the atp-operated clamp of human dna topoisomerase iiα: hyperstimulation of atpase by...

UNCORRECTED PROOF

The ATP-operated Clamp of Human DNATopoisomerase IIa: Hyperstimulation of ATPase by“Piggy-back” Binding

Spencer Campbell and Anthony Maxwell*

Department of BiochemistryUniversity of LeicesterLeicester LE1 7RH, UK

We have constructed a series of clones encoding N-terminal fragments ofhuman DNA topoisomerase IIa. All fragments exhibit DNA-dependentATPase activity. Fragment 1–420 shows hyperbolic dependence of ATPaseon DNA concentration, whereas fragment 1–453 shows hyperstimulationat low ratios of DNA to enzyme, a phenomenon found previously withthe full-length enzyme. The minimum length of DNA found to stimulatethe ATPase activity was ,10 bp; fragments $32 bp manifest the hyper-stimulation phenomenon. Molecular mass studies show that fragment1–453 is a monomer in the absence of nucleotides and a dimer in thepresence of nucleotide triphosphate. The results are consistent with therole of the N-terminal domain of topoisomerase II as an ATP-operatedclamp that dimerises in the presence of ATP. The hyperstimulation effectcan be interpreted in terms of a “piggy-back binding” model for protein–DNA interaction.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: DNA gyrase; supercoiling; anti-tumour drugs; DNA–proteininteractions*Corresponding author

Introduction

The DNA topoisomerases have evolved to over-come the topological complexities of DNA.1 Theypass one DNA segment through a transient breakin another DNA segment, and are therefore ableto alter the topological state of DNA. The import-ance of DNA topoisomerases is testified to bytheir ubiquity in nature; they have been found inall organisms studied to date, and are involved inthe maintenance of the level of intracellular DNAsupercoiling, removing supercoils that build upahead of and behind transcription and replicationcomplexes, and the decatenation of daughter

chromosomes following replication. Topoisomer-ases are able to cleave and subsequently reseal thephosphodiester backbone of a DNA strand, whichinvolves a transesterification reaction between anactive-site tyrosine hydroxyl group and a phos-phoryl group on the DNA strand, forming a phos-photyrosine linkage. In this manner, the bondenergy is conserved and no energy cofactor isrequired. Conformational changes within theenzyme–DNA covalent complex allow the DNAends to be separated, which creates a gate throughwhich another DNA strand can be passed. On thebasis of their mechanisms of action and alignmentof their amino acid sequences, topoisomerases canbe grouped into four families: types IA and IB,and types IIA and IIB. Type II enzymes aregenerally dimeric, and can cleave both strands ofa double-helical DNA molecule; their reactionsgenerally require the hydrolysis of ATP. As aconsequence of their essential roles in cells, topo-isomerases have become important drug targets,e.g. eukaryotic topoisomerase (topo) II is the targetof a variety of anti-tumour drugs, includingamsacrine, epipodophyllotoxins and merbarone,2,3

bacterial DNA gyrase is the target of a range ofanti-bacterial agents.4

Type IIA enzymes are evolutionarily andstructurally related, each possessing two distinct

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

Present addresses: S. Campbell, De NovoPharmaceuticals, Compass House, Vision Park, ChiversWay, Histon, Cambridge CB4 9ZR, UK; A. Maxwell,Department of Biological Chemistry, John Innes Centre,Norwich Research Park, Colney Lane, Norwich NR47UH, UK.

E-mail address of the corresponding author:[email protected]

Abbreviations used: ADPNP, 50-adenylyl-b,g-imidodiphosphate; GyrA, DNA gyrase A protein; GyrB,DNA gyrase B protein; DMS, dimethylsuberimidate;DSG, disuccinimidyl glutarate; BS3,bis(sulphosuccinimidyl) suberate; IPTG, isopropyl-b-D-thiogalactopyranoside; topo, topoisomerase.

doi:10.1016/S0022-2836(02)00461-8 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 320, 171–188

UNCORRECTED PROOF

catalytic centres: a DNA cleavage and rejoiningsite, and a site for ATP hydrolysis.5 – 9 The enzymesdiffer in their molecular mass and subunit compo-sition, e.g. Escherichia coli DNA gyrase consists oftwo subunits (GyrA and GyrB), whereaseukaryotic type II enzymes are homodimers, eachmonomer can be regarded as a fusion of GyrB andGyrA. Homology between eukaryotic and pro-karyotic enzymes is closest in the region containingthe active site for DNA cleavage (corresponding tothe N-terminal domain of GyrA) and the ATPaseactive site (corresponding to the N-terminaldomain of GyrB).

The mechanism of eukaryotic topo II involvesthe binding of the enzyme to two segments ofDNA. The gate (or G) segment (,25 bp) is cleavedin both strands with a four base stagger betweenthe break sites, leading to the formation of covalentbonds between the 50 phosphate groups at thebreak site and the active-site tyrosines. The trans-ported (or T) segment is captured by an ATP-operated clamp (comprising the N-terminaldomains of the two subunits) that presents the Tsegment to the double-stranded break in the G seg-ment, facilitating strand passage. Resealing of thebreak changes the linking number of the DNA by2, in the case of intramolecular reactions (e.g.DNA relaxation), or catenation/decatenation, inthe case of intermolecular strand passage.10

The mechanism of topo II is now understood inoutline, but the role of ATP hydrolysis remains tobe clarified. ATP hydrolysis normally drives reac-tions that are energetically unfavourable; in thecase of DNA gyrase, the requirement for ATPhydrolysis is clear. Gyrase introduces negativesupercoils into DNA, an energetically unfavour-able reaction that is coupled to ATP hydrolysis.Eukaryotic topo II cannot supercoil DNA butrelaxes DNA in an ATP-dependent reaction. Giventhat this is an energetically favourable reaction, itis unclear why ATP is required. Studies on thedecatenation, unknotting and relaxation reactionsof type II enzymes have shown that topo II (froma variety of sources) is apparently able to generatenon-equilibrium distributions of topoisomers.11

This behaviour can be rationalised by invokingthe free energy of ATP hydrolysis to drive the reac-tions away from equilibrium. A tracking mechan-ism has been proposed to account for theseobservations.11 Other mechanisms suggested toexplain this phenomenon include, kinetic proof-reading12,13 and the generation of a sharp bend inthe G segment upon topo II binding.14

The ATPase reaction of topo II has been analysedusing both full-length enzyme and the isolatedN-terminal domain. Studies on full-length topo IIprepared from HeLa cells, Drosophila melanogasterand calf thymus showed the enzyme to possess aDNA-dependent ATPase activity;15 – 17 the degreeof stimulation by DNA was threefold to 17-fold.The ATPase reaction of yeast (Saccharomycescerevisiae ) topo II has an intrinsic ATPase activitythat is stimulated 19-fold by DNA.18 Pre-steady-

state kinetics have shown that the enzyme bindsand hydrolyses two ATP molecules per reactioncycle. One of the ATP molecules is hydrolysedrapidly, prior to the rate-limiting step of the reac-tion (ADP release). The second ATP molecule ishydrolysed only after the release of the first ADPmolecule.19,20

The ATPase activity of human topo IIa is stimu-lated by DNA and shows apparent Michaelis–Menten kinetics.21 Although the ATPase activity islower than that of yeast topo II, it is more active indecatenation, implying more efficient coupling ofthe ATPase to DNA strand passage. Using plasmidpBR322 as the DNA cofactor, the reaction showshyperstimulation by DNA at a base-pair to enzymedimer ratio of 100–200:1. When DNA fragmentswere used as cofactors, the reaction required,100 bp to stimulate the activity and fragments of,300 bp showed hyperstimulation. This behaviourcan be rationalised in terms of the enzyme requir-ing fragments that can bind to both the DNA gateand the ATP-operated clamp in order for theATPase reaction to be stimulated. Hyperstimula-tion was suggested to be a consequence of the sat-uration of DNA with enzyme, i.e. possibly due toprotein–protein interaction between adjacent topoII dimers.21 The physiological significance of thisphenomenon is unknown but could be related tothe role of topo II as part of the chromosome scaf-fold where protein–protein interactions betweentopo II dimers are likely to occur.22,23

The ATPase reaction of DNA gyrase has beeninvestigated, focusing particularly on the ATPasereaction of the 43 kDa N-terminal ATPase domainof GyrB.24,25 It was found that this domain is amonomer in the absence of nucleotide butdimerises in the presence of ATP or ADPNP(50-adenylyl-b,g-imidodiphosphate). The ATPaseactivity is distinctly non-Michaelian, and theATPase and binding data are consistent with theactive form of the enzyme being a dimer, anddimerisation being the rate-limiting step of theATPase reaction. In conjunction with the X-raycrystal structure of this domain complexed withADPNP,26 these data support the idea that theN-terminal domain of GyrB and, by implication,the N-terminal domain of other type II topo-isomerases, acts as an ATP-operated clamp in thetopoisomerase reaction cycle.

To study the ATPase activity of human topo IIa,Gardiner et al. made an N-terminal 52 kDafragment, comprising residues 1–439, plus 17additional residues introduced during cloning.27

This fragment showed a DNA-stimulated ATPaseactivity that obeyed Michaelis–Menten kineticsand showed a hyperbolic dependence of ATPaseactivity on the concentration of DNA. No hyper-stimulation of the ATPase activity was observed.The results were interpreted in terms of the role ofthis domain as an ATP-operated clamp; the inter-action of the protein with the T segment wassuggested to be responsible for the DNA depen-dence. This protein was found to be relatively

172 ATPase of Human Topoisomerase IIa

UNCORRECTED PROOF

insoluble. Olland & Wang28 made two N-terminalfragments of yeast topo II. Fragment 1–409 waspredominantly monomeric and had a low-levelATPase activity that was DNA-independent (at100 mM NaCl). Fragment 1–660 was found to bepredominantly dimeric and had an ATPase activitythat was stimulated by DNA. At 50 mM NaCl, theATPase activity showed hyperstimulation at lowratios of DNA to protein. The DNA-dependenceof the ATPase activity was attributed to interactionwith the G segment. Recently, Hu et al.29 have madean N-terminal construct of human IIa consisting ofthe first six amino acid residues of yeast topo II fol-lowed by residues Ser29-Lys425 of the humanenzyme and a C-terminal -His6 tag. This fragmentshows DNA-dependent ATPase activity, but nohyperstimulation was reported.

In order to investigate further the ATPaseactivity of topo II, we have made a series of con-structs encoding N-terminal fragments of humantopo IIa. We interpret the behaviour of thesefragments in terms of the role of the N-terminaldomain of topo II as an ATP-operated clamp.We observe hyperstimulation of the ATPaseactivity at low ratios of DNA to enzyme, whichcan be explained by “piggy-back” binding, atheoretical model for ligand–protein–DNAinteraction.30

Results

Cloning and expression of N-terminalfragments of human topo IIa

The N-terminal domain of topo II constitutes anATP-operated clamp, which is responsible forbinding the T segment during the topoisomerasereaction.31 A number of constructs have previouslybeen made comprising this domain and theATPase reaction has been studied.27 – 29,32 However,the properties of these protein fragments differdepending on the construct. In order to rationalisethese differences and to further understand the

ATPase activity of this domain, we made a seriesof N-terminal constructs of human topo IIa. Theresultant proteins contained N-terminal thio-redoxin fusions in order to overcome the solubilityproblems encountered previously.

The exact location of the domain boundarybetween the N-terminal domain and the centralDNA breakage–reunion domain is uncertain, so anumber of constructs were made (Table 1). Therationale for these constructs was as follows. Resi-due Gln420 corresponds to the final residue of theN-terminal fragment of yeast topo II (Gln410), asdetermined by proteolysis.33 Residue Lys425 corre-sponds to the final residue of the N-terminaldomain of Escherichia coli GyrB (Arg393).34 Theclones expressing residues 1–453, 454 and 455were constructed in the light of evidence fromlimited proteolysis of human DNA topoisomeraseIIb.35 This enzyme, which shows a high level ofsequence homology to the a isoform, is cleaved bySV8 protease following residue Glu470, which cor-responds to residue Glu454 in the a isoform.These constructs were made initially as fusion pro-teins with enterokinase cleavage sites.36 However,we found that the enterokinase cleavage stepcaused cleavage within the topo IIa sequence; soalternative fusion constructs were made involvingfactor Xa cleavage (Table 1). All the purified fusionconstructs showed DNA-dependent ATPaseactivity, with fragments 1–420 (47.9 kDa) and1–453 (51.5 kDa) showing the highest ATPaseactivity for the short and long constructs, respect-ively. Therefore, these two proteins were cleavedwith factor Xa and re-purified; both proteinsretained their DNA-dependent ATPase activityand were subjected to further study.

ATPase activity of N-terminal fragments

In the absence of DNA, the rate of ATP hydroly-sis of the topoIIa(1–420) fragment is linearlydependent upon enzyme concentration (as wasfound with the 52 kDa fragment in the work byGardiner et al.27), having a turnover number of

Table 1. ATPase activity of N-terminal fragments of human topo IIa

Kinetic parameters

HyperstimulatedATPase activity No DNA

250 bp/dimermolecule

50 bp/dimermolecule

N-terminalfragment(amino acidresidues)

DNA-dependentATPase activity

Fusionprotein

Cleavedprotein

Vmax

(nM/s)KM

(mM)Vmax

(nM/s)KM

(mM)Vmax

(nM/s)KM

(mM)

1–420 U £ £ 1.54(^0.06)

0.28(^0.04)

8.67(^0.33)

0.35(^0.04)

NT NT

1–425 U £ NTa NT NT NT NT NT NT1–453 U U U 9.05

(^0.33)0.69

(^0.06)88.13

(^6.97)0.90

(^0.19)137.85

(^10.79)0.76

(^0.15)1–454 U U NT NT NT NT NT NT NT1–455 U U NT NT NT NT NT NT NT

a Not tested.

ATPase of Human Topoisomerase IIa 173

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0.012 s21 (per dimer) at 2 mM ATP (data notshown). By contrast, the ATPase activity oftopoIIa(1–453) exhibits a greater than first-orderdependence on enzyme concentration (Figure 1),indicative of an oligomerisation process occurringduring the course of the ATPase cycle. Non-lineardependence of ATPase activity upon enzyme con-centration was seen with the 43 kDa fragment ofE. coli DNA gyrase24 and the N-terminal domainof eukaryotic topo II,28,29 and is assumed to be a

result of a dimerisation step in the ATPase cycle(Figure 1(b)).

The ATPase activities of both the 1–420 and the1–453 fragment were found to be DNA-dependent;under these conditions, the rates of both fragmentswere linearly dependent upon enzyme concen-tration, implying that the dimeric form of theenzyme is stabilised in the presence of DNA.Figure 2 shows the ATPase activities of fragments1–420 and 1–453 in the presence and in the

Figure 1. ATPase activity oftopoIIa(1–453) as a function ofenzyme concentration. (a) TheATPase activity of the N-term-inal fragment topoIIa(1–453)was determined at various con-centrations of enzyme understandard conditions in theabsence of DNA. The lineshown is a theoretical curvebased on the scheme andequation given by Ali et al.24

(b) A model for the ATPasecycle. In this scheme, ATP isshown binding to the mono-meric protein (orange), whichinduces a conformationalchange that allows the enzymeto dimerise. The active dimercomplex hydrolyses ATP,which leads to product releaseand dissociation into the mono-meric form.24


UNCORRECTED PROOF

absence of DNA at a range of concentrations ofATP; from these data, apparent KM and kcat valuescan be determined (Table 1). For the shorterfragment (1–420) in the presence of 250 bp/dimermolecule, DNA causes kcat to increase approxi-mately sixfold, whilst KM is largely unaffected; thevalues of kcat and KM are comparable with thosefor the 52 kDa fragment.27 For the longer fragment(1–453), the presence of DNA at 250 bp/dimermolecule causes kcat to increase approximately ten-fold, whilst KM is largely unaffected. Surprisingly,the presence of DNA at 50 bp/dimer moleculeleads to a larger increase in kcat (,15-fold), aphenomenon observed with full-length topo IIaand termed “hyperstimulation”.21 Table 1 showsthat the longer protein fragments exhibited hyper-stimulated ATPase activity, whereas the shorterones did not. The values of kcat in the absence ofDNA and at 250 bp/dimer molecule are similar tothose determined for the full-length enzyme:0.055 s21/dimer in the absence of DNA and0.59 s21/dimer in the presence of excess DNA; theKM of the full-length enzyme in the presence ofexcess DNA is 0.56 mM, which compares to

0.90 mM seen here with topoIIa(1–453) in thepresence of DNA at 250 bp/dimer molecule. Over-all, it can be seen that the 1–453 fragment displayscatalytic properties similar, in terms of ATP turn-over, to that of the full-length enzyme, whereasthe shorter 1–420 fragment displays catalyticproperties similar to that of the 52 kDa protein.27

The ATPase activity of the shorter fragmenttopoIIa(1–420) was determined as a function ofpBR322 concentration (Figure 3(a)). This fragment,like the 52 kDa protein, does not show any hyper-stimulation of the ATPase rate at low ratios ofDNA to enzyme, and requires a large excess ofDNA to maximally stimulate the ATPase activity.In Figure 3(b), the ATPase activity of topoIIa(1–453) is shown as a function of the concentration ofpBR322 DNA. The ATPase activity is stimulatedby DNA up to a maximum, which occurs at,50 bp/dimer molecule, whereupon the ATPaserate decreases as the concentration of DNAincreases, reaching a plateau at ,150 bp/dimermolecule, above which the ATPase rate remainsconstant. This pattern of ATPase rate versus theratio of base-pair to dimer is very similar to that

Figure 2. ATPase activities oftopoIIa(1–420) and topoIIa(1–453)as a function of ATP concentrationin the presence and absence ofDNA. (a) The ATPase activity of 1–420 was determined at 250 nMwith various concentrations of sub-strate (ATP) under standard con-ditions in the absence and in thepresence of DNA (relaxed pBR322)at 250 bp/dimer molecule. (Thetotal concentration of Mg2þ wasmaintained at 1 mM greater thanthe concentration of ATP in theassay.) The data were fitted to theMichaelis–Menten equation bynon-linear regression. The derivedvalues of KM and kcat are given inTable 1. (b) The ATPase activity of1–453 was determined at 250 nMwith various concentrations of ATPin the absence and in the presenceof DNA (relaxed pBR322) at 50 bp/dimer molecule and 250 bp/dimermolecule, as described for (a).


UNCORRECTED PROOF

seen with the full-length enzyme,21 albeit with thepeak and plateau levels at different ratios of base-pair to dimer. It is clear from these data that thepresence of residues 421–453 is having a signifi-cant effect on the intrinsic ATPase activity of theN-terminal fragment of human topoisomerase IIa,and results in the hyperstimulated ATPase rate,which peaks at a base-pair to dimer ratio of 50using pBR322.

ATPase activity as a function of DNA length

To further investigate the DNA length-depen-dence of the DNA-stimulated ATPase activity oftopoIIa(1–453), DNA fragments of various lengthsfrom 8 bp to 140 bp were used in ATPase assays.All DNA molecules were based symmetricallyaround the preferred DNA gyrase cleavage site inpBR322, the “990” site.37 To determine the mini-

mum length of DNA required to stimulate theATPase activity, assays were performed with DNAfragments of 8–24 bp. These experiments wereconducted at 37 8C and 25 8C, because the shorterfragments are likely to melt at 37 8C. At 37 8C(Figure 4(a)), addition of DNA fragments of 12–24 bp stimulates the ATPase rate, although the12 bp and 14 bp DNA fragments are required inlarge excess over the enzyme in order to maximallystimulate the rate. The 10 bp DNA fragment, how-ever, causes only a slight increase in ATPase in thepresence of excess DNA; this fragment is likely todissociate into single strands at this assay tempera-ture. At 25 8C, the longer DNA fragments give thesame pattern of stimulation of the ATPase rate asat 37 8C, although the actual rates are reducedbecause of the lower temperature. The 10 bp frag-ment stimulates the ATPase rate to a greater extentthan was seen at 37 8C, presumably because more

Figure 3. ATPase activity as afunction of DNA concentration.The ATPase activity of (a)topoIIa(1–420), and (b) topoIIa(1–453) were determined at 250 nMunder standard conditions withvarious concentrations of DNA(relaxed pBR322 in (a), and relaxedpBR322 and a 140 bp fragment in(b)), expressed as bp/dimermolecule.


UNCORRECTED PROOFFigure 4. ATPase activity of topoIIa(1–453) as a function of the concentration of short DNA fragments. ATPase activity was determined at 250 nM under standardconditions at (a) 37 8C and (b) 25 8C, with various concentrations of DNA fragments (8–24 bp) as indicated, expressed as DNA molecules/enzyme dimer.

UNCORRECTED PROOF

of the DNA is in the double-stranded form. An8 bp fragment causes a slight stimulation of theATPase activity at 25 8C, similar to that seen withthe 10 bp fragment at 37 8C. Therefore, it seemslikely that a DNA fragment of 8–10 bp is sufficientto stimulate the ATPase activity of topoIIa(1–453).However, it is possible that a longer DNA frag-ment, i.e. 12–14 bp, is required to maximallystimulate the ATPase activity.

In order to investigate the length-dependence ofthe hyperstimulation effect, ATPase experimentswere performed with a range of longer DNA frag-ments (Figure 5). Experiments were carried outwith DNA fragment concentrations from twoenzyme dimers/DNA fragment (i.e. excessenzyme), up to four DNA fragments/dimer (i.e.excess DNA). In the case of the 20 and 30 bp frag-ments, increasing the amount of DNA presentcaused an increase in the rate of ATP hydrolysis.However, for fragments of 40 bp and longer,increasing the concentration of DNA caused theATPase rate to decrease. Therefore, the hyper-stimulation of the ATPase activity would appearto require a DNA fragment of ,40 bp or greater.

To determine the exact length of DNA requiredto cause hyperstimulation, DNA fragments of 28–44 bp were used in ATPase assays (Figure 6). Theenzyme concentration was kept constant and theDNA concentration was varied up to a 20-foldexcess of DNA fragment per enzyme dimer. Thehyperstimulation effect is absent with a DNA frag-ment of 28 bp, peaks with a DNA fragment of38 bp, and is diminished, but not abolishedaltogether, with a DNA fragment of 44 bp. Further-more, at high concentrations of the longer DNAfragments ($32 bp), the ATPase rate drops signifi-cantly. This effect was not seen with shorter DNA

fragments (,30 bp), although with these frag-ments the ATPase rate did not reach the samelevel as that seen with high concentrations ofpBR322 (Figure 3(b)). Thus, the length of the DNAfragment determines whether the hyperstimulationphenomenon occurs, and the plateau level ofDNA-stimulated ATPase activity.

To further explore the hyperstimulationphenomenon, a 140 bp fragment was used. TheATPase activity of topoIIa(1–453) in the presenceof different concentrations of the 140 bp fragmentis shown in Figure 3(b), alongside the data forpBR322. The ATPase rate rises sharply withincreasing concentrations of the 140 bp fragment,up to a maximum at ,40 bp/dimer molecule,after which the ATPase rate decreases with increas-ing DNA concentration, and reaches a plateau at,200 bp/dimer molecule. The initial sharp riseand then fall in the ATPase rate is similar to thatseen in the presence of pBR322 at low ratios ofbase-pair to dimer. However, high concentrationsof the 140 bp DNA fragment cause the ATPaserate to fall to a level that is significantly lowerthan that seen with high concentrations ofpBR322, and represents only an ,75% stimulationover the intrinsic ATPase rate; this level is compar-able to that for the short DNA fragments shown inFigure 6.

Interaction with nucleotides and DNA

The ATPase data described above suggest thatthe 1–453 fragment dimerises during the ATPasecycle and, in the dimeric form, interacts withDNA. To explore these ideas further, we embarkedon a series of studies to probe the interaction of thisfragment with nucleotides and DNA.

Figure 5. ATPase activity oftopoIIa(1–453) as a function of theconcentration of DNA fragments.The ATPase activity was deter-mined at 250 nM enzyme understandard conditions with variousconcentrations of DNA fragmentsas indicated, expressed as numberof DNA molecules/dimer.


UNCORRECTED PROOF

The oligomeric state of the protein was investi-gated using three techniques: protein cross-linking,gel-filtration and analytical ultracentrifugation.Three protein cross-linking agents were used totry to resolve the oligomeric state of the protein inthe presence and absence of nucleotides andDNA: dimethyl suberimidate (DMS, an imidoestercross-linker), disuccinimidyl glutarate (DSG, anNHS-ester cross-linker) and bis(sulphosuccinimi-dyl) suberate (BS3, an NHS-ester cross-linker).This approach has been used with the 43 kDa frag-ment of GyrB24 and the 52 kDa fragment of humantopo IIa.27 With all three cross-linking agents, nohigh molecular mass cross-linked species wereseen in the absence of nucleotide, whereas cross-linked species were seen in the presence of

ADPNP (data not shown). With ATP and ADPplus phosphate, cross-linked bands with electro-phoretic mobility similar to that seen with ADPNPwere seen, but these were present at lower concen-trations. On the basis of only the electrophoreticmobility of the cross-linked bands, it is not possibleto say for certain whether they represent dimers(the most likely explanation) or a mixture of oligo-meric species. No high molecular mass specieswere seen with any of the cross-linking agents inthe presence of DNA (relaxed pBR322) at 50 bp/dimer molecule, or at 250 bp/dimer molecule(data not shown), which suggests that the inter-action of the N-terminal fragment topoIIa(1–453)with DNA alone does not cause the protein todimerise.

Figure 6. ATPase activity of topoIIa(1–453) as a function of the concentration of long DNA fragments. The ATPaseactivity was determined at 250 nM under standard conditions with various concentrations of DNA fragments (28–44 bp) as indicated, and expressed as number of DNA molecules/dimer: (a) 28–34 bp fragments; (b) 36–44 bpfragments.


UNCORRECTED PROOF

Gel-filtration was used to determine the nativemolecular mass of the protein topoIIa(1–453)alone or complexed with ADPNP. The protein wasincubated with or without 2 mM ADPNP for onehour and applied to a pre-equilibrated SephacrylS-200 HR (16/60) FPLC column in the presence orin the absence of 0.1 mM ADPNP; fractions werecollected and analysed by SDS-PAGE. The proteinalone eluted as a single main peak, whereas, inthe presence of ADPNP, two main peaks wereobserved that eluted earlier than the peak seen forprotein alone, indicating an ADPNP-inducedincrease in molecular mass. The molecular mass ofthe single peak for protein alone was estimated tobe ,83 kDa, and ,136 kDa, and ,231 kDa for thetwo peaks in the presence of ADPNP. Given thatno high molecular mass species were seen incross-linking studies without nucleotide, it is feas-ible that the single peak eluting at ,83 kDa corre-sponds to the monomeric species. The peak at,136 kDa would therefore be consistent with theprotein being a dimer, and the peak at ,231 kDacould correspond to a tetramer (or a higher-orderoligomeric species). However, the single peakeluting at ,83 kDa could correspond to amonomer–dimer equilibrium.

Samples were prepared for analytical ultra-centrifugation following gel-filtration. Fractionscorresponding to the most prominent peak foreach sample (i.e. the peak at ,83 kDa for proteinalone and the peak at ,136 kDa for protein–ADPNP) were used in analytical ultracentrifuga-tion experiments. The molecular mass values thatwere obtained for the protein ^ADPNP were52,204(^1460) Da and 103,338(^2150) Da, respect-ively. These studies show conclusively that, underthese conditions, the protein alone is a monomerwhereas in the presence of ADPNP it is a dimer.

The interaction of topoIIa(1–453) with DNA wasinvestigated using filter-binding. Samples ofprotein were pre-incubated in the presence andabsence of relaxed [3H]pBR322 or ADPNP and,

after 30 minutes, either DNA or ADPNP wasadded, or no addition was made, and the sampleswere incubated for a further 30 minutes; sampleswere then analysed by nitrocellulose filter-binding.The interpretation of the results (Figure 7) is com-plicated by the fact that multiple proteins can bindto the same DNA molecule. Protein alone bindsDNA with a relatively low affinity, and DNA bind-ing does not reach saturation at 1 mM protein(Figure 7, white squares). Where the protein isfirst incubated with DNA, and then ADPNP isadded to dimerise the protein, the extent of DNAbinding is increased and begins to saturate at,1 mM protein (Figure 7, grey circles). When theprotein clamp is first locked with ADPNP, andthen DNA is added (Figure 7, black triangles), theextent of DNA binding is similar to that seenwhen DNA is added before the ADPNP. This resultsuggests that the nature of the protein–DNA inter-action is the same whether the DNA is addedbefore or after the protein dimerises and theclamp is locked.

Limited proteolysis of a protein in its native statecan be used to probe the higher-order structure ofthe protein, and to monitor ligand-induced confor-mational changes in protein structure. This tech-nique was previously used to show that yeasttopo II undergoes allosteric interdomainal move-ments following ATP binding.33 Furthermore,limited proteolysis of the 43 kDa N-terminal frag-ment of E. coli DNA gyrase revealed that thepresence of ADPNP protects a 33 kDa N-terminalfragment from the further digestion seen in theabsence of nucleotide.25 The difference in proteoly-sis pattern seen in these two cases is interpretedas being caused by the ADPNP-induced dimerisa-tion of the N-terminal domains.31

To probe the conformation of topoIIa(1–453) inthe presence of nucleotides and DNA, the proteinwas first incubated with ligand for one hour, thentrypsin was added and samples were removedover a two hour period and analysed by

Figure 7. Filter-binding assay forDNA binding by topoIIa(1–453).Samples (50 ml) containing up to1 mM protein were incubated at25 8C in the presence or absence ofrelaxed [3H]pBR322 (3.62 nM) orADPNP (2 mM). After 30 minutes,either DNA or ADPNP was addedas above, or no addition was made,and the samples were incubatedfor a further 30 minutes at 25 8C,before being subjected to filter bind-ing. The amount of tritiated DNAretained on the filter disc was deter-mined by scintillation counting.


UNCORRECTED PROOF

SDS-PAGE (Figure 8). In the absence of ligand,most of the protein is degraded to small peptideswithin two hours. With the protein–ADPNPcomplex, however, the protein is more resistant toproteolysis and two protein fragments of ,40 kDaand ,35 kDa are protected from further digestion(Figure 8). In the presence of ATP, the proteolysispattern is distinct from that in the presence ofADPNP, indicating that the protein is more sus-ceptible to proteolysis in the presence of ATP. Thispartial protection is to be expected, as ATPhydrolysis would lead to dissociation of enzymedimers and loss of the protein conformation lead-ing to proteolytic protection. Incubation with ADPalone or ADP plus phosphate (data not shown)produced a pattern of proteolytic digestion similarto that seen with ATP. The presence of DNA(relaxed pBR322) at 50 bp/dimer molecule and250 bp/dimer molecule did not alter the proteoly-sis pattern from that seen with protein alone, andthe presence of ADPNP and DNA did not alterthe proteolysis pattern from that seen withADPNP alone (data not shown).

To enable the protein bands to be identified, thefive bands numbered 1 to 5 (Figure 8) were sub-jected to N-terminal sequence analysis. In theabsence of any ligand or the presence of ATP, thehighest molecular mass band seen (,38 kDa, no.2) had an amino acid sequence whose first sixamino acid residues correspond to residues 37–42of topo IIa (Figure 8). In the presence of ADPNP,the two protected bands at ,40 kDa (no. 4) and,35 kDa (no. 5) had an amino acid sequence corre-sponding to residues 26–31. This suggests that thestretch of residues from 26–36 is protected fromproteolytic removal in the protein–ADPNP com-plex. Such protection is consistent with the crystalstructure of the 43 kDa fragment of DNA gyrasein the presence of ADPNP,26 where the N-terminalresidues form an extending “arm” that interactswith the adjacent enzyme molecule in the dimer.

Discussion

It is well established that the N-terminal(ATPase) domain of type IIA DNA topoisomerasesforms an ATP-operated clamp that captures a seg-ment of DNA (the T segment) during the topo-isomerase reaction. Evidence for this has comefrom the X-ray structure of the N-terminal domainof GyrB26 and from the results of experiments byRoca & Wang, which examined DNA binding byyeast topo II in the presence of ADPNP.31,38,39 How-ever, a number of issues surrounding the functionof this domain remain unresolved.

1. Is DNA (the T segment) stably bound withinthe clamp during the topoisomerase cycle?

2. How is the ATPase reaction stimulated byDNA?

3. What is the origin of the hyperstimulatedATPase activity seen with full-length topo IIand with N-terminal fragments?

4. How is ATP binding and hydrolysis withinthis domain coupled to strand passage?

The work described here goes some way towardaddressing these issues. Previously the structureof the N-terminal (43 kDa) domain of GyrB com-plexed with ADPNP suggested that there is acavity formed by the two monomers that couldbind DNA. However, biochemical experimentshave failed to show stable binding of DNA by thisdomain.24,40 Experiments with human topo IIa,27,29

yeast topo II,28 and, more recently, E. coli topo IV(J. E. Riley & A. D. Bates, personal communi-cation), have shown that DNA can stimulate theATPase reaction of the isolated ATPase domain ofthese enzymes; however, it is not clear where theDNA is bound. Evidence in support of the DNAbeing bound within the internal cavity comesfrom site-directed mutagenesis experiments withDNA gyrase41 and topo IV (J. E. Riley & A. D.Bates, personal communication), which showed

Figure 8. Limited proteolysis of topoIIa(1–453). Tryp-tic digests were performed in the presence of: no ligand,2 mM ATP or 2 mM ADPNP. Aliquots were removedafter 30 and 60 minutes, and analysed by SDS-PAGE ona 15% polyacrylamide gel, along with a sample of puri-fied protein (1–453). A duplicate gel was blotted onto aPVDF membrane and the indicated bands were sub-jected to N-terminal sequence analysis. The N-terminalamino acid sequence of each excised band is indicated,along with the corresponding residue number in thehuman DNA topo IIa sequence.


UNCORRECTED PROOF

that mutation of an Arg residue that protrudes intothe cavity abolished DNA-dependent ATPaseactivity. In this regard, it is worth noting the effectsof deleting residues 350–407 from topo IIa.42 Onthe basis of homology with GyrB, this regionforms the wall of the cavity that exists in thedimeric form of the ATP-operated clamp.26 Theenzyme bearing this deletion hydrolyses ATP effi-ciently and can carry out DNA cleavage/religation,but lacks topoisomerase activity.42 This suggeststhat amino acid residues 350–407 are essential forcommunication between the ATPase and DNAbreakage–reunion domains.

In this work, we have constructed a range ofN-terminal fragments of human topo IIa. Bymaking a series of fusion constructs, we foundthat we were able to overcome the insolubilityproblems previously experienced with thisdomain.27 We found that a representative “short”construct (1–420) shows DNA-dependent ATPaseactivity but does not exhibit hyperstimulation athigh ratios of enzyme to DNA. A representative“long” construct (1–453) also shows DNA-dependent ATPase activity and, in addition, exhi-bits hyperstimulation at high ratios of enzyme toDNA. These results are consistent with previousobservations with fragments of human topo IIaand yeast topo II,27 – 29 and suggest that themagnitude of the DNA stimulation of the ATPaseactivity and the hyperstimulation phenomenonare dependent on the size of the N-terminal frag-ment; longer fragments show behaviour reminis-cent of the full-length protein.21,28 It is not clearwhether the hyperstimulation phenomenon is uni-versal for all type II topoisomerases; experimentson full-length yeast topo II did not reveal anyhyperstimulation effect.19

Kinetic studies of the ATPase activity oftopoIIa(1–453) showed that the presence of DNAinduces a change from a parabolic to a lineardependence of ATPase rate upon enzyme concen-tration. This suggests that the protein exists pre-dominantly in the monomeric form in the absenceof DNA, which is the same as was observed forthe 43 kDa domain of E. coli DNA gyrase24 andN-terminal fragments of eukaryotic topo II.28,29 Incontrast, in the presence of DNA, the linear depen-dence of ATPase activity upon protein concen-tration for topoIIa(1–453) and the N-terminalfragment of yeast topoisomerase II, but not for the43 kDa domain of E. coli DNA gyrase (which doesnot have a DNA-stimulated ATPase activity),suggests that DNA may induce the enzyme todimerise. However, in cross-linking studies, nohigh molecular mass species were seen whencross-linking was performed in the presence ofDNA, whereas cross-linked species were seenwith ADPNP. The interaction of enzyme mono-mers with DNA, leading to dimerisation, mayoccur only transiently, particularly in the absenceof ATP, and thus dimers may not have beendetected by cross-linking. Molecular mass studiessupport the idea that topoIIa(1–453) is a monomer

in the absence of nucleotide and a dimer in thepresence of ADPNP.

At a constant concentration of enzyme the rate ofATP hydrolysis was hyperbolically dependentupon the concentration of ATP. The intrinsicATPase rate of topoIIa(1–453) was found to bestimulated ,15-fold by DNA at 50 bp/dimermolecule and , tenfold by DNA at 250 bp/dimermolecule. The shorter fragment topoIIa(1–420)has a lower intrinsic ATPase activity that is stimu-lated ,sixfold by excess DNA. Direct comparisonof the results for the full-length enzyme21 and the52 kDa fragment27 with the results shown here arecomplicated by the non-linear dependence of theintrinsic ATPase rate upon the concentration ofenzyme. However, the longer N-terminalfragment topoIIa(1–453) is distinct from theshorter N-terminal fragments (topoIIa(1–420) andthe 52 kDa protein) in terms of the level of intrinsicand DNA-dependent ATP hydrolysis and itsaffinity for substrate, and displays catalytic proper-ties similar to that of the full-length enzyme. Thedifferent properties of these two N-terminal frag-ments is presumably due to the longer C terminusof topoIIa(1–453), which increases the intrinsicATPase rate and alters the DNA-stimulation andsubstrate affinity of the enzyme. These residuesmay stabilise the enzyme in a configuration that ismore competent to hydrolyse ATP, and alter theinteraction of the enzyme with DNA either directlyor indirectly.

A question raised by the crystal structure of theN-terminal domain of GyrB is, what length ofDNA is required to occupy the proposed DNA-binding cavity? The intrinsic ATPase activity ofthe N-terminal fragment topoIIa(1–453) wasfound to be stimulated by a DNA fragment of,10 bp, although a DNA fragment of ,12–14 bpmay be required for full stimulation (Figure 4).This is consistent with the size and shape of thecavity in the structure of the 43 kDa protein–ADPNP complex,26 and favours the idea thatDNA stimulates the ATPase activity by bindingwithin the cavity rather than interacting with theoutside of the protein. Given the above, it is inter-esting to note that a DNA fragment of 40 bp is notsufficient to stimulate the ATPase activity of thefull-length enzyme (except at .1000-fold excess);a DNA fragment of .100 bp is required.21 In thefull-length enzyme, DNA-stimulation of theATPase rate may be dependent upon both the Tand G segments binding, which may be favouredby a fragment of DNA in which the T and G seg-ments are contiguous, i.e. it requires a DNA frag-ment of .100 bp.21

We investigated the length of DNA required toexhibit the hyperstimulation phenomenon. Frag-ments of 28 bp or 30 bp did not show this effect,whereas fragments $32 bp did (Figure 6).Maximum hyperstimulation occurred with afragment of 38 bp. These data suggest that ,10 bpis sufficient to occupy the clamp cavity butthat ,40 bp is required to accommodate two


UNCORRECTED PROOF

clamps side-by-side (Figure 9). DNA fragments.40 bp show lower levels of hyperstimulation,presumably due to the clamps being lesscrowded on such fragments. Consistent with thisidea, the 60 bp fragment appears to show a higherlevel of hyperstimulation than the 50 bp fragment(Figure 5); we interpret this as a 60 bp fragment

being just large enough to accommodate threeclamps.

The similarity in the hyperstimulation patternseen with the fragment topoIIa(1–453) and thefull-length human enzyme21 suggests that thehyperstimulation effect is principally a function ofthe N-terminal domains of eukaryotic type II

Figure 9. A model for the DNA-dependent ATPase activity of the N-terminal fragment topoIIa(1–453). Enzymemolecules are shown in orange. (a) In the absence of DNA the enzyme has an intrinsic ATPase activity, dependentupon ATP-induced dimerisation. (b) With a short DNA (black) molecule (,10–12 bp) DNA stimulation of the ATPaseactivity occurs. (c) With a longer fragment (,40 bp) hyperstimulation occurs as a consequence of crowding of enzymedimers on DNA. (d) With relaxed pBR322 at 50 bp/dimer molecule, the DNA is saturated with enzyme dimers thatinteract with one another to give a hyperstimulated ATPase rate. (e) With excess DNA, interactions between enzymedimers occurs with much lower probability and a basal DNA-stimulated ATPase rate is seen. Green arrows indicatepotential cooperative interactions. (The dimers are shown in the same orientation, but their actual orientation isunknown.)


UNCORRECTED PROOF

topoisomerases. The hyperstimulated ATPase ratecan be attributed to protein–protein interactionsbetween adjacent dimers bound to DNA, asdepicted in Figure 9. There are two results relatingto the hyperstimulation effect that are difficult toexplain. Firstly, the extent of DNA required toyield maximal hyperstimulation varies dependingon the DNA size. With pBR322 (Figure 3(b)), themaximum effect is seen at a DNA to enzyme ratioof ,50 bp/molecule; we interpret this as thelength of DNA occupied by one enzyme dimer(clamp). With a 140 bp fragment (Figure 3(b)), thislength is reduced to ,40 bp. However, with shortfragments (Figure 6), maximal hyperstimulationoccurs with a fragment of 38 bp, implying thateach clamp is occupying ,19 bp. One obviousdifference between the DNA fragments andpBR322 is that, in the former case, end effects canoccur, i.e. there will be enzyme dimers that caninteract only with one adjacent dimer. WithpBR322, at saturation, all dimers have two neigh-bours and a maximal cooperative effect wouldoccur. With a 38 bp fragment, the two bounddimers have only one neighbour and the minimalcooperative effect would be observed. Such “end-effects” could account for the apparent differencesbetween binding-site size between long and shortDNA molecules. Another possible manifestationof this end-effect is the different magnitudes ofhyperstimulation with different DNAs, asjudged by the sizes of the “humps” in Figures 5and 6. The short fragments tend to have smallhumps, whereas the long fragments have largerones, perhaps indicative of greater cooperativity.In Figure 5, the magnitude of the hyperstimulationis much greater for a 60-mer than for a 40-mer,consistent with the greater potential forcooperativity with three dimers bound comparedwith two.

Secondly, the plateau level of ATPase stimulationwith excess DNA varies with the size of the DNA.For example, with pBR322 it is ,0.2 s21, whereasit is ,0.05 s21 with the 140 bp fragment (Figure3(b)). With short DNA fragments (12–30 bp) theATPase plateaus at ,0.1 s21, whereas with slightlylonger fragments (32–44 bp) the plateau level is,0.05 s21. Although the variation in these plateauvalues is only a factor of ,4, it appears to be con-sistent. One possibility that might explain thesedifferences is the nature of the DNA sequence towhich the protein is bound. We have assumedthat T segment-clamp interactions are independentof DNA sequence. However, it is possible that thelocal sequence influences the extent of the ATPasestimulation. In the case of pBR322, the enzyme hasthe opportunity of binding to a range of sites thatmight be more effective at stimulating ATPaseactivity than the sequence surrounding the 990sites.

The phenomenon of hyperstimulation of theATPase activity of topo II and of its N-terminalfragments at limiting DNA concentration is wellestablished but its origins are unclear. In Figure 9,

we suggest that this effect is caused by protein–protein interactions between adjacent bound pro-tein dimers (clamps). This is manifested principallyas a change in apparent kcat (Table 1). This changein enzymic behaviour of proteins bound to DNAwas observed previously and has been examinedtheoretically in a so-called piggy-back bindingmodel.30 In this model, the binding of a ligand(enzyme) to a one-dimensional lattice (DNA) inthe presence of a second (“rider”) ligand (ATP),which binds only to the first ligand, was studied.The model suggests that multiple cooperativeinteractions between bound ligands can occur,which vary depending upon whether the riderligand is bound. Modelling of the reaction rates asa function of the concentration of polymer (DNA)under conditions with a range of cooperativityparameters produced a variety of (apparent)binding isotherms, including examples with thehump-backed appearance seen in Figure 3(b).30

However, the humps were not as dramatic asthose shown in Figure 3(b). The piggy-backbinding model30 involved two assumptions:(1) that the rate constant (kcat) was small comparedto the rate constants of the binding reactions; and(2) the value of kcat was not affected by theinteractions with neighbouring enzyme molecules.It is possible to relax these assumptions and useMonte Carlo simulations to determine whetherthe data in Figure 3(b) can be reproduced.Modelling studies are currently underway toexplore these possibilities (Y. Chen, personalcommunication).

The actual cooperative effects responsible for thehyperstimulation seen in this work cannot bederived uniquely from the data presented here.However, one possibility that is consistent withwhat is known about topo II and the properties ofits N-terminal (ATPase) domain, is that negativecooperativity exists between adjacent bounddimers that destabilises DNA binding. This wouldlead to dissociation of the protein from the DNAand more rapid release of ADP and Pi. It is thoughtthat the rate-limiting step of the gyrase and topo IIDNA-dependent ATPase (and topoisomerase)reactions is product release,19,24,43 therefore suchnegative cooperativity would lead to an increasein apparent kcat.

Conclusions

We have studied the ATPase reaction of theN-terminal domain of human topo IIa. This hasled to a number of novel findings. Firstly, we haveshown that the ATPase characteristics dependupon the length of the N-terminal fragment. Ashort fragment (1–420) has a linear dependence ofATPase activity on enzyme concentration andshows no hyperstimulation at high ratios of pro-tein to DNA. A longer fragment (1–453) shows agreater than first-order dependence of ATPase onthe concentration of enzyme and hyperstimulation


UNCORRECTED PROOF

at high ratios of protein to DNA. These resultsrationalise apparently conflicting results fromprevious studies, and suggest that T segment bind-ing to the ATPase domain is sufficient to stimulatethe ATPase activity and to manifest the hyper-stimulation effect. Secondly, we have found that aDNA fragment of ,10 bp is sufficient to stimulatethe DNA-dependent ATPase activity. This can beinterpreted as the minimum-sized T segment thatwill bind in the ATP-operated clamp (the cavityformed by the two monomers of the N-terminaldomain) and stimulate ATPase activity. Thirdly,we find that DNA fragments $32 bp cause hyper-stimulation of ATPase activity, consistent with amodel in which adjacent protein dimers bound onDNA interact through “crowding” (Figure 9). Wecan interpret these results using a theoreticalmodel (piggy-back binding) previously developedto explain the behaviour of gyrase bound toDNA.30 We speculate that the origin of the hyper-stimulation is negative cooperativity betweenadjacent protein dimers leading to stimulation ofADP/Pi release, thus accelerating the rate-limitingstep of the reaction. In vivo, it is feasible thatcrowding of topo II might occur in the context ofits proposed role as a chromatin scaffold protein.44

The elevated ATPase activity may reflect increasedcatalytic activity connected with its role in main-taining chromosome structure and function. Thebinding of proteins to a one-dimensional lattice(e.g. DNA) is not uncommon in nature (e.g. RecA,single-strand binding proteins) and the obser-vations made here are likely to be applicable toother biological systems (e.g. kinesin–microtubuleinteractions).

Materials and Methods

DNA preparation

Relaxed plasmid pBR322 DNA was a gift from MrsA. J. Howells. A 140 bp fragment was derived frompBR322 by PCR as described.45 Short double-strandedDNA fragments of 8–44 bp were made by annealingcomplementary oligonucleotides (PNACL, University ofLeicester) based symmetrically around the preferredDNA gyrase binding and cleavage site within pBR322(the 990 site).37

Cloning and protein expression

Expression plasmids were constructed using a lig-ation-independent cloning (LIC) technique,36 using apET LIC vector kit from Novagen. The system usedproduces proteins with N-terminal fusions tothioredoxin and a histidine tag, which can be removedwith factor Xa to generate the native proteins. Expressionplasmids were transformed into competent E. coliBL21(DE3) cells and protein expression was induced bythe addition of IPTG to a final concentration of 0.1 mMat mid-log phase, and growth continued for a further16 hours at 20 8C. Cells were harvested bycentrifugation and the cell pellet was resuspended in50 mM Tris–HCl (pH 7.5), 10% (w/v) sucrose, frozen in

liquid nitrogen, and stored at 270 8C. After thawing,protease inhibitors were added: 4-(2-aminoethyl)-benzenesulphonyl fluoride-hydrochloride to 0.5 mM,benzamidine to 1 mM, soybean trypsin inhibitor to5 mg/ml. The cells were disrupted by passage threetimes through a pre-cooled French pressure cell at8000–12,000 psi (1 psi < 6.9 kPa), and by sonicationusing two cycles of sonication (30 seconds on/30 secondsoff) at 10 m amplitude. The cell debris was pelleted bycentrifugation at 40,000 rpm (Sorvall TFT 50.38 rotor)and the supernatant was applied to a pre-equilibratedTALON Co2þ-based immobilised metal affinity column(Clontech) and allowed to bind with gentle agitation forone hour. The column was washed several times withfive bed volumes of high-salt wash buffer (50 mM Tris–HCl (pH 8.0), 500 mM NaCl), and subsequently withlow-salt wash buffer (50 mM Tris–HCl (pH 8.0),250 mM NaCl) plus 5 mM imidazole. The fusion proteinwas then eluted with several washes of low-salt washbuffer plus 50 mM imidazole. Fractions were analysedby SDS-PAGE, and peak fractions were pooled. Thepooled fractions were diluted fivefold with 50 mMsodium phosphate buffer (pH 7.0), 0.5 mM DTT, 0.5 mMEDTA and then loaded onto a HiTrap SP ion-exchangechromatography column (Pharmacia) using a pump at aflow-rate of ,5 ml/minute at 4 8C. The column wasthen washed with 50 mM phosphate buffer (pH 7.0),50 mM NaCl, 0.5 mM DTT, 0.5 mM EDTA, and theneluted with a stepwise salt gradient of 75 mM to 1 MNaCl in the same phosphate buffer. Peak fractions, typi-cally .95% pure as determined by SDS-PAGE, werepooled and dialysed overnight at 4 8C in storage buffer(50 mM Tris–HCl (pH 8.8), 100 mM NaCl, 50% (v/v) gly-cerol, 0.5 mM DTT, 0.5 mM EDTA), and stored at 220 8C.

Proteolytic cleavage of fusion proteins was performedusing biotin-labelled factor Xa (Boehringer Mannheim).Following proteolytic cleavage, the sample was incu-bated with an immobilised streptavidin–agarose resin(200 ml of a 50% (w/v) suspension of the resin per100 mg of factor Xa) for one hour at 4 8C to bind the factorXa. The sample was then centrifuged to pellet the resin,and the supernatant was removed. The topo IIa frag-ment was separated from the fusion peptide using theTALON resin (as before), and dialysed into storagebuffer and stored at 220 8C.

ATPase assays

ATPase assays were performed using an ADP-sensitive linked enzyme assay.46 ATPase reactions(under “standard” conditions) were performed in avolume of 150 ml at 37 8C in the following buffer (unlessotherwise stated): 50 mM Tris–HCl (pH 7.5), 10 mMNaCl, 5% (v/v) glycerol, 0.4 mM phosphoenolpyruvate,0.2 mM NADH, 5 ml pyruvate kinase/lactate dehydro-genase (in 50% (w/v) glycerol, 100 mM KCl, 10 mMHepes (pH 7.0), 0.1 mM EDTA), 2 mM Mg-ATP (in sodiumacetate buffer at pH 7.5), 0.5 mM DTT, 0.05 mM EDTA.DNA, where present, was added at the indicated concen-tration or ratio (bp:enzyme dimer). Reactions wereinitiated by the addition of enzyme and monitored con-tinually for one hour in an absorbance plate reader (eithera Bio-Tek EL340 Bio-Kinetics reader or a Molecular DevicesSpectraMax Plus Microplate Spectrophotometer). Thechange in absorbance is related to ADP production usingA1 mM

340 ¼ 6:22 cm21: The initial rate of ATP hydrolysis wasdetermined, following a five minute equilibration period,using a minimum of ten minutes of data collection.


UNCORRECTED PROOF

Protein cross-linking

Cross-linking of protein was performed using threecross-linking reagents (Pierce): dimethyl suberimidate(DMS), disuccinimidyl glutarate (DSG) and bis(sulpho-succinimidyl) suberate (BS3). Stock solutions of cross-linkers were prepared as follows: DMS, 20 mg/ml inwater; DSG, 5 mg/ml in dimethyl sulphoxide (DMSO);BS3, 10 mg/ml in water. Protein (7.5 mM final concen-tration) was incubated in the presence or in the absenceof nucleotide (2 mM) or relaxed pBR322 at 50 bp/dimermolecule for one hour at 25 8C in 50 mM Tris–HCl (pH8.8), 100 mM NaCl, 10% (v/v) glycerol, 0.5 mM DTT,0.5 mM EDTA. Cross-linker was then added at a tenfolddilution of the stock solution and the samples incubatedat 25 8C for a further 16 hours. Samples were analysedby SDS-PAGE on a 10% (w/v) polyacrylamide gel.

Molecular mass studies

A Sephacryl S-200 HR (16/60) FPLC column(Pharmacia) was equilibrated overnight at a flow-rate of0.2 ml/minute in gel-filtration buffer (50 mM Tris–HCl(pH 8.8), 100 mM NaCl, 5 mM MgCl2, 10% (w/v)glycerol, 0.5 mM DTT, 0.5 mM EDTA) in the presence orin the absence of 0.1 mM ADPNP. The column wascalibrated using a set of molecular mass markers andBlue dextran to determine the void volume of thecolumn. Protein (0.4 mg/ml in gel-filtration buffer) wasincubated for one hour at 25 8C in the presence or in theabsence of 2 mM ADPNP. A 500 ml aliquot of the sample(,200 mg) was loaded onto the column and eluted withgel-filtration buffer at a flow-rate of 0.8 ml/minute inthe presence or in the absence of 0.1 mM ADPNP. Theabsorbance at 280 nm was monitored, and 1 ml fractionswere collected and analysed by SDS-PAGE.

Analytical ultracentrifugation was carried out byA. Leech (University of East Anglia). Samples were pre-pared by gel-filtration in a manner identical with thatdescribed above, except that the elution buffer used was50 mM Tris–HCl (pH 8.8), 100 mM NaCl, 5% (w/v)glycerol, 5 mM MgCl2, 0.5 mM EDTA ^ 0.1 mMADPNP. Fractions corresponding to the most prominentpeak in each sample were pooled and concentrated to,0.1 mg/ml and stored on ice. Analytical ultracentri-fugation was carried out in a Beckman Optima XL-1Analytical Ultracentrifuge using an eight-hole rotor anda double-sector cell with an optical length of 12 mm.Equilibrium sedimentation runs were performed initiallyat 15,000 rpm for 28 hours. The concentration distri-bution for the protein within the cell was determinedusing an optical absorbance system.

Limited proteolysis

Limited proteolysis was carried out using trypsin(Sigma). Samples contained protein (4 mM, 0.2 mg/ml)in 50 mM Tris–HCl (pH 8.8), 100 mM NaCl, 5 mMMgCl2, 10% (v/v) glycerol, 5 mM DTT, 0.5 mM EDTA.Samples were incubated for one hour at 25 8C in thepresence or in the absence of nucleotide (2 mM) orrelaxed pBR322 at 50 or 250 bp/dimer molecule. Trypsinwas then added to the samples to a final concentration of15 mg/ml and the reactions incubated at 25 8C. Sampleswere removed at various times and analysed by SDS-PAGE on a 15% polyacrylamide gel.

N-terminal sequencing of proteins was carried out byK. Lilley (University of Leicester). Protein samples to be

sequenced were subjected to SDS-PAGE, then blottedonto a polyvinylidene fluoride (PVDF) membrane totransfer proteins. The membrane was stained to identifyprotein bands, and the band of interest was excised, andthe protein isolated and subjected to N-terminalsequence analysis by Edman degradation using an ABI476 protein sequencer.

Filter-binding

Protein–DNA interactions were assessed using afilter-binding technique. Samples (50 ml) containing upto 1 mM protein in 50 mM Tris–HCl (pH 8.8), 20 mMNaCl, 5 mM MgCl2, 10% (v/v) glycerol, 0.5 mM DTT,0.5 mM EDTA, 0.1 mg/ml bovine serum albumin (BSA)were incubated at 25 8C in the presence or absence of3.62 nM relaxed [3H]pBR322 (a generous gift from A. J.Howells) or 2 mM ADPNP. After 30 minutes, eitherDNA or ADPNP was added as above, or no additionwas made, and the samples were incubated for a further30 minutes at 25 8C. A 25 mm filter disc (0.45 mm NC 45membrane filter, Schleicher & Schuell) was washed with2 £ 500 ml of binding buffer (50 mM Tris–HCl (pH 8.8),20 mM NaCl, 5 mM MgCl2, 10% (v/v) glycerol, 0.5 mMDTT, 0.5 mM EDTA). The sample was added to 200 mlof binding buffer, and this was filtered through the filterdisc at a flow-rate of approximately 1 ml/minute. Thefilter disc was washed with 2 £ 500 ml of binding buffer,and was then left to dry in air for approximately 30minutes. Then 4 ml of scintillation liquid was addedand the amount of tritiated DNA retained on the filterdisc was calculated by scintillation counting.

Acknowledgements

This work was supported by grants from the BBSRCand the Wellcome Trust. S.C. was supported by astudentship from the BBSRC. We thank Yi-Der Chen,Janet Lindsley and Mark Szczelkun for their insightfulcomments on the manuscript.

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UNCORRECTED PROOF

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Edited by J. Karn

(Received 2 January 2002; received in revised form 3 May 2002; accepted 8 May 2002)


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