 170 kDa topo II subunit.


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Fig. 8.   Formation of salt-stable topo II/DNA complex in CsCl density gradient ultracentrifugation. The major peak at the bottom of each gradient corresponds to DNA without any bound protein, whereas the lighter species are complex of DNA with different numbers of protein molecules. The binding reactions were carried out with circular pCaSpeRhs83 DNA and wild-type topo II (A-C), K359Q (D-F), or K359E (G-I) in the presence of ATP (B, E, and H), AMPPNP (C, F, and I), or no cofactors (A, D, and G). The minor species observed between major bands (see C and F) correspond to topo II complexed with dimer DNA, which is present in the plasmid DNA preparations.


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Fig. 9.   No topo II clamp complex is formed with linear DNA in CsCl density gradient ultracentrifugation. In contrast to the multiple peaks corresponding to multiple molecules of topo II complexed with circular DNA, very little complex was observed with linear DNA under identical conditions. The reaction conditions were similar to those described for Fig. 8, except that XhoI-digested pCaSpeRhs83 DNA was used in all experiments. Binding reactions contained either wild-type topo II (A-C), K359Q (D-F), or K359E (G-I) in the presence of cofactor ATP (B, E, and H) or AMPPNP (C, F, and I) or no cofactor (A, D, and G). The minor peak observed in A-C and F is likely from the single strand DNA cleavage complex in which one of topo II subunits is covalently linked to the single strand break in a linear duplex molecule.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A key question in understanding the mechanism of topo II is to elucidate the role of ATP binding and hydrolysis in the overall process of DNA strand passage reaction. One approach is to identify the crucial residues in topo II that interact with ATP and analyze their functions. Examining the structure of the 43-kDa fragment of gyrB shows three regions of the enzyme that directly interact with the beta  and gamma  phosphates of the bound ATP: His38-Asn46, His99-Gly119, and Pro330-Asp338. The first region is part of a helix in which a conserved Glu42 was identified to be critical to the ATPase and DNA supercoiling activities of Escherichia coli gyrase (45). The second region, which forms a loop and part of a short helix, contains a GXXGXG motif found in a variety of ATP binding proteins (46, 47). Located here are two lysine residues, Lys103 and Lys110, which were specifically modified with an ATP analog in affinity labeling studies (48). Changing the Gly144 of the GXXGXG motif in the yeast enzyme to Ala and the mutations of Lys103 in gyrase result in the complete loss of ATP-dependent topo II activities (49, 50). The third region is a loop of Pro330-Asp338 that is a part of the domain that forms the 20 Å hole. Within this loop, both Gln335 and Lys337 are in contact with oxygens in the gamma -phosphate of the bound AMPPNP. The unique molecular architecture of this loop suggests a potential mechanism to relay the signaling in the binding and hydrolysis of ATP to induce the conformational changes in the rest of the topo II molecule (31).

Our data presented here demonstrate that Lys359 in Drosophila topo II, the homologous residue of Lys337 in gyrB, is critical for the catalytic activities of topo II. Although a conservative substitution with arginine does not inactivate the enzyme, changing it to either glutamine or glutamate greatly reduces both the strand passage and DNA-dependent ATPase activity. Because these mutant proteins have chromatographic behavior identical to the wild-type enzyme during the purification steps and form dimers based on the CsCl density gradient experiments, we believe that there is no gross misfolding in the mutant proteins. Furthermore, the apparent similarity in DNA cleavage activity, cleavage site specificity, and sensitivity toward topo II-targeting drugs indicates that these mutants still retain part of the wild-type topo II functions. Therefore, the reduced strand passage activity seems to be due to a defect in specific steps involving ATP-dependent functions rather than in the formation of a protein-mediated DNA gate. There is a correlation among the ATP-dependent activities in the mutant proteins. For instance, although the K359E mutant is less active in the unknotting assays than the K359Q mutant, it has also lost most of the ATP-stimulation in both DNA cleavage and protein-clamp assays. This defect in ATP-induced response could be either because the mutation has abolished the ability of topo II to bind ATP or because the defect is in a step subsequent to the binding of ATP. Our data favor the latter possibility because the residual strand passage activity in the mutant proteins is still ATP-dependent (Fig. 3), and the Km of ATP in this reaction for both mutant proteins is comparable with the wild-type enzyme (data not shown). Furthermore, at least for K359Q protein, ATP can stimulate both the DNA cleavage reaction and protein clamp formation.

In an interesting contrast with the wild-type topo II, Lys359 mutants can form a salt-stable protein clamp complex with circular DNA even in the presence of ATP or in the absence of any nucleotide cofactors. The clamp closure for mutant proteins with ATP could be due to their lower ATPase activity, thereby prolonging the ATP-bound state. However, both mutant proteins have their ATPase activities reduced to a similar extent, whereas the formation of clamp complex with K359Q mutant is more efficient than K359E, suggesting the possible involvement of other factors, such as the efficiency of ATP signaling in clamp closure. It is possible that there is a dynamic equilibrium between the open-form and closed-form of the protein clamp. In the presence of DNA, this equilibrium can be affected upon the binding of nucleotide cofactor. For wild-type enzyme, binding of ATP can shift this equilibrium toward the closed clamp conformation and initiate the subsequent conformational changes that are necessary for the strand passage reaction (5). Replacing Lys359 with either Gln or Glu interferes with the ATP-signaling mechanism, and the equilibrium between open/closed clamp complex. The fact that K359Q and K359E could form protein clamps in the absence of nucleotide cofactor suggests that the basal equilibrium is shifted toward closed clamp conformation in these mutant proteins. Although the site-specific mutants presented in this paper are unique in that they showed altered response of clamp closure to triphosphate cofactors in addition to the loss of strand passage activity, the complexity in the clamp closure process suggests that future site-specific mutagenesis experiments can identify other critical residues involved in the movement of this intricate macromolecular machinery.

    ACKNOWLEDGEMENTS

We thank Dr. Harvey Sage for assistance in the analytical ultracentrifuge runs and Dr. Jane Richardson for discussion of the use of kinemage.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM29006.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, Box 3711 DUMC, Duke University Medical Center, Durham, NC 27710. Tel.: 919-684-6501; Fax: 919-684-8885; E-mail: hsieh{at}biochem.duke.edu.

1 The abbreviations used are: topo, topoisomerase; gyrB, DNA gyrase B protein; gyrA, DNA gyrase A protein; AMPPNP, 5'-adenylylbeta ,gamma -imidodiphosphate.

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Abstract
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Discussion
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