DNA gyrase is a bacterial type II topoisomerase that couples the free energy of ATP hydrolysis to the introduction of negative supercoils into closed-circular DNA. (For recent reviews, see (1, 2, 3) .) In the absence of ATP, DNA gyrase relaxes negatively, but not positively, supercoiled DNA. In contrast, eukaryotic and T-even phage type II topoisomerases, which are structurally related to DNA gyrase (4) , catalyze ATP-dependent relaxation of both positively and negatively supercoiled DNA, but do not supercoil DNA(5) . The topoisomerase reactions of all type II enzymes involve the passage of a double-stranded DNA segment through a transient double-strand break, which is then resealed to give changes in the DNA linking number in steps of two.

DNA gyrase from Escherichia coli contains two subunits, A (GyrA) ()and B (GyrB), which are assembled in an AB complex(6, 7) . GyrA has a molecular weight of 97,000 and is essential for DNA breakage and reunion. An intermediate step in this reaction involves the covalent attachment of Tyr of GyrA at the broken 5`-end of each DNA strand(8) . GyrB has a molecular weight of 90,000 and carries a site for ATP binding and hydrolysis(9, 10, 11, 12) . Affinity labeling studies using the ATP analog, PLP-AMP, have identified Lys and Lys as possible active site residues(13) . The presence of Lys at the nucleotide binding site was later confirmed by crystallographic studies on an N-terminal fragment of GyrB with bound ADPNP(14) , an ATP analog which cannot be hydrolyzed by gyrase.

The mechanism of energy coupling between ATP hydrolysis and DNA supercoiling is largely unknown. Limited supercoiling of relaxed closed circular DNA occurs upon the binding of ADPNP, suggesting that conformational changes associated with nucleotide binding can induce one supercoiling event while catalytic supercoiling requires hydrolysis of ATP and dissociation of products(15, 16) . Once formed, the gyrase-DNA-ADPNP complex does not readily dissociate. Binding of ADPNP to a gyrase-DNA complex is slow, and involves cooperative interactions between the two nucleotide binding sites in the gyrase tetramer(16) . Modification of only one of these sites appears to inhibit both ATP hydrolysis and DNA supercoiling(13) .

In this work, we introduce mutations into GyrB at Lys and Lys and study the effects of these changes on the ATP-dependent and ATP-independent reactions of DNA gyrase. Evidence is presented indicating that Lys, which is not conserved in the equivalent region of the eukaryotic type II topoisomerases, is essential for the ATP-dependent activities of gyrase. Results of supercoiling experiments combining wild-type and Lys mutant GyrB subunits in the reconstituted enzyme are discussed in terms of a mechanism requiring participation by both nucleotide binding sites of the gyrase AB tetramer.

EXPERIMENTAL PROCEDURES

Materials

GyrA protein was purified from E. coli strain N4186 by the method of Mizuuchi et al.(19) . Oligonucleotides for mutagenesis and sequencing primers were synthesized using an Applied Biosystems model 380B DNA synthesizer. DNA sequencing was performed using Sequenase Version 2.0 from U. S. Biochemical Corp.

Site-directed Mutagenesis

Purification of GyrB Proteins

Enzyme Assays

ATPase activity was measured using two methods. The first followed P liberation from [-P]ATP in a reaction containing 50 mM Hepes-NaOH, pH 7.5, 24 mM KCl, 10 mM potassium phosphate, pH 7.5, 6 mM MgCl, 6.5% (w/v) glycerol, 1.8 mM spermidine, 5 mM dithiothreitol (buffer 1) and 0.4 mM [-P]ATP. Additions to selected assay samples included 33 µg/ml novobiocin, 15 µg/ml linear pBR322 DNA, and 10 µg/ml GyrA protein. The reactions were initiated by addition of 5 µg/ml GyrB protein. After a 1-h incubation at 25 °C the reactions were terminated by the addition of 20 mM EDTA. Hydrolysis products were separated by thin-layer chromatography on polyethyleneimine-cellulose plates (Bakerflex) developed in 0.8 M acetic acid, 0.8 M LiCl. The dried plates were then exposed to Kodak XAR-5 x-ray film. The second method, for kinetic studies of DNA-dependent ATPase activity, was an ATP-regenerating spectrophotometric assay used as described previously(13) . The apparent values for K and k were determined from double reciprocal plots of the turnover rate for ATP hydrolysis against the concentration of ATP (0.1-1.0 mM). Prior to the assay, 1.2 µM GyrA, 1 µM GyrB, 50 µg/ml linear pUC9 DNA, and 0.2 mg/ml bovine serum albumin were preincubated for 1 h at room temperature in buffer 1. ATPase reactions were initiated by the addition of 10 µl of enzyme to 90 µl of the ATPase assay mixture. Binding studies using ADPNP (Sigma) and [-P]ADPNP (ICN) were carried out as described elsewhere(16) .

RESULTS

Mutated Proteins

Wild-type and mutant GyrB proteins were expressed in E. coli as described under ``Experimental Procedures.'' Cells producing the Lys GyrB mutants grew at the same slow rate as those producing the wild-type protein. On the other hand, cells producing the Lys mutants grew more rapidly, indicating that excess production of the Lys mutants may be less toxic to the cells than overproduction of the wild-type GyrB protein. Expression of all of the GyrB proteins was very high, averaging about 30% of the total cell protein. However, based on the supercoiling assay, the specific activity of the wild-type GyrB protein in the cell lysate was much lower than expected. Further investigation led to the finding that wild-type GyrB protein could be resolved into two peaks, which eluted at 0.2 and 0.3 M NaCl on a heparin-agarose column. The first peak had a much higher specific supercoiling activity than the second, which may largely consist of improperly folded protein. Wild-type GyrB prepared from a strain carrying the same plasmid as used here was previously found to contain a large amount of low-activity protein, which showed increased activity after renaturation from guanidine hydrochloride solution(12) . All of the mutant GyrB proteins were similarly partitioned into two peaks on heparin-agarose columns. DEAE-Sepharose chromatography of the more active wild-type peak fractions produced a nearly homogenous GyrB preparation with a specific activity close to that previously reported(19) . This wild-type GyrB and corresponding DEAE-Sepharose fractions containing the mutant GyrB proteins were used for the experiments which follow.

ATP-independent Activities

Figure 1: Oxolinic acid-induced cleavage of DNA. DNA gyrase (5 nM) was incubated with linear pBR322 DNA (2 nM) and oxolinic acid (50 µg/ml) at 25 °C for 1 h in the presence or absence of 1.1 mM ATP as indicated. The reactions were terminated by addition of SDS followed by digestion with proteinase K, as described under ``Experimental Procedures.'' Lane 1 contains no enzyme. Gyrase tetramers were reconstituted using wild-type (WT) or mutant GyrB proteins as shown. The 0.8% agarose gel was run in 90 mM Tris borate, 1 mM EDTA. L is linear pBR322 DNA.

ATP-dependent Activities: Supercoiling

Figure 2: Extent of supercoiling by wild-type DNA gyrase and by gyrase reconstituted using K110V and K110E GyrB proteins. Lane 1 contains 2.5 nM intracellularly supercoiled pBR322 DNA. Lanes 2-20 contain 2.2 nM relaxed pBR322 DNA which has been incubated alone (lane 2) or with gyrase reconstituted using wild-type (WT) or mutant GyrB subunits at the indicated concentrations and incubation times. Gyrase solutions (0.6 µM) containing equimolar amounts of gyrase subunits A and wild-type or mutant B were preincubated for 30 min at 25 °C before dilution and addition to the supercoiling assay mix containing DNA and 1.4 mM ATP. Electrophoresis was carried out using a 1.2% agarose gel in 40 mM Tris base, 30 mM NaHPO, 1 mM EDTA and 40 µg/ml chloroquine. Relaxed DNA (Rel) migrates with high mobility in this system, due to increased positive writhe in the presence of chloroquine. The positions of intracellularly negatively supercoiled (SC) and open circular (OC) pBR322 DNAs are shown. The arrow indicates the direction of resolution of more negatively supercoiled topoisomers.

ATP Hydrolysis

Figure 3: Kinetic analysis of the ATPase activity of wild-type gyrase and of gyrases containing mutations at Lys of GyrB. At a constant enzyme and DNA concentration, the turnover rates for ATP hydrolysis by wild-type, K110V, or K110E gyrases were measured in the presence of varying concentrations of ATP by an enzyme-linked spectrophotometric assay, as described under ``Experimental Procedures.''

Using a more sensitive radioactive assay (see ``Experimental Procedures''), the effects of the various mutations on the hydrolysis of [-P]ATP were studied. The K103E, K103I, or K103T GyrB proteins had very low levels of ATPase activity, either in the presence or absence of GyrA and DNA. This residual ATP hydrolysis was largely insensitive to novobiocin suggesting that most, if not all, of the activity can be attributed to contaminating ATPases in the preparations. In assays using the K110V or K110E enzymes, DNA-dependent novobiocin-sensitive ATPase activity was observed at levels 2-3-fold lower than with the wild-type enzyme (data not shown).

Cleavage Site Preference

ADPNP Binding

Figure 4: Binding of ADPNP to wild-type and mutant GyrB proteins and to their complexes with GyrA and DNA. GyrB proteins (0.5 µM) were preincubated alone or with 0.5 µM GyrA protein and 50 µg/ml linear pBR322 DNA in 35 mM Tris-HCl (pH 7.5), 24 mM KCl, 10 mM potassium phosphate (pH 7.5), 5 mM dithiothreitol, 1.8 mM spermidine, 6 mM MgCl, 0.5 mg/ml bovine serum albumin, and 6.5% glycerol for 30 min at 25 °C. 50 µM [-P]ADPNP was added and the incubation at 25 °C was continued for 5 h or 25 h. Unbound nucleotide was removed by rapid gel filtration and stoichiometry of binding was determined as described by Tamura et al.(16) .

Reconstitution of Mixed Gyrase Tetramers

Fig. 5shows the result of the reconstitution experiment. Included in the figure are three theoretical curves, each predicting a different outcome depending on the mechanism. The straight line (curve 1) is subject to two interpretations. First, curve 1 represents the anticipated results if the GyrB protein in solution is already a dimer or if assembly is not random but strongly favors two GyrB subunits of the same type per tetramer. Tetramers containing one active and one inactive GyrB monomer would be unlikely to form; the specific supercoiling activity of the wild-type enzyme would be simply diluted. Alternately, curve 1 would also be obtained if tetramers containing one inactive and one active B monomer are formed, but the activity of the mixed tetramer is proportional to the active GyrB content; that is, a mixed tetramer would have half the activity of a tetramer containing two active B subunits. The upper curve (curve 2) predicts the results if assembly of the gyrase tetramers is random, and catalytic supercoiling can occur with enzyme having one active and one inactive GyrB subunit; it is assumed that an enzyme tetramer containing only one active GyrB subunit has full catalytic activity. This result could also be achieved if a conformational change following ATP binding to the active GyrB subunit of a mixed tetramer promoted ATP binding at the inactive GyrB of the tetramer despite the inactivating Lys mutation, thus generating a second active site. Last, the lower curve (curve 3) represents the predicted outcome for random assembly into tetramers when enzyme containing two active GyrB subunits is the only species capable of catalyzing DNA supercoiling. The data points are in close agreement with curve 3, in strong support of the model that DNA supercoiling requires the participation of both ATP binding sites in the enzyme AB complex.

Figure 5: Correlation between DNA supercoiling activity and percentage of inactive GyrB protein used in reconstitution of the gyrase tetramer. Wild-type and K103I mutant GyrB proteins were combined in 1.6 µM solutions at molar ratios of 10:0, 9:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:9, and 0:10. Mixed gyrase tetramers were formed by adding an equimolar amount of GyrA protein to each GyrB solution and incubating for 30 min at 25 °C. A series of dilutions of each tetramer preparation was assayed for supercoiling activity. Experimentally determined supercoiling activities of the mixed tetramers () are expressed as percentages of the activity of tetramers containing 100% wild-type GyrB protein. Theoretical curves 1, 2, and 3 are described under ``Results''.

DISCUSSION

ATP-dependent type II topoisomerases possess an amino acid sequence motif predictive of ATP binding. This motif, a highly conserved glycine-rich region (residues 114-119 of GyrB from E. coli) of the sequence GXXGXG, is found in all known bacterial, phage, and eukaryotic type II topoisomerases (Table 2). This region, which is also found on a variety of other ATP binding proteins, has been postulated to form part of a flexible loop structure involved in conformational changes following nucleotide binding(23, 24, 25) . A possible role in nucleotide binding for the region of GyrB comprising amino acids 103-119 was initially proposed from the results of affinity labeling studies, in which Lys and Lys were specifically modified(13) . The importance of this region was shown in more detail in the crystal structure of a 43-kDa N-terminal domain of GyrB containing bound ADPNP(14) . The structure shows that residues 96-117 form a loop followed by a short helix composed of residues 118-126. The phosphates of the bound nucleotide are in contact with glycines 114, 117, and 119 of the ATP-binding motif at the base of the helix. Furthermore, the -amino group of Lys forms a salt bridge with the -phosphate of the bound ADPNP molecule, and Tyr forms a hydrogen bond with N3 of the adenine ring. When the amino acid sequence of E. coli GyrB residues 96-127 is compared with the corresponding region of all other bacterial GyrB proteins for which sequences have been determined, a very high degree of conservation is found. A separate comparison of these regions from the eukaryotic type II topoisomerases also shows very high sequence homology. However, comparing the gyrases to the eukaryotic enzymes reveals variations in this region which may be significant (Table 2). The sequences of residues 96-104 are quite different in the two classes of enzyme; in particular, the Gly and Gly residues of gyrase, which should contribute to the flexibility of the loop, are replaced by serine, and Lys, which is conserved in all GyrB species, is replaced by asparagine in all of the eukaryotic type II topoisomerases. The Tyr residue found in all GyrB proteins is replaced by lysine, glutamine or serine in the eukaryotic type II topoisomerases. Lys is commonly found in enzymes from both groups; however, substitutions at this position have been identified in both gyrases and eukaryotic type II topoisomerases. Mutation of the corresponding lysine to alanine in yeast topoisomerase II had little effect on the activity(26) . Thus, in eukaryotic type II topoisomerases, neither the equivalent of Lys nor of Lys is required. It is interesting that this segment of E. coli ParE, a component of bacterial topoisomerase IV, very closely resembles GyrB in overall sequence, while the phage T2 and T4 gene 39 proteins include characteristics of both gyrase and eukaryotic type II topoisomerase. Like gyrase, topoisomerase IV and the T-even phage type II topoisomerases are multimers of two or more different subunits, but they resemble the homodimeric eukaryotic type II topoisomerases in activity.

Our present results suggest that Lys is essential for the ATP-dependent activities of DNA gyrase. Mutations at this site resulted in loss of these functions but did not affect ATP-independent activities. The ADPNP binding results, along with the implication of Lys in nucleotide binding from the crystal structure, support the hypothesis that it is the ability to bind ATP which is lost. However, we have previously proposed that ATP and its analog ADPNP initially form a rapidly reversible complex with gyrase, followed by a conformational change to a tightly bound state (16) . It is possible that mutations at Lys do not altogether prevent nucleotide binding, but prevent or alter the conformational changes that follow; our spin column assay for ADPNP binding may not detect bound ADPNP if dissociation of the initial complex is very fast. We can nevertheless conclude that these substitutions at Lys alter the nucleotide binding behavior of gyrase in such a way that little or no tightly bound enzyme-nucleotide complex is formed and binding cannot be demonstrated by the methods previously used with the wild-type enzyme.

The mutations of Lys indicate that this residue is not directly involved in ATP binding. Enzyme reconstituted with GyrB containing valine or glutamic acid substituted for Lys retains all activities of gyrase. However, the increase in the K for ATP of these mutants, together with the reduced level of ADPNP binding, suggests that ATP binds with lower affinity. The K and k values must be interpreted with caution; cooperative binding of two nucleotide molecules to the two binding sites in each gyrase tetramer, leading to conformational changes in the protein prior to hydrolysis(11, 16, 27) casts doubt upon the validity of a steady-state approach to gyrase kinetics. However, by using gyrase as the preformed AB-DNA complex and holding the concentration of this complex constant, we obtain apparent parameters for the wild-type and mutant GyrB proteins which provide a useful means for comparing their relative ATPase activities.

Ali et al.(11) have proposed that the rate-limiting step for the ATPase activity of gyrase is not nucleotide binding, but hydrolysis and release of products. However, if the rate of nucleotide binding is considerably slower for the Lys mutants, as suggested by Fig. 4, then ATP binding may become rate-limiting. The binding rate of ADPNP to the wild-type gyrase-DNA complex has recently been found to be dependent on the topology of the substrate DNA, with binding being more rapid if the DNA is negatively supercoiled(28) . This increase in nucleotide binding rate at higher levels of supercoiling may be less pronounced with the Lys mutants. It is therefore possible that with these mutants the ATP-independent DNA relaxation activity would make a greater contribution to the equilibrium superhelical density (Fig. 2).

Our results on the Lys mutants are consistent with the structural information on the 43 kDa fragment of GyrB which shows that Lys does not form contacts with bound ADPNP(14) . It is probable that amino acid substitutions for Lys cause a slight perturbation of the protein conformation involved with ATP binding or subunit interactions. This is not surprising in view of the close proximity of Lys to residues known to interact with ATP such as Tyr, Gly, Gly, and Gly.

The Lys mutants have provided a unique tool for obtaining gyrase AB tetramers containing a predictable distribution of active and inactive GyrB subunits. They offer an alternative to partial inactivation using nucleotide analogs or other inhibitors which might themselves cause or prevent conformational changes when bound to the protein, perhaps blocking interactions between the active and inactivated subunits. We have exploited the fact that the GyrB mutants at Lys, while devoid of ATP-dependent reactions when reconstituted with GyrA and DNA, appear to retain the capacity to assemble into an enzyme complex capable of carrying out ATP-independent reactions. In the analysis, it was assumed that wild-type and mutant GyrB proteins in a mixture can assemble randomly and equivalently into the enzyme AB complex. The observed supercoiling activities closely follow the predicted theoretical curve of a mechanism in which both GyrB subunits must be functional in ATP binding and hydrolysis to catalyze DNA supercoiling.

The interdependent action of the two ATP binding sites in DNA supercoiling by gyrase has been previously proposed. Thermodynamic calculations of the free energy change required to decrease the DNA linking number by two at the high-supercoiling limit of gyrase action are consistent with the concerted hydrolysis of two molecules of nucleotide(29, 30, 31, 32) . Furthermore, measurements of the inhibition of ATPase and DNA supercoiling activities of gyrase following reaction with the ATP affinity reagent, PLP-AMP, indicate that modification of only one of the two ATP binding sites can lead to inactivation(13) . A cooperative model for ATP binding was proposed based on the rates of ADPNP binding in the presence of ATP(16) . This model predicts the interdependence of the GyrB subunits for catalyzing ATP-driven reactions and agrees well with the present results from the subunit mixing experiments. Lastly, there is structural information which supports the functional interaction of the two ATP binding sites. The crystal structure of the N-terminal fragment of GyrB with ADPNP bound shows that the fragment exists as a dimer with dyad symmetry(14) . Each subunit contains an N-terminal extension that goes from close proximity to one nucleotide binding site to direct contact with the bound nucleotide on the other subunit. The physical contact between the two subunits provides a possible way of coupling ATP hydrolysis at one site to the participation of the ATP binding site on the other subunit.

Related studies of the yeast type II topoisomerase have come to different though not necessarily contradictory conclusions. ATP binding to the two ATPase sites of the DNA-bound homodimer of this enzyme appears to be cooperative(26) . At low ATP concentrations, at which the coupling of ATP hydrolysis to relaxation activity is most efficient, an average of 1.9 ± 0.5 ATP molecules are hydrolyzed for each DNA transport event(26) . However, in a yeast heterodimer consisting of one wild-type subunit and one mutant allele defective in ATP binding, binding of ADPNP resulted in a concerted conformational change in both wild-type and mutant subunits(26, 33, 34) . This suggests the possibility that binding only one ATP to an enzyme dimer might suffice to drive a DNA transport event. However, strand-passage activity by the heterodimer was not studied.

Further comparison of the yeast heterodimer with our present work is complicated because the inactivating mutation at Gly of yeast topoisomerase II does not correspond to Lys of gyrase, but to Gly in the gyrase ATP binding motif. It remains possible that in a Gly gyrase mutant an induced conformational change similar to that seen in the yeast topoisomerase II mutant/wild type heterodimer would be found.

FOOTNOTES

*

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Present address: Department of Protein Biochemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, PA 19406.

¶

To whom correspondence should be addressed: Laboratory of Molecular Biology, Bldg. 5, Rm. 241, NIDDK, National Institutes of Health, Bethesda, MD 20892-0540. Tel.: 301-496-5888; Fax: 301-496-0201.

()

The abbreviations used are: GyrA, DNA gyrase A protein; GyrB, DNA gyrase B protein; PLP-AMP, pyridoxal 5`-diphospho-5`-adenosine; ADPNP, 5`-adenylyl-,-imidodiphosphate.

ACKNOWLEDGEMENTS

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