Identification of Four GyrA Residues Involved in the DNA Breakage–Reunion Reaction of DNA Gyrase

doi: 10.1016/S0022-2836(02)00048-7 available online at http://www.idealibrary.com on

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J. Mol. Biol. (2002) 318, 351–359

Identification of Four GyrA Residues Involved in the DNA Breakage –Reunion Reaction of DNA Gyrase Susan C. Hockings and Anthony Maxwell* Department of Biochemistry University of Leicester Leicester LE1 7RH, UK

DNA supercoiling by DNA gyrase involves the cleavage of a DNA helix, the passage of another helix through the break, and the religation of the first helix. The cleavage –religation reaction involves the formation of a 50 -phosphotyrosine intermediate with the GyrA subunit of the gyrase (A2B2) complex. We report the characterization of mutations near the active-site tyrosine residue in GyrA predicted to affect the cleavage – religation reaction of gyrase. We find that mutations at Arg32, Arg47, His78 and His80 inhibit DNA supercoiling and other reactions of gyrase. These effects are caused by the involvement of these residues in the DNA cleavage reaction; religation is largely unaffected by these mutations. We show that these residues cooperate with the active-site tyrosine residue on the opposite subunit of the GyrA dimer during the cleavage – religation reaction. q 2002 Elsevier Science Ltd. All rights reserved

*Corresponding author

Keywords: topoisomerase; supercoiling; quinolone; DNA cleavage

Introduction DNA gyrase regulates DNA topology in the cell by a three-step mechanism. DNA is cleaved in both strands, the DNA ends are then pulled apart to allow passage of another DNA helix through the break and, lastly, the cleaved helix is aligned and religated. The interaction of gyrase with the DNA “gate” segment, as the cleaved DNA is called, is not well understood, except that the active-site tyrosine residue (Tyr122) forms a 50 -phosphotyrosine intermediate with the cleaved DNA strands.1,2 Escherichia coli DNA gyrase is an A2B2 tetramer composed of GyrA and GyrB subunits.3 Each subunit can be divided into two domains. The 90 kDa GyrB protein has an N-terminal 43 kDa domain and a C-terminal 47 kDa domain.4 – 6 The 43 kDa domain has ATPase activity required for enzyme turnover. ATP, or ADPNP, binding closes the Present addresses: S.C. Hockings, Department of Chemistry, Washington University, St. Louis, MO 63130, USA; A. Maxwell, Department of Biological Chemistry, John Innes Centre, Colney Lane, Norwich NR4 7UH, UK. Abbreviations used: ADPNP, 50 -adenylyl b,g-imidodiphosphate; A2B2, DNA gyrase tetramer; GyrA, DNA gyrase A protein; GyrB, DNA gyrase B protein; PK/LDH, pyruvate kinase/lactate dehydrogenase; PEP, phosphoenolpyruvate; topo II, DNA topoisomerase II; ts, temperature-sensitive. E-mail address of the corresponding author: [email protected]

dimer interface of the N-terminal domains of the GyrB dimer, which forms a protein clamp; ATP hydrolysis is required for enzyme turnover. By analogy with other type II topoisomerases, the 47 kDa domain interacts with GyrA and holds the transported helix prior to strand passage.7 GyrA is a 97 kDa protein that has an N-terminal 64 kDa domain and a C-terminal 33 kDa domain. The X-ray crystal structure of a 59 kDa fragment of the 64 kDa domain has been solved.8 This domain contains the active-site tyrosine residue and the binding surface for the cleaved DNA helix, in addition to a large cavity capable of holding the transported DNA after strand passage. The C-terminal domains of GyrA are proposed to wrap DNA around gyrase in a positive superhelical sense, consistent with the large DNA-binding site observed for gyrase.9 – 11 The preferred reaction of DNA gyrase is DNA supercoiling. The GyrA dimer is thought to assemble with a DNA helix to be cleaved (the gate or “G” segment) and two GyrB subunits.12 ATP binding closes the GyrB clamp and traps the DNA segment to be transported.13 The gate segment is cleaved at sites four bases apart, forming 50 -phosphotyrosine intermediates, and the two ends are separated. The transported helix (the “T” segment) passes from GyrB through the gate DNA into GyrA. The cleaved DNA is then realigned and religated. ATP hydrolysis and release allow the opening of the GyrB clamp. In the absence of ATP, gyrase can carry out DNA relaxation, which is

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

352

DNA Cleavage Reaction of Gyrase

Figure 1. Ribbon representation of the crystal structure of the GyrA 59 kDa fragment dimer.8 (a) Side view, and (b) close-up top view of the DNA cleavage site. Each monomer is coloured red or blue, and Tyr122 and the mutated residues are shown as space-filling representations. Tyr122 in the blue subunit is coloured light blue and it is coloured orange in the red subunit. The mutated residues are: Arg32, purple; Arg47, black; His78, green; and His80, yellow.

essentially the reverse of supercoiling; the transported segment enters through the GyrA subunits and is passed to GyrB.14 Quinolone drugs and Ca2þ can stabilize the cleavage intermediate in gyrase-catalysed DNA supercoiling. Quinolones target gyrase and are thought to form a complex including drug, DNA and both gyrase subunits.15 In vitro assays reveal double-stranded cleavage upon the addition of SDS. In vivo, the complex is thought to block the translocation of enzymes along the DNA, which leads to cell death.16,17 The mechanism of Ca2þ cleavage is unknown, but it is thought to shift the cleavage– religation equilibrium towards the cleaved state.18 DNA cleavage assays are well established for studying drug-resistance mutations in gyrase, but the religation reaction has only recently become accessible experimentally.19 The religation assay, developed originally for another type II enzyme, topoisomerase (topo) IV from E. coli, depends upon the ability to make a population of the cleaved DNA intermediate and slow the religation rate for observation. Previous work with yeast topo II suggests that some of the residues near the active-site tyrosine residue are required for cleavage – religation activity.20 Liu & Wang observed that plasmids expressing alanine mutations at His735 (His78 in GyrA) or His736 (His80 in GyrA) complemented a temperature-sensitive (ts) strain. At Arg704, equivalent to GyrA Arg46, the alanine mutation did not complement the ts strain and showed decreased activity in vitro; in the GyrA crystal structure, Arg46 is buried.8 Expression of an alanine mutation at Arg690 of topo II, which corresponds to GyrA Arg32, was found not to com-

plement the ts strain. Furthermore, neither relaxation nor DNA cleavage in vitro were observed with this mutant protein. Liu & Wang concluded that residues His735, His736, and Arg704 were not required for enzyme activity because alanine mutations at those residues complement the ts strain or showed activity in vitro. These results pose questions as to whether the equivalent residues are involved in the cleavage – religation reaction in DNA gyrase. While there are many similarities between yeast topo II and gyrase, there are significant differences. Gyrase has the C-terminal domains of GyrA, which wrap a DNA segment around the protein so that the binding site is about six times larger than that of yeast topo II, and the cleaved and transported segments of DNA are preferentially on the same DNA molecule.11 Yeast topo II is essentially a fusion of the two gyrase proteins such that gyrase has more flexibility in allowing the GyrB and GyrA dimers to dissociate. Gyrase has the unique ability to relax DNA in an ATP-independent reaction. We have used site-directed mutagenesis to make five mutations in GyrA near the active-site tyrosine residue to identify other residues involved in interactions with the DNA gate segment. Two of the mutations are Arg to Gln mutations, which may allow partial function if the residue is required for activity. While none of the mutations produces an inactive gyrase, they do have greatly decreased activity relative to wild type. The residues are shown to be involved primarily in the cleavage step of the cleavage – religation reaction, and to cooperate with the active-site tyrosine residue from the opposite GyrA subunit.

353

DNA Cleavage Reaction of Gyrase

Table 1. Supercoiling and relaxation activity

Relative supercoiling rate (%) Relative relaxation rate (%)

Wild-type

R32A

R32Q

R47Q

H78A

H80A

100 100

0.9 25

2 33

0.8 50

0.4 20

6 100

Results Design and in vivo properties of mutants The X-ray crystal structure of the 59 kDa domain of GyrA was used to identify conserved residues, among type II topoisomerases, on the putative DNA-binding surface of the enzyme, near the active-site tyrosine residue, and with functional groups likely to interact with DNA.8 The residues that met these criteria were Arg32, Arg47, His78, and His80 (Figure 1). Site-directed mutagenesis was used to introduce the mutations Arg32 to Ala (R32A) and Gln (R32Q), Arg47 to Gln (R47Q), His78 to Ala (H78A), and His80 to Ala (H80A), into full length GyrA. In vivo activity of the mutants was determined by their ability to complement the GyrAts strain KNK453.21 Derivatives of plasmid pPH322 expressing wild-type GyrA and all five mutants were transformed into KNK453. Transformants were selected for ampicillin resistance at 30 8C, the permissive temperature. Viability was tested at 42 8C, the non-permissive temperature, in the presence of IPTG to induce GyrA expression. Wild-type GyrA and all five of the mutants were able to complement KNK453 in the presence of IPTG. The ability to complement the ts strain demonstrates that the five mutants are functional in vivo but it does not address the question of whether they have wild-type activity. Therefore, the proteins were purified to . 95% purity by fast protein liquid chromatography (FPLC) and further characterized using in vitro assays. Mutant GyrA proteins are less active than wildtype Typical yields of wild-type and mutant GyrAs were . 5 mg/l of bacterial culture. Based on previous work and results presented in the accompanying paper,23,24 the level of wild-type GyrA homodimers in samples of mutant GyrA proteins is insignificant (calculated to be , 0.04%) and can be disregarded in the following experiments. Table 2. Kinetics of ATP hydrolysis GyrA

21 kapp cat (s )

Kapp M (mM)

WT R32A R32Q R47Q H78A H80A

1.4 ^ 0.1 0.21 ^ 0.01 0.41 ^ 0.02 0.26 ^ 0.02 0.33 ^ 0.02 0.64 ^ 0.04

0.52 ^ 0.11 0.56 ^ 0.07 0.52 ^ 0.07 0.52 ^ 0.09 0.43 ^ 0.08 0.49 ^ 0.09

The preferred reaction of DNA gyrase is DNA supercoiling; reactions were studied kinetically in vitro for the mutants and wild-type GyrA in A2B2 complexes with excess GyrB. Comparisons of the rates of supercoiling are shown in Table 1. The mutants all have slower rates than wild-type gyrase; H80A is the most active in supercoiling (6% of wild-type activity) while H78A is the least active (0.4%). Consistent with the idea that a glutamine residue might be able to substitute partially for arginine while alanine cannot, R32Q is more active than R32A. Thus, the mutant enzymes are still active, as demonstrated by in vivo complementation assays, but their activity is decreased greatly relative to that of wild-type GyrA. In vitro, gyrase can convert negatively supercoiled DNA into the relaxed form, in the absence of ATP. The mutants were compared to wild-type on the basis of the amount of mutant enzyme that was required to achieve equivalent levels of relaxation (Table 1). H80A required the same protein concentration as wild-type while the other four mutants required two to five times the protein concentration needed for wild-type. As in the supercoiling experiments, H78A was the least active and R32Q was more active than R32A. The effects of these mutations on DNA relaxation activity are much less profound than their effects on supercoiling. Turnover of gyrase is regulated by ATP hydrolysis and release of ADP; in the wild-type enzyme, the release of ADP by GyrB is the rate-limiting step.6 We used ATPase assays to determine whether the mutations had altered the rate-limiting step for these proteins. ATPase assays with varying ATP concentrations can be fitted to the Michaelis – Menten equation (Table 2). As gyrase is strictly a non-Michaelian enzyme,25 the derived KM and kcat values are apparent values; Kapp is a measure of M the apparent affinity of the enzyme for ATP, which is very similar for wild-type and for the mutants (Table 2). The mutant enzymes have lower kapp cat values, suggesting that, in contrast to the wildtype enzyme, the cleavage – religation reaction may have become the rate-limiting step for these proteins. A trivial explanation for the loss in activity of the mutant proteins is that they have a decreased affinity for DNA. Using nitrocellulose filterbinding assays, we showed that H80A binds DNA with an affinity equivalent to that of wild-type and the other four mutants show less than twofold decreases in affinity (data not shown), eliminating this possibility.

354

DNA Cleavage Reaction of Gyrase

cleavage efficiencies of the mutants are the same (Table 3). Religation rates are not altered significantly

Figure 2. Ciprofloxacin cleavage by mutant GyrA proteins. Negative controls of DNA alone, GyrA alone, and GyrB alone are shown along with cleavage by wild-type and mutant GyrA proteins. Concentrations and GyrA proteins, assayed in A2B2 complexes, are indicated; the DNA concentration was 3.5 nM. Samples were analysed on a 1% agarose gel containing 5 mg/ml chloroquine. Gels were scanned to determine the amount of linear DNA, expressed as a percentage of the wild-type cleavage after correcting for the different enzyme concentrations (Table 3); sn, singly nicked plasmid; lin, linear pBR322, and rel, relaxed pBR322.

DNA cleavage efficiency is reduced for the mutants DNA cleavage can be studied using quinolone drugs or Ca2þ to trap the cleaved DNA complex. An example of ciprofloxacin-stabilised cleavage is shown in Figure 2. From the amount of cleaved product and the enzyme concentration, we determined the decrease in the extent of cleavage relative to the wild-type enzyme with each cleavage agent (Table 3). The mutant proteins required significantly more enzyme to achieve cleavage levels similar to that of the wild type with two quinolones, ciprofloxacin and oxolinic acid, and Ca2þ. As before, H80A is the most active mutant, and R32Q is more active than R32A. However, H78A is not the least active mutant, R32A is the least effective at DNA cleavage. The results are not ligand-specific since ciprofloxacin and Ca2þ produce similar results. The mutants cleave less efficiently with oxolinic acid but the relative

Observing the religation reaction requires starting from an equilibrium population of the cleaved complex. Religation can then be observed kinetically following the addition of NaCl, which allows cleaved covalent complexes to be religated, while preventing the formation of new protein –DNA interactions and new cleaved intermediates.19 We adapted the religation assay to DNA gyrase and used it to study the rate of religation. Religation is a fast reaction that cannot be observed easily at room temperature or 16 8C under these conditions, so reactions were carried out at 4 8C to slow the reaction to an observable rate. The results reveal a two-step reaction (representative results are shown in Figure 3). First, the cleaved complex is converted to a complex with one DNA strand cleaved, producing a nicked product, which is then converted to the circular product with intact DNA strands. The rate of each of these two steps could be calculated for four of the mutants and wild type enzyme.26 Within error, we obtained the same rates from at least two experiments with each A2B2 complex. (We were not able to make a large enough population of the R32A cleaved complex to characterise the kinetics of religation, but it appears similar to R32Q.) Interestingly, the rates for the first step are 50% faster for R32Q, H78A, and H80A than for wild-type, while R47Q is the same as wild-type. The rate for the nicked to closed reaction was the same for all the mutants and wildtype. These results demonstrate that the mutations have not altered the religation step of the reaction significantly. Therefore, they alter the cleavage – religation reaction of the enzyme by decreasing the cleavage activity. Transactivation of DNA cleavage We used an active-site mutant, Y122S, that can bind but is unable to cleave DNA,27 to determine whether residues 32, 47, 78, and 80 cooperate with the Tyr122 on the same or opposite subunit in the GyrA dimer.20 GyrA forms a strong homodimer, but by combining two types of GyrA subunits, denaturing them in guanidinium hydrochloride and refolding them out of urea, we were able to make populations that contained some of the original homodimers in addition to a population of heterodimers. We tested the recovery of active

Table 3. Relative DNA cleavage (%) Cleavage agent

WT

R32A

R32Q

R47Q

H78A

H80A

Ciprofloxacin Oxolinic acid Ca2þ

100 100 100

4^1 1.8 ^ 0.3 3.1 ^ 0.3

10 ^ 3 5^1 11 ^ 2

9^2 4^1 6.2 ^ 0.9

11 ^ 3 3.6 ^ 0.9 5.5 ^ 0.8

32 ^ 6 14 ^ 3 29 ^ 5

DNA Cleavage Reaction of Gyrase

355

Figure 3. Representative religation experiments are shown for 50 nM wild-type GyrA ((a) and (d)), 300 nM R47Q ((b) and (e)), and 150 nM H80A ((c) and (f)). (a)– (c) Experiments were analysed on 1% agarose gels containing 5 mg/ml chloroquine. Reaction times (in minutes) are indicated. Gel bands correspond to cleavage reactions: top to bottom, singly nicked plasmid, linear pBR322, and relaxed pBR322. (d)– (f) The intensity of the nicked and linear bands was quantified and corrected for the nicked population in the DNA alone. The results are plotted as percentages of maximum total cleavage at time zero. Open circles are the linear product and filled squares are the singly nicked product. The results kwere fitted to equations determined for the forward cleavage reaction by Kampranis & Maxwell:26 k2 1 linear DNA ! singly nicked DNA ! closed circular DNA: The linear product is fitted to: ½lin ¼ ½lin0 e2k1 t and the singly nicked product is fitted to: k1 ½sn ¼ ½lin0 ðe2k1 t 2 e2k2 t Þ k2 2 k1

enzyme by submitting wild-type GyrA to this procedure. We observed the same cleavage efficiencies with wild-type GyrA that was untreated and wildtype GyrA that was denatured and refolded, i.e. the recovered enzyme appeared to retain full activity. We combined wild-type GyrA with each of the five mutants or Y122S, and we combined each of the five mutants with Y122S. In a ciprofloxacin cleavage assay, the Y122S heterodimers can produce only nicked products, since there is only one tyrosine residue available for cleavage. If Y122S cooperates with the residues on the same subunit, the mutations at 32, 47, 78, and 80, which

are inefficient at cleavage, should decrease the amount of nicked product. If the Y122S cooperates with the opposite residues at 32, 47, 78, and 80, the mutations at those sites will still permit the production of nicked product; the wild-type residue at 32, 47, 78, or 80 on the Y122S subunit cooperating with the active-site tyrosine residue on the other. The results of ciprofloxacin cleavage assays with the heterodimers are shown in Figure 4. The populations with wild-type gyrase and a mutant are dominated by the efficient cleavage by wild-type GyrA homodimer to produce mostly linear DNA; the predicted wild-type homodimer

356

Figure 4. Ciprofloxacin cleavage with GyrA heterodimers, assayed as (gyrase) A2B2 complexes. Individual GyrA proteins were subjected to refolding: wild-type (50 nM), Y122S, R32A, and H78A (all 200 nM), along with heterodimer populations with either wild-type GyrA, designated WT·mutant (100 nM), or Y122S, designated Y122S·mutant (200 nM). Heterodimer populations consist of homodimers of each GyrA protein in addition to the heterodimer, predicted to be in a 1:1:2 ratio. Samples were analysed on a 1% agarose gel containing 5 mg/ml chloroquine: sn, singly nicked plasmid; lin, linear pBR322; and rel, relaxed pBR322.

concentration is sufficient to produce the observed cleavage. Similarly, the linear DNA observed in the Y122S-mutant reactions is consistent with mutant homodimer cleavage for the predicted homodimer concentration. In the Y122S-mutant reactions, a significant portion of the DNA was singly nicked, strongly supporting the notion that the active-site tyrosine residue is on the opposite subunit to that of the mutant residues, consistent with a model where they interact with the 30 -OH group of the DNA, i.e. the opposite side of the DNA break to the 50 -phosphotyrosine intermediate.

Discussion The X-ray crystal structure of the 59 kDa fragment of GyrA provides structural information that is being used to further elucidate the mechanism of DNA gyrase. Structure-based incorporation of cysteine residues to cross-link GyrA dimers has provided insight into the movement of the transported DNA segment during supercoiling and relaxation, and the relationship between gate

DNA Cleavage Reaction of Gyrase

opening and DNA cleavage.14,28 However, another mechanistic aspect of the gyrase reaction, the roles of individual amino acid residues in the cleavage – religation process, is still not understood. We report the results of experiments designed to test the role of conserved residues in the cleavage – religation reaction. We have made five mutations in GyrA that impair but do not destroy the activity of the enzyme. There are now several crystal structures of the DNA cleavage site region of type II topoisomerases;7,8,29 our mutations are based on the gyrase structure, which is believed to represent the enzyme prior to DNA cleavage. The yeast topo II structures are thought to represent the open conformation of the enzyme during strand passage,7 and an intermediate state29 between the closed conformation observed for gyrase and the open topo II conformation. We mutated residues Arg32, Arg47, His78, and His80, which are highly conserved among type II topoisomerases and contain functional groups likely to interact with DNA. Based on accessibility to the proposed DNA-binding site in the gyrase structure, we mutated Arg47, which extends up to the DNAbinding surface. In contrast, Liu & Wang20 included Arg704 in their alanine mutations; in the gyrase structure, the equivalent residue, Arg46, is buried. The yeast R704A mutation did not complement the ts strain and was active in in vitro assays. Liu & Wang concluded that Arg704 is involved in an ion pair, which plays an architectural role, but is not involved in the reaction directly. The default mutation in this type of study is generally Ala, but we made Arg to Gln mutations at Arg32 and Arg47, which might retain function not provided by Ala. All five mutants, R32A, R32Q, R47Q, H78A, and H80A, complement the GyrA ts strain, despite the supercoiling activities being as low as 0.4% in one case. In the accompanying paper, we find that none of the GyrB mutants complements the GyrB ts strain, even though the supercoiling activities were as high as 20% in one case.24 We do not understand the reasons for these differences, but it demonstrates that there is not a straightforward correlation between in vivo and in vitro activities. The R32A mutant is able to complement the ts strain and perform supercoiling, while the equivalent mutation in yeast, R690A, is unable to perform these functions.20 There are several possible explanations for this observation. Gyrase might provide nearby residues that are better able to substitute for R32A. Alternatively, the yeast enzyme might rely on DNA interactions with Arg690 in active site recognition. The larger binding site of gyrase suggests that other regions of the DNA –protein interface may be important for defining the cleavage site in addition to the active site. If the DNA interactions with the 33 kDa domains of GyrA are present, GyrA might allow some variation in the DNA – protein interactions in the active site. The greater activity of R32Q in all

DNA Cleavage Reaction of Gyrase

quantified reactions compared to R32A suggests that the guanidino functional group is involved in the reactions. However, R32Q does not display wild-type characteristics; Arg32 is likely to participate in other interactions that R32Q cannot. Furthermore, the position of the glutamine side chain might alter the DNA-binding surface. Arg47 extends up to the DNA-binding surface of GyrA. The decreased activity of R47Q can be explained in two ways. The shorter side-chain of Gln relative to Arg may mean that R47Q does not reach the DNA-binding surface. Arg47 could interact with the DNA backbone at the phosphate group adjacent to the cleavage site or with GyrB. Alternatively, the conformation of the glutamine side-chain may interrupt the packing of GyrA in that region and thus interfere with protein – protein and protein– DNA interactions required for activity. Histidine residues are proposed to participate in the reaction to increase the nucleophilicity of the attacking group, either tyrosine or a hydroxyl group, and participate in hydrogen transfer.30 These functions could be water-mediated or performed by lysine residues, as in some type I topoisomerases. Our results suggest that His78 and His80 are involved in the cleavage – religation reaction. The H78A mutation is more detrimental to activity, so it seems to be more important in the reaction. The His78 and His80 side-chains are free in the 59 kDa GyrA crystal structure, but they are involved in interactions in the topo II crystal structures. The intermediate structure29 shows His736 (His80 in GyrA) involved in hydrophobic packing with Ile, Met, and Tyr residues. In the open structure,7 His735 (His78 in GyrA) forms a saltbridge with a glutamate residue. The function of these contacts may be to provide alternate interactions to prevent religation when the DNA is pulled apart. His78 and His80 are very close together and can probably substitute for each other, at least to some degree. Since mutations at these positions impact on cleavage much more than on religation, they may activate the tyrosine residue for attack, rather than the 30 -hydroxyl group. The fact that the mutation at His80 leads to significant reduction in supercoiling activity with no effect on relaxation (Table 1) suggests that DNA cleavage is not the rate-limiting step in the relaxation reaction. One possibility is that the opening of the DNA gate limits the rate of the relaxation reaction. Experiments have been described that allow the separation of the cleavage and religation reactions for type I and II topoisomerases using suicide substrates.31 These experiments have not been feasible with gyrase because of the larger DNAbinding site and the resistance to cleavage near a nick in the DNA.32 While the experiments cannot be translated to gyrase, the models they propose are worth considering in light of the religation experiment reported here. In the suicide substrate experiments, the cleavage reaction produces a

357

short DNA fragment, typically three bases, that is able to diffuse away. Religation can occur when a single strand is introduced. These results suggest that the interactions with the 30 end of the DNA are more important in cleavage than during religation. All five gyrase mutations have a slower cleavage reaction, which affects supercoiling, relaxation, and ATPase rates, and decreases cleavage activity. However, they show approximately wild-type religation rates, consistent with a model where the DNA interactions with these residues are more important in the cleavage part of the reaction than religation. Transactivation is shown for all five mutants; residues 32, 47, 78, and 80 all interact with the Tyr122 on the opposite subunit of the GyrA dimer. Both of the GyrA subunits are required to cleave each strand of the DNA helix. This suggests a regulatory mechanism that ensures cleavage of both strands of a DNA double helix. Alignment of the DNA and both GyrA monomers is required, in addition to the GyrB monomers, to allow cleavage and the supercoiling reaction to proceed. By using the GyrA dimer interface to cleave the DNA, the realignment of the interface would align the DNA ends for religation. The transactivation experiments required the formation of GyrA heterodimers; GyrA, like topo II, forms a very stable dimer.33 To form heterodimers, the proteins either need to be co-expressed or denatured and recombined. We chose the latter method, because previous work had shown the ability to refold denatured GyrA.14,18,28 By analogy to other reactions of this type, where a phosphate group interacts with a tyrosine residue to form a phosphotyrosine intermediate, we can begin to propose a reaction mechanism for cleavage and religation.30 Our proposal is consistent with previous results that suggest that the GyrB region of type II topoisomerases contributes to the DNA cleavage site34 (see the accompanying paper24). Two moieties are proposed to promote nucleophilic attack on the phosphorus atom due to an inductive effect upon the electrons around it, and stabilise the charge on the oxygen atoms during the transition states.30 In type IA topoisomerases, these are proposed to be two Arg residues. We propose that these moieties in gyrase are Arg32 and magnesium ions coordinated by GyrB.24 As described above, His78 is actively involved in the reaction, perhaps supported by His80. This leaves another acid/base moiety with functionality similar to that of His78, as yet undetermined in gyrase. It could be water, or an amino acid residue (either Lys or His), which could be supplied by GyrB. In the accompanying paper24 we show that amino acid residues in GyrB are involved in the cleavage/religation reaction of gyrase, probably by coordination of two metal ions and we propose a scheme consistent with the results from both papers.

358

Materials and Methods Materials DTT was from Melford Laboratories; XL-10 Gold competent cells were from Stratagene. DNA oligonucleotides used for site-directed mutagenesis and sequencing were obtained from PNACL (University of Leicester). GyrB, GyrA mutant Y122S, and negatively supercoiled and relaxed forms of plasmid pBR322 were gifts from Mrs A. J. Howells (University of Leicester).

Site-directed mutagenesis The Quikchange protocol (Stratagene) was used to introduce amino acid changes into full-length GyrA by site-directed mutagenesis of plasmid pPH3.22 The identity of the mutants was confirmed by restriction digests and DNA sequence analysis performed by PNACL (University of Leicester). GyrA proteins were purified as described.35 GyrA proteins were expressed in XL-10 Gold cells with 2% (w/v) glucose in the medium; expression was induced with 100 mM IPTG. Wild-type GyrA and R32A were pure after the Hi-Load Q column, but R32Q, R47Q, H78A, and H80A were further purified on a phenyl-Sepharose column (Amersham Pharmacia).

Complementation tests Plasmid pPH3 and its mutant derivatives were transformed individually into E. coli KNK453,21 which carries a temperature-sensitive gyrA mutation. Single colonies were picked and replica-plated onto gradient plates containing 0 – 100 mM IPTG to induce GyrA expression on pPH3. One set of plates was incubated at 30 8C and the other set at 42 8C.

Enzyme assays Supercoiling assays were performed as described.18 ATP-independent relaxation was carried out as described for supercoiling, except that ATP and spermidine were omitted, the MgCl2 concentration was increased to 8 mM, and negatively supercoiled pBR322 replaced relaxed pBR322. Cleavage assays were performed as described for supercoiling, except that ATP was omitted. Quinolone cleavage included either 200 mM ciprofloxacin or 500 mM oxolinic acid. In Ca2þ-stabilized cleavage assays, 4 mM MgCl2 was replaced by 4 mM CaCl2. Cleavage reactions were performed at 25 8C for 30 minutes. Reactions were terminated and electrophoresed as described.18 The DNA concentration was 3.5 nM for supercoiling, relaxation, cleavage, and religation experiments. Enzyme concentrations varied and are given in the Figure legends. Chloroquine (5 mg/ml) was present in the gel and running buffer for cleavage assays. Band intensities were quantified using the GeneTools software on a Syngene Gel Documentation system. ATPase assays were performed using a PK/LDH linked assay,6 with the modifications described by Williams & Maxwell,14 except that the PEP concentration was 3.2 mM. Filter-binding assays were carried out as described.12 Religation assays were initiated with Ca2þ cleavage as described above; after 30 minutes at 25 8C the reactions were transferred to 4 8C. Time-courses were carried out at 4 8C and started with the addition of NaCl to 300 mM. Time-points were stopped with SDS and

DNA Cleavage Reaction of Gyrase

proteinase K as described for cleavage assays,18 and analysed by electrophoresis. The subunits were recombined for the transactivation experiments by denaturing the proteins together in EB (50 mM Tris – HCl (pH 7.5), 100 mM KCl, 10% (w/v) glycerol, 1 mM EDTA, 2 mM DTT) without glycerol and 8.6 M guanidinium hydrochloride for three hours at 37 8C. Glycerol was then added to restore a concentration of 10%. The proteins were dialyzed into EB with 8 M urea overnight at 4 8C. The proteins were refolded out of urea by dialysis overnight at 4 8C into EB. Proteins were concentrated using a Nanosep concentrator (Pall Filtron). Ciprofloxacin cleavage assays were performed as described above.

Acknowledgments S.C.H. is a recipient of a Burroughs Wellcome Fund Hitchings-Elion Fellowship. We thank Alison Howells for providing proteins and DNA, and David Hooper for E. coli strain KNK453. We thank Timothy Lohman, Christian Noble, Melisa Wall and Nicola Williams for comments on the manuscript.

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Edited by J. Karn (Received 14 September 2001; received in revised form 17 January 2002; accepted 6 February 2002)