Helicase-defective RuvBD113E promotes RuvAB-mediated branch migration in Vitro

Article No. jmbi.1999.3187 available online at http://www.idealibrary.com on J. Mol. Biol. (1999) 293, 505±519

Helicase-defective RuvBD113E Promotes RuvAB-Mediated Branch Migration in Vitro

Helen George1, Christine MeÂzard1, Andrzej Stasiak2

and Stephen C. West1*

1Genetic RecombinationLaboratory, Imperial CancerResearch Fund, Clare HallLaboratories, South MimmsHerts, EN6 3LD, UK2Laboratoire d'AnalyseUltrastructurale, UniversiteÂ deLausanne, 1015, LausanneSwitzerland

Present address: C. MeÂzard, LaboMoleÂculaire de la Recombinaison, UCurie, Section de Recherche, 26 rue75248 Paris 05, France.

Abbreviations used: ds, double-stgapped circular plasmid DNA; ss, s

E-mail address of the [email protected]

0022-2836/99/430505±15 $30.00/0

In Escherichia coli, the RuvA and RuvB proteins interact at Holliday junc-tions to promote branch migration leading to the formation of hetero-duplex DNA. RuvA provides junction-binding speci®city and RuvBdrives ATP-dependent branch migration. Since RuvB contains sequencemotifs characteristic of a DNA helicase and RuvAB exhibit helicaseactivity in vitro, we have analysed the role of DNA unwinding in relationto branch migration. A mutant RuvB protein, RuvBD113E, mutated in heli-case motif II (the DExx box), has been puri®ed to homogeneity. Themutant protein forms hexameric rings on DNA similar to those formedby wild-type protein and promotes branch migration in the presence ofRuvA. However, RuvBD113E exhibits reduced ATPase activity and isseverely compromised in its DNA helicase activity. Models for RuvAB-mediated branch migration that invoke only limited DNA unwindingactivity are proposed.

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Keywords: recombination; DNA repair; Holliday junction; ATPase;DNA unwinding
*Corresponding author
Introduction

DNA helicases are a diverse class of enzymesthat use the energy derived from NTP hydrolysisto unwind duplex DNA (Lohman & Bjornson,1996). The Escherichia coli RuvB protein, which isinvolved in homologous genetic recombinationand recombinational repair, belongs to an emerg-ing class of helicases that possess a common hex-americ ring structure. Other members includeE. coli DnaB and Rho, bacteriophage T7 gp4 andT4 gp41, the simian virus 40 (SV40) large T anti-gen, plasmid-encoded RepA and bovine papillomavirus type 1 E1 protein (Egelman, 1996; Fouts et al.,1999; Scherzinger et al., 1997). Based on their struc-tural similarities, it has been suggested that thehexameric helicases share a common mechanism ofaction.

ratoire de GeÂneÂtiqueMR 144, Institutd'Ulm,

randed; gDNA,ingle-stranded.ing author:

E. coli RuvB, together with RuvA and RuvC, isinvolved in the processing of Holliday junctionintermediates into mature recombinant products(reviewed by West, 1997). RuvA (22 kDa) andRuvB (37 kDa) are co-expressed from a DNAdamage-inducible (SOS) operon and together pro-mote ATP-dependent branch migration of Hollidayjunctions. RuvC (19 kDa) is a junction-speci®cendonuclease that catalyses Holliday junction res-olution. Recent studies have shown that branchmigration and resolution can be coupled by theformation of a RuvABC-Holliday junction complex(Davies & West, 1998; van Gool et al., 1998, 1999;Whitby et al., 1996; Zerbib et al., 1998).

Because high levels of RuvB, a DNA-dependentATPase (Iwasaki et al., 1989a), can catalyse branchmigration in the absence of RuvA, RuvB is thoughtto be the motor of the RuvAB branch migrationcomplex (Mitchell & West, 1996). At reduced RuvBconcentrations, however, branch migration requiresRuvA, which exhibits a high af®nity for Hollidayjunctions and promotes the speci®c assembly ofthe RuvAB complex at the junction (Iwasaki et al.,1992; Parsons et al., 1992; Parsons & West, 1993).The RuvA tetramer, which has 4-fold symmetry,holds the junction in an open square-planar con-®guration, an arrangement that facilitates branch

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506 Helicase-defective RuvBD113E

migration (Parsons et al., 1995a; Rafferty et al.,1996). The junction can be sandwiched betweentwo RuvA tetramers (Roe et al., 1998; Yu et al.,1997) or be bound by only one tetramer, presum-ably leaving the other face open for interactionwith RuvC (Hargreaves et al., 1998).

RuvB is a hexameric ring protein that formsdoublets which encircle duplex DNA (Stasiak et al.,1994). In the presence of RuvA, a tripartite RuvAB-Holliday junction complex has been observed byelectron microscopy. In this complex, RuvA bindsthe crossover and is ¯anked by two RuvB hexame-ric rings which are threaded on opposite arms ofthe junction (Parsons et al., 1995a). The RuvB ringsare oriented in a bipolar manner, with their moreconvex surfaces facing outwards (Yu et al., 1997).Models for branch migration suppose that the twoRuvB rings exert equal and opposite forces on theDNA (Parsons et al., 1995a). However, because therings are tethered by RuvA, simple translocationalong the DNA is restricted. Consequently DNA isdrawn into the RuvAB complex and pumped outthrough the centre of each hexameric ring (shownschematically in Figure 9(a)).

Sequence comparisons indicate that DNA andRNA helicases contain seven conserved motifs(Gorbalenya & Koonin, 1993), two of which arepresent in all helicase families and correspond tothe NTP binding Walker A and B boxes (Walkeret al., 1982). Six of the seven helicase motifs havebeen identi®ed in RuvB and, of these, motifs I, II,III and VI are well conserved (MeÂzard et al., 1999).Using standard DNA helicase substrates (i.e. asingle-stranded DNA circle carrying a shortannealed fragment), RuvAB was shown to exhibita 50-30 DNA helicase activity (Tsaneva et al., 1993).Although this helicase activity requires RuvA,RuvB alone is capable of transiently unwindingcovalently closed circular duplex DNA (Adams &West, 1995). Based on these observations, it wassuggested that ATP-dependent DNA unwindingby RuvB is an essential step in the mechanism ofbranch migration.

The mechanism by which hexameric DNA heli-cases promote strand separation is unknown. Thecrystal structure of a monomeric helicase, Bacillusstearothermophilus PcrA, shows that the nucleotide-binding site is located in a cleft between twodomains and is surrounded by the signature heli-case motifs (Subramanya et al., 1996). There areremarkable similarities between the core of PcrAand the core of E. coli RecA, in that all the mainresidues in the nucleotide-binding sites arespatially conserved (Story & Steitz, 1992;Subramanya et al., 1996). Indeed, structural andsequence homologies between the RecA ATP-bind-ing fold and a number of other helicases led to theproposal that many helicases have a common`RecA-like' structural core (Bird et al., 1997). TheWalker A and B boxes within this core, corre-sponding to the highly conserved helicase motifs I(GxGKT) and II (DExx), are thought to be involved

in nucleotide binding and Mg2�-dependent NTPhydrolysis, respectively (Pause & Sonenberg, 1992).

To investigate the role of DNA helicase activityin RuvAB-mediated branch migration, we intro-duced an aspartate to glutamate substitution intohelicase motif II of RuvB. Here we describe thepuri®cation of the resultant mutant protein,RuvBD113E, and its characterisation both in vivo andin vitro. We show that RuvBD113E can promotebranch migration despite being severely compro-mised in its DNA helicase activity.

Results

Phenotypic effects of RuvBD113E expression

Mutagenesis studies of the mammalian trans-lation initiation factor eIF-4A, an RNA helicase,showed that replacement of the ®rst aspartate resi-due in the DEAD box with glutamate led to anuncoupling of its ATPase and helicase activities,resulting in a helicase-defective protein (Pause &Sonenberg, 1992). To analyse the role of DNA heli-case activity in RuvAB-mediated branch migration,oligonucleotide site-directed mutagenesis was usedto make a similar substitution at this highly con-served amino acid in E. coli RuvB (Figure 1(a)).The mutant protein is designated RuvBD113E.

ruv mutants are sensitive to UV light, ionisingradiation and mitomycin C treatment (Otsuji et al.,1974). To determine the phenotype of strainsexpressing RuvBD113E, plasmid pME3 carryingruvBD113E under control of the lac promoter wastransformed into E. coli strain HI24 (ruvB4). In thisstrain, the promoter was derepressed, due to theabsence of lacIq, resulting in constitutive over-expression of RuvBD113E. The level of RuvBD113E

was estimated by SDS-PAGE and Western blottingto be �1000-fold higher than that of chromosomalRuvB (data not shown). Over-expression of themutant protein in HI24 resulted in partial comple-mentation of the ruvB4 mutation, seen by the par-tial restoration of UV resistance (Figure 1(b), leftpanel). Interestingly, over-expression of RuvBD113E

in the wild-type control strain AB1157 resulted inthe cells becoming UV sensitive (Figure 1(b), rightpanel). This dominant negative effect was notobserved when wild-type RuvB was over-expressed.

To study the effects of RuvBD113E at low levels ofexpression, cells carrying pME3 were co-trans-formed with pREP4, a low copy number plasmidwhich constitutively expresses lac repressor. In thisbackground, RuvBD113E expression was only fourto eightfold higher than chromosomal RuvB (datanot shown). In this case, RuvBD113E failed to comp-lement the ruvB mutation in HI24, even at low UVdoses, and dominant negative effects were notobserved in AB1157 (data not shown). Wild-typeRuvB expressed at the same copy number comple-tely restored UV resistance to HI24.

Figure 1. RuvBD113E; location ofthe amino acid substitution, in vivocomplementation studies and puri-®cation of RuvBD113E protein.(a) Alignment of part of the E. coliRuvB sequence (residues 55-76 and104-124) with the correspondingregions of 12 homologues. Identicaland similar amino acids are boxedin black and grey, respectively. Thehighly conserved helicase motifs Iand II are indicated along with theposition of the site-directed E. coliRuvBD113E substitution. (b) UVsurvival curves of E. coli strainHI24 (ruvB4) and its wild-typecontrol AB1157 following trans-formation with plasmids pME3(ruvBD113E; *), pGTI19 (ruvB�; &)or pUC19 (}). (c) SDS-PAGE show-ing the puri®cation of RuvBD113E

protein. Lane a, puri®ed RuvB(2 mg); lane b, crude cell lysate; lanec, phosphocellulose column ¯owthrough after Polymin P precipi-tation; lanes d-f, fractions elutedfrom DEAE-Bio-Gel, hydroxylapa-tite and MonoQ. Proteins were visu-alised by staining with Coomassieblue. The positions of the molecularmass markers are indicated.

Helicase-defective RuvBD113E 507

Purification of RuvBD113E

To characterise the biochemical properties ofRuvBD113E, the protein was over-expressed inE. coli strain FB820, a �ruvAB derivative of JM101,and puri®ed to homogeneity (Figure 1(c)). TheruvB deletion was essential to avoid contaminationwith wild-type protein. Being a JM101 derivative,the strain contained a lacIq repressor gene allowingIPTG-inducible expression of RuvBD113E. Aftereight hours induction, the amount of solubleRuvBD113E in FB820 comprised approximately30 % of total cell protein (lane b). RuvBD113E waspuri®ed by Polymin P precipitation followed bychromatography on phosphocellulose (lane c),DEAE-Biogel (lane d), hydroxylapatite (lane e) andMono Q (lane f). During puri®cation, RuvBD113E

displayed a different elution pro®le on DEAE-Biogel to wild-type RuvB, eluting at 175-200 mMKCl as opposed to 240-270 mM.

DNA binding by RuvBD113E

To determine whether RuvBD113E binds DNAnormally, the ability of wild-type and mutant pro-teins to bind form I (supercoiled) dsDNA was com-pared. Initially, reactions were carried out in thepresence of 15 mM Mg2� and 1 mM ATPgS, con-ditions that favour the stable interaction of RuvBwith dsDNA (MuÈ ller et al., 1993). The resultingprotein-DNA complexes were ®xed with glutaral-dehyde and analysed by agarose gel electrophor-esis. Wild-type RuvB formed de®ned protein-DNA

complexes exhibiting a retardation proportional tothe protein concentration, until saturation wasreached at a stoichiometry of one RuvB hexamerper �26 bp. With RuvBD113E, however, very littlebinding was observed under these conditions (datanot shown).

Related experiments, which will be discussedlater, indicated that the poor DNA bindingobserved with RuvBD113E was due to the presenceof ATPgS. When binding reactions were carriedout using 0.5 mM ATP and varying concentrationsof ATPgS, however, DNA binding by the mutantprotein was observed (Figure 2(a)). Unlike thewild-type protein which shows only slight bindingto dsDNA in the presence of ATP alone (lane b),RuvBD113E exhibited signi®cant DNA bindingunder these conditions (lane j). However, whereasincreasing the concentration of ATPgS stimulatedDNA-protein complex formation by the wild-typeprotein (lanes c-i), similar increases reduced DNAbinding by RuvBD113E (lanes k-q). These resultsindicate that RuvBD113E forms complexes withdsDNA in the presence of ATP, but that DNAbinding is inhibited by ATPgS.

Previous studies have shown that RuvA facili-tates the binding of RuvB to duplex DNA underconditions where only weak interactions are nor-mally detected, i.e. at low concentrations of RuvBand/or 410 mM Mg2� (MuÈ ller et al., 1993). To testwhether RuvA facilitates the loading of RuvBD113E

onto DNA, band-shift assays were conducted inthe presence of RuvA at 5 mM Mg2�. Both wild-

Figure 2. Duplex DNA bindingby RuvBD113E. (a) Band-shift assayswere conducted for RuvB andRuvBD113E, as described inMaterials and Methods, in the pre-sence of 0.5 mM ATP and with theindicated concentrations of ATPgS.Protein-DNA complexes were ®xedwith glutaraldehyde and analysedby 0.8 % agarose gel electrophor-esis. (b) DNA binding by RuvAand RuvBD113E. Reactions were car-ried out as described for (a), exceptthat the Mg2� concentration wasreduced to 5 mM and RuvB orRuvBD113E were premixed withRuvA as indicated.


type and mutant RuvB bound the dsDNA, but thedegree of retardation was dependent on theATPgS/ATP ratio (Figure 2(b)). Both RuvB andRuvBD113E bound DNA in the presence of ATPalone (lanes c and k). The binding of both proteinswas stimulated by the presence of ATPgS, withmaximum retardation at a ratio of 1:2 (wild-type)and 2:1 (mutant) ATP:ATPgS (lanes h and n).However, further increases in the concentration ofATPgS severely inhibited the binding of RuvBD113E

to DNA (lanes o-r). These experiments show thatRuvBD113E forms stable protein-DNA complexes inthe presence of RuvA.

Formation of hexameric rings by RuvBD113E

Complexes of RuvBD113E and linear duplexDNA, formed in the presence of 15 mM Mg2� and1 mM ATPgS, were ®xed with glutaraldehyde andvisualised by electron microscopy. Doublet ringswere observed that were indistinguishable fromthe double hexameric rings seen with wild-typeRuvB (compare Figure 3(a) and (b)). However,with RuvBD113E, fewer rings were associated withthe DNA, consistent with the reduced DNA bind-ing observed in the presence of ATPgS(Figure 2(a)). A large highly structured aggregateof the mutant protein was also observed(Figure 3(c)). This appeared to be free of DNA andconsisted of regular ®lament-like structures thatassociated in a side-by-side manner. This self-association of RuvBD113E may be responsible for

the apparent reduced DNA binding af®nity inATPgS.

ATPase activity of RuvBD113E

The DNA-dependent ATPase activity ofRuvBD113E was compared with wild-type RuvB inthe presence and absence of RuvA (Figure 4). Therate of ATP hydrolysis by RuvBD113E was aboutthreefold lower than that observed with RuvB(4.8 mol ATP/min per mol RuvB). It has beensuggested that the hydrolysis of ATP might beinvolved in the dissociation of RuvB from DNA(MuÈ ller et al., 1993), hence the need for ATPgS toobserve stable RuvB-DNA complexes. The reducedATPase activity exhibited by RuvBD113E couldaccount for its ability to bind DNA in the presenceof ATP alone (Figure 2(a)). In the presence ofRuvA, the rate of ATP hydrolysis by RuvBD113E

was stimulated about fourfold, a rate enhancementsimilar to that observed with wild-type RuvB(Figure 4).

DNA helicase activity

The primary aim associated with the construc-tion of RuvBD113E was to uncouple its ATPase andhelicase activities by substituting glutamate forAsp113. To determine whether RuvBD113E exhibitsa defect in DNA helicase activity, we measured thedisplacement of a 32P-labelled 66 nt oligonucleotidefrom circular single-stranded DNA. With wild-type

Figure 3. Electron microscopic visualisation of RuvB(a) and RuvBD113E (b) and (c) in the presence of duplexDNA. Samples were prepared as described in Materialsand Methods, and observed at a magni®cation of70,000 �.

Figure 4. ATPase activity of RuvBD113E. Comparisonof the ATPase activities of RuvB and RuvBD113E in thepresence and absence of RuvA. Large scale reactionswere initiated by the addition of 4 mM RuvB (&) or4 mM RuvBD113E (*) and performed as described inMaterials and Methods. In each case, RuvB was pre-mixed with RuvA (1 mM). In control reactions, RuvAwas omitted (& and *). At the indicated times,samples were removed and analysed by thin-layerchromatography to determine the percentage of ATPhydrolysed. Background levels of ADP (2.2 % and2.6 %), observed in the absence of RuvB, have beendeducted.


RuvB, in the presence of RuvA, ef®cient displace-ment was observed (Figure 5(a), lanes b-g). In con-trast, RuvBD113E failed to displace the 66-mer (lanesh-m). Indeed, concentrations of RuvBD113E (1.3 mM)much greater than those required by the wild-typeprotein failed to promote strand displacement(data not shown). Similar results were observed

with a DNA helicase substrate carrying anannealed 40-mer (data not shown).

To determine whether RuvBD113E releases shorterfragments, helicase substrates carrying annealed 20or 30 nt long oligonucleotides were prepared. Eachsubstrate was used in reactions containing combi-nations of RuvA, RuvB and RuvBD113E, as shownin Figure 5(b). In accord with previous ®ndings(Tsaneva et al., 1993), neither RuvA alone (lanes band k) nor RuvB alone (lanes d and m) promotedstrand displacement. In the presence of RuvA,however, wild-type RuvB ef®ciently displaced theannealed oligonucleotides (lanes c and l), whereasRuvABD113E displaced only small amounts of the30 nt long fragment (lanes n and p). These resultsindicate that the mutant protein is effectively heli-case-defective. However, we found that a 20-mercould be displaced by RuvABD113E (lanes e and g),although time course studies revealed that the rateof displacement of the 20-mer by RuvBD113E was�30-fold lower than that observed with wild-typeRuvB (Figure 6).

Transient DNA unwinding catalysedby RuvBD113E

It was shown that RuvAB can transientlyunwind covalently closed DNA, as detected by atopological assay (Adams & West, 1995). In thisassay, the localised unwinding of relaxed plasmidDNA by RuvAB results in the introduction of com-pensating positive supercoils that are removed byeukaryotic topoisomerase I. Upon deproteinisation,

Figure 5. DNA helicase activityof RuvBD113E. (a) Displacement of a66 nt fragment annealed to circularssDNA. Annealed substrate con-taining a 32P-labelled 66-mer waspremixed with RuvA (30 nM) andthen incubated with the indicatedamounts of RuvB or RuvBD113E.Products were deproteinised andanalysed by 8 % neutral PAGE.Lane n, heat denatured control.(b) Displacement of 20 and 30 ntfragments. Reactions were carriedout as described for (a).


rewinding of the DNA in the regions opened byRuvAB leads to negative supercoiling, generatingspecies which migrate faster on an agarose gelthan the relaxed substrate. Due to the transientnature of this unwinding reaction, its detectionrequires the presence of ATPgS as well as ATP,which is thought to trap the RuvAB complexes onthe DNA shortly after ATP-dependent unwindinghas occurred (Adams & West, 1995).

In light of the inhibitory effects of ATPgS onDNA binding by RuvBD113E, unwinding assayswere conducted in the presence of ATP andincreasing concentrations of ATPgS. In controlexperiments in which 32P-labelled relaxed DNA(Figure 7(a), lane b) was incubated with RuvABand the reaction supplemented with topoisomeraseI, DNA unwinding was observed (lanes f-l). Sincethe unwinding products (topoisomers) migrate atspeci®c positions depending on the number ofsupercoils introduced, electrophoretic mobility

represents a direct measure of the amount ofunwinding. As observed previously (Adams &West, 1995), maximum unwinding by RuvABoccurred at an ATP:ATPgS ratio of �1:2 (lanes i-k).Under the same conditions, unwinding byRuvABD113E was not detected (lanes q-t). Instead,unwinding was observed in the absence (lane m)and at low concentrations (lanes n-p) of ATPgS.Maximum unwinding was observed at an ATP/ATPgS ratio of 10:1 (lane n), signi®cantly differentfrom that observed with the wild-type protein.

The abilities of RuvAB and RuvABD113E to pro-mote DNA unwinding, under optimum reactionconditions, are compared in Figure 7(b). Thedegree of unwinding observed for wild-type andmutant RuvB was comparable and, in both cases,directly proportional to the protein concentration(compare lanes e-i with j-n).

At elevated concentrations of Mg2� (12 to30 mM), conditions that favour RuvB binding to

Figure 6. Rate of displacement of a 32P-labelled 20 ntfragment annealed to circular ssDNA. (a) The annealedsubstrate was incubated with the Ruv proteins at 37 �C,and at the times indicated aliquots were taken and thereactions were stopped. DNA products were deprotei-nised and analysed as described in the legend toFigure 5. (b) Quanti®cation of the gel shown in (a).(&) RuvB, (*) RuvBD113E. Displaced 20 nt fragmentsare expressed as a percentage of total 32P-labelled DNA.Background amounts of 20-mer observed in the absenceof protein have been subtracted.


DNA, wild-type RuvB can unwind DNA in theabsence of RuvA (Adams & West, 1995). However,over a range of experimental conditions, we failedto detect unwinding by RuvBD113E in the absenceof RuvA (data not shown).

Branch migration catalysed by RuvBD113E

Since RuvBD113E is severely compromised in itsability to promote strand separation, but is able tobind and transiently unwind DNA in the presenceof ATP/ATPgS, we tested its ability to promotebranch migration. Recombination intermediates(a-structures) were prepared by RecA-mediatedstrand exchange between gapped circular plasmidDNA (gDNA) and 32P-labelled linear duplex DNAcontaining a heterologous block (Eggleston et al.,

1997). In the presence of ATP and Mg2�, RuvAB(Figure 8(b), lanes c-h) or RuvB alone (Figure 8(a),lanes b-f) promoted branch migration through2.6 kb leading to the dissociation of the a-structureand release of 32P-labelled linear duplex DNA.RuvB-mediated branch migration required stoichi-ometric amounts of protein and elevated(515 mM) Mg2� concentrations, which favourDNA binding in the absence of RuvA (Mitchell &West, 1996). In contrast, RuvBD113E alone failed topromote branch migration, even at high proteinconcentrations (Figure 8(a), lanes g-l). In thepresence of RuvA, RuvBD113E catalysed branchmigration, albeit with reduced ef®ciency comparedto the wild-type protein (Figure 8(b), lanes i-n).The reduced ef®ciency of branch migrationobserved with RuvBD113E may be a consequence ofits reduced ATPase activity. Quanti®cation of thedata shown in Figure 8(a) and (b) is presented inFigure 8(c).

Discussion

Helicase motif II (DExx), corresponding to theWalker B box (Walker et al., 1982), is highly con-served in RNA and DNA helicases (Gorbalenya &Koonin, 1993). The ®rst two negatively chargedresidues (Asp and Glu) of this motif are known tobe important for Mg2�-coordinated ATP hydroly-sis. The aspartate has also been implicated in thecoupling of ATPase and helicase activities (Pause& Sonenberg, 1992). To investigate the role ofDNA unwinding in RuvAB-mediated branchmigration, Asp113 of E. coli RuvB protein wasreplaced with glutamate by site-directed mutagen-esis. The mutant protein, RuvBD113E, was over-expressed, puri®ed to homogeneity and subjectedto a detailed biochemical analysis.

RuvBD113E was pro®cient in DNA binding, hex-americ ring formation, interaction with RuvA andDNA-dependent ATPase activity. The mutant pro-tein was defective in extensive DNA helicaseactivity (as measured by the strand displacementassay) but promoted RuvAB-mediated branchmigration. These in vitro results were re¯ectedin vivo where the over-expression of RuvBD113E wasfound to partially complement the UV-sensitivephenotype of a ruvB mutant strain, indicating thatthe protein was able to promote some cell recoveryfollowing DNA damage. Using a specialised topo-logical assay, transient unwinding of relaxed plas-mid DNA by RuvABD113E was detected. Takentogether, these results suggest that RuvAB-mediated branch migration proceeds by a mechan-ism that does not require extensive helicaseactivity.

An uncoupling of ATPase and helicase activitieswas observed previously with eIF-4A after intro-duction of the same aspartate to glutamate substi-tution (Pause & Sonenberg, 1992). Mutations atother positions that result in uncoupling have beenreported for bacteriophage T7 gp4 (Washington

Figure 7. Transient unwinding ofcovalently closed duplex DNAby RuvBD113E. (a) Dependence ofDNA unwinding by RuvAB orRuvABD113E on the ATP:ATPgSratio. Uniformly 32P-labelled relaxedplasmid DNA was incubated withRuvA (2 mM) and RuvB/RuvBD113E

(2.4 mM), as described in Materialsand Methods, in buffer containing0.5 mM ATP and the indicatedconcentrations of ATPgS. Wheatgerm topoisomerase I was addedwhere indicated, and the productswere deproteinised and analysed by0.7 % agarose gel electrophoresis.(b) Comparison of the unwindingactivities of RuvAB andRuvABD113E. Assays were con-ducted as in (a), using the indicatedamounts of RuvB/RuvBD113E andATP/ATPgS as shown. In (a) and(b), lane a contains supercoiledDNA.


et al., 1996), E. coli Rho (Pereira & Platt, 1995),mammalian eIF-4A (Pause & Sonenberg, 1992),and a number of viral helicases (Graves-Woodward et al., 1997; Gross & Shuman, 1995;Jindal et al., 1994). In the Bacillus stearothermophilusPcrA structure, the nucleotide binding site islocated in a cleft between two domains and isideally situated to mediate conformational changesbetween the domains in response to nucleotidebinding and hydrolysis (Subramanya et al., 1996).Many of the mutated residues, including the aspar-tate of helicase motif II, are located at the domaininterface and it has been proposed that contacts atthis site are important for the coupling of ATPhydrolysis and DNA unwinding (Subramanyaet al., 1996). X-ray crystallographic studies of RecA(Story & Steitz, 1992) and H-ras p21 (Pai et al.,1990) indicate that the highly conserved aspartatealso plays a role in NTP hydrolysis. Thus,mutations of this residue are often associated withdisrupted NTP hydrolysis in addition to helicasedefects (Dombroski et al., 1988; Gross & Shuman,

1995; MeÂzard et al., 1997; Pause & Sonenberg, 1992;Washington et al., 1996). Replacing aspartate withglutamate maintains the negative charge butincreases the size of the side-chain. The ability ofRuvBD113E to hydrolyse ATP, albeit threefoldreduced, could be due to interactions between thelarger side-chain of the glutamate residue andATP-bound Mg2�, as proposed for eIF-4AD182E

(Pause & Sonenberg, 1992).In contrast to RuvB, which binds DNA in the

presence of ATPgS, DNA binding by RuvBD113E

occurred preferentially in the presence of ATP.This difference was also found in the transientunwinding assay where ATPgS is normally neededto trap RuvB or RuvAB on DNA. Since ATPhydrolysis is thought to be involved in the dis-sociation of RuvB from DNA (MuÈ ller et al., 1993),these differences may be due to the reducedATPase activity of the mutant protein. However,we cannot rule out the possibility that the Asp toGlu substitution causes a conformational change inthe protein, such that the hexameric rings assemble

Figure 8. Branch migration activity of RuvBD113E. (a)32P-labelled recombination intermediates were incubatedwith the indicated concentrations of RuvB or RuvBD113E.Products were deproteinised and analysed by 1.2 %agarose gel electrophoresis. (b) RuvAB-mediated branchmigration reactions were carried out as described in (a),except the intermediates were premixed with RuvA(15 nM) prior to the addition of RuvB or RuvBD113E.(c) Quanti®cation of the branch migration reactionsshown in (a) and (b). (&) RuvB, (*) RuvBD113E. Linearduplex products are expressed as a percentage of total32P-labelled DNA. Background amounts of linear duplexDNA observed in protein-free reactions have beensubtracted.


on the duplex DNA in a more locked con®gurationthan the wild-type protein.

Using recombination intermediates made byRecA, RuvABD113E was shown to promote branchmigration. Although activity was reduced com-pared to wild-type protein, presumably due to itsimpaired ATPase activity, RuvABD113E was capableof driving branch migration through �2.6 kb. Thisis a signi®cant result given that its DNA helicaseactivity was severely compromised such that itwas unable to promote the separation of a 30-merfrom ssDNA. Interestingly, the E. coli RecG pro-tein, which also promotes branch migration in vitro,only exhibits a very weak DNA helicase activity

(Whitby et al., 1994). More extensive DNA unwind-ing may, however, be required for branchmigration through heterologous DNA sequences(Adams & West, 1996; Iype et al., 1994; Parsonset al., 1995b) and substrates containing DNAlesions (Tsaneva et al., 1992b), and a defect in thisprocess may contribute to the UV-sensitive pheno-type associated with RuvBD113E.

This study demonstrates that extensive helicaseactivity is not required for RuvAB-mediatedbranch migration. However, the precise mechan-ism by which RuvB promotes the passage of DNAthrough its rings remains unclear. Most likely,RuvB acts as a translocating motor, using theenergy derived from ATP hydrolysis to fuel itsmovement relative to the DNA. The replicativehexameric helicases T7 gp4, DnaB and T4 gp41encircle and translocate along one strand of a repli-cation fork, excluding the other DNA strand fromthe centre of the ring (Hacker & Johnson, 1997;Jezewska et al., 1997; Raney et al., 1996). This isclearly not the case with RuvB, which is known toencircle both DNA strands (Stasiak et al., 1994), butit is unknown whether translocation occurs alongdsDNA or whether the duplex is transientlyopened as it passes through the ring. Interestingly,RuvBD113E alone failed to promote branchmigration, despite being able to bind DNA andhydrolyse ATP in the absence of RuvA. SinceRuvBD113E showed the same RuvA dependency inthe transient unwinding assay, the results mayindicate that some DNA opening within the RuvBrings is necessary for branch migration(Figure 9(a)).

Based on our knowledge of RuvB, and on obser-vations made with other hexameric helicases, wepropose three possible models for DNA transloca-tion by RuvB (Figure 9(b)). The ®rst two modelsinvoke transient strand separation with ssDNAinteracting with one subunit of the RuvB hexamer;the third model may be equally appropriate fortranslocation along fully base-paired dsDNA.Models (i) and (ii) involve a ``bind-release'' mech-anism in which successive rounds of DNA bindingand release are coupled to cycles of ATP bindingand hydrolysis, respectively. In the ``cyclingmodel'' (Figure 9(b), model (i)), translocation iscoupled to a rotational movement of the singlestrand within the RuvB hexamer. Since RuvB isdimeric in solution (Tsaneva et al., 1992a), it ispossible that the hexamer comprises a trimer ofdimers and that translocation occurs as a strand ispassed sequentially from dimer to dimer aroundthe ring. In such a model, each dimer is presumedto be asymmetric with one catalytic and one non-catalytic subunit. It has been shown that DnaB(Bujalowski & Klonowska, 1993), Rho (Geiselmann& von Hippel, 1992; Stitt, 1988) and T7 gp4(Hingorani & Patel, 1996; Hingorani et al., 1997)contain three high af®nity and three low af®nityATP-binding sites, indicating the presence of threeasymmetric dimers. A model where the ssDNA isrotated between three catalytic subunits, each at

Figure 9. Models for RuvAB-mediated branch migration. (a) Schematic of the RuvAB complex. The RuvA tetrameris bound to the crossover point with the RuvB hexameric rings threaded on opposite (heteroduplex) arms of theunfolded junction. The RuvB rings, which lie in opposite orientations, pump DNA out through their central cavitiesby a translocation mechanism which may involve separation of the duplex DNA. The diagram is not drawn to scale.The branch migration complex may contain two RuvA tetramers which bind to opposite faces of the junction.(b) Models for DNA translocation by RuvB. (i) ``Cycling model'', in which the duplex DNA is opened within theRuvB ring and a single strand (indicated in cross-section by a yellow star) is passed sequentially around the hexamer,from the catalytic subunit (indicated in red) of one dimer to the next. DNA binding and release are thought to becoupled to ATP binding and hydrolysis, respectively. The predicted state of the bound nucleotide at each catalyticsubunit during translocation is indicated. (ii) ``Shuttling model'', a similar mechanism to (i) but involving the ¯ippingof the ssDNA between two adjacent catalytic subunits. (iii) ``Inchworm model'', whereby a single catalytic subunitcontains two non-identical DNA binding sites. Conformational changes of the two DNA binding sites, in response toATP binding and hydrolysis, result in translocation of the DNA (indicated in side view by a yellow bar). In eachmodel, the arrows indicate the direction of movement of the DNA. Where opening is thought to occur, the positionof the other DNA strand is not indicated, but presumably resides in the central cavity of the RuvB ring.


different stages of catalysis depending on the stateof the bound nucleotide, has been proposed for T7gp4 and shows remarkable similarity to the cyclicalbinding change mechanism of F1-ATPase(Abrahams et al., 1994; Boyer, 1993; Hingorani et al.,

1997). However, mixed hexamer experiments con-ducted with the ATPase-defective RuvB mutantRuvBD113N (MeÂzard et al., 1997) may not be consist-ent with the cycling model presented in Figure 9(b),model (i), leading us to propose the ``shuttling


model'' shown in Figure 9(b), model (ii). This is avariation of the ®rst model, except in this case theunwound ssDNA is ¯ipped between two adjacentsubunits upon cycles of ATP binding and hydroly-sis. The model is supported by the ®nding thatRuvAB-mediated branch migration occurs withcomparable ef®ciency in reactions containing eitherATP or a 1:2 ratio of ATP/ATPgS (Mitchell &West, 1996). Furthermore, kinetic studies show thatunder single turnover conditions each RuvB hex-amer hydrolyses only two molecules of ATP,suggesting that only two subunits are involved ineach translocation cycle (Marrione & Cox, 1995).Assuming RuvB is a trimer of dimers, these twocatalytic subunits may reside in the same dimer.

The third model requires two non-identical bind-ing sites in a single catalytic subunit (Figure 9(b),model (iii)). Wigley and colleagues have proposedan ``inchworm'' mechanism for the action of themonomeric helicase PcrA (Velankar et al., 1999).The leading site (in domain 2A) and tail site (indomain 1A) of PcrA are located either side of acleft containing the nucleotide binding pocket(Subramanya et al., 1996; Velankar et al., 1999). Thebinding and hydrolysis of ATP at this site resultsin conformational changes of the two domains,thereby powering movement of the protein alongthe DNA in an inchworm fashion. The leading siteis thought to reach forward to the forked region,where it binds and unwinds the duplex DNA. Thetail site tracks along behind the leading site, grip-ping the single strand as it is formed (Velankaret al., 1999).

RuvB could adopt a similar mechanism of DNAtranslocation, the main difference being that bothDNA binding sites are present in a single catalyticsubunit of the hexamer (Figure 9(b) (iii)). It is notknown whether ssDNA (produced by transientunwinding) or dsDNA are bound at these sites.RuvB (Stasiak et al., 1994), DnaB (San Martin et al.,1995) and T7 gp4 (Egelman et al., 1995) have allbeen shown to possess bilobed subunits, resultingin hexameric rings with a large and small tier.Interestingly, a recent study shows that the DNAbinding site of DnaB contains two subsites withdifferent af®nities for ssDNA (Jezewska et al.,1998). The strong ssDNA binding site is in thevicinity of the small domain of the protein, and theweak binding subsite is located in the largedomain. The strong binding site is thought to bindthe 50 ssDNA tail whereas the incoming duplex isthought to be accommodated within the weak site.These results are consistent with the structuralpolarity of RuvB and T7 gp4, both of which areknown to pump DNA out through the small endof the hexameric ring (Egelman et al., 1995; Yu et al.,1997). We suggest that RuvB may contain twonon-identical DNA binding sites, one in each lobe.Such a mechanism would allow directional trans-location along DNA resulting in the formation ofheteroduplex DNA.

Materials and Methods

Bacterial strains

E. coli strain FB820, a �ruvAB derivative of JM101,was provided by Dr F. Benson. It was generated by P1transduction from the ruvAB deletion strain HRS1004(Iwasaki et al., 1989b). HI24 is a ruvB4 derivative ofAB1157 (Otsuji et al., 1974; Sharples et al., 1990).

Sequence comparisons

Homologues of E. coli RuvB have been identi®ed inMycobacterium leprae (Smith et al., 1997), Mycobacteriumtuberculosis (Cole et al., 1998), Haemophilus in¯uenzae(Fleischmann et al., 1995), Pseudomonas aeruginosa(Hishida et al., 1996), Thermus thermophilus (Tong &Wetmur, 1996), Thermotoga maritima (Tong & Wetmur,1996), Synechocystis sp (Kaneko et al., 1995), Mycoplasmagenitalium (Fraser et al., 1995), Mycoplasma pneumoniae(Himmelreich et al., 1996), Bacillus subtilis (Swiss-Protdatabase accession number O32055), Helicobacter pylori(Tomb et al., 1997) and Borrelia burgdorferi (accessionnumber P70828). Protein sequences were aligned usingPileUp (Wisconsin Package version 10.0, Genetics Com-puter Group, Madison, WI).

Site-directed mutagenesis

A mutation in E. coli ruvB was introduced by site-directed mutagenesis into plasmid pGTI19 (Sharpleset al., 1990), using the Transformer site-directedmutagenesis kit (Clontech). The oligonucleotide50-GACGGTGGATCTCTTCAATAAACAGCACGTCATGCGG-30 altered codon 113 from GAU (Asp) to GAA(Glu). The site-directed mutation in the resultant plas-mid, pME3, was veri®ed by sequencing the entire ruvBgene using the PRISM2 Ready Reaction Dye-Deoxy2

terminator cycle sequencing kit (Perkin Elmer) and anABI model 373A automated sequencer.

UV sensitivity

UV sensitivities were measured as described byMeÂzard et al. (1997).

Proteins

E. coli RecA, RuvA and wheat germ topoisomerase Iwere puri®ed as described (MeÂzard et al., 1999). RuvBwas expressed in E. coli FB820 and puri®ed as described(Tsaneva et al., 1992a), except that the cleared extractwas loaded directly onto a DEAE-Biogel column. E. colitopoisomerase I was puri®ed as described (Lynn &Wang, 1989) using the over-expression vector pJW312-Sal, kindly provided by Professor J. Wang (Harvard Uni-versity). Protein concentrations are expressed in moles ofmonomer.

Purification of RuvBD113E

RuvBD113E was over-expressed from plasmid pME3 inE. coli strain FB820. A fermenter culture (11 litres), con-taining 100 mg/ml ampicillin, was grown with aerationat 37 �C. At an A650 of 3.0, the cells were induced byaddition of 1 mM IPTG. After a further eight hours ofgrowth the cells were harvested as described (Tsanevaet al., 1992a). Plasmid pME3 contains ruvBD113E under


control of a lac promoter. To achieve optimumexpression of the mutant RuvB protein, 0.2 % (w/v) glu-cose was included in the pre-cultures used to inoculatethe fermenter culture.

RuvBD113E was puri®ed using a modi®cation of theprotocol developed for wild-type RuvB (Tsaneva et al.,1992a). Following cell lysis and high speed centrifu-gation, 10 % (v/v) Polymin P (pH 7.5) was added to thesupernatant (total volume 230 ml) to a ®nal concen-tration of 0.5 %. After stirring for 30 minutes, the suspen-sion was centrifuged and the pellet resuspended in115 ml R-buffer (20 mM Tris-HCl (pH 7.5), 0.1 mMEDTA, 10 % (v/v) glycerol, 0.5 mM dithiothreitol) con-taining 400 mM NaCl using a Waring blender (lowspeed, 2 � 30 seconds), stirred for ten minutes and cen-trifuged at 10,000 rpm for 15 minutes. The pellet wassuspended as before in 46 ml R-buffer containing 1.1 MNaCl. Following centrifugation, this latter step wasrepeated to maximise recovery of RuvBD113E. The super-natants obtained with the 1.1 M NaCl cuts were pooledand passed through a phosphocellulose column (50 ml),equilibrated with R-buffer containing 1.1 M NaCl, toremove residual Polymin P. The ¯ow through was dia-lysed against TEGD (20 mM Tris-HCl (pH 7.5), 1 mMEDTA, 10 % glycerol, 0.5 mM dithiothreitol) containing50 mM KCl and loaded onto a DEAE-BioGel column(50 ml volume) equilibrated with the same buffer. Thecolumn was developed with a 50 mM-300 mM KCl line-ar gradient in TEGD. RuvBD113E eluted at a lower saltconcentration (175-200 mM KCl) than wild-type RuvB(240-270 mM KCl). Pooled fractions were then applied toa hydroxylapatite column (40 ml) in buffer containing10 mM potassium phosphate and 200 mM KCl, andeluted as a sharp peak by washing with the same phos-phate buffer but with the salt omitted. Finally, the pro-tein was further puri®ed and concentrated on a Mono QFPLC column in TEGD buffer (�100 mM KCl) andeluted with a 100 mM-600 mM KCl gradient. Peak frac-tions containing RuvBD113E (340-420 mM KCl) werepooled and dialysed against storage buffer (50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 40 % glycerol, 100 mM KCl,0.5 mM dithiothreitol). Approximately 25 mg of pureprotein were obtained from 55 g of wet cells. Theconcentration of RuvBD113E was determined from itsabsorption at 280 nm using an extinction coef®cient of1.64 � 104 Mÿ1 cmÿ1.

DNA substrates and nucleotides

Form I Bluescript KS (Stratagene), pDEA-7Z (Shahet al., 1994), pAKE-7Z (Eggleston et al., 1997) plasmidDNAs were prepared using Qiagen maxipreps. �X174virion DNA was purchased from New England Biolabs.Gapped duplex pDEA-7Z DNA (gDNA) and (30-32P)-labelled linear duplex pAKE-7Z DNA were prepared asdescribed (MeÂzard et al., 1997). Form I 32P-labelledpFB585 (Van Dyck et al., 1998) was prepared by growthin E. coli JM109 cells in the presence of [32P]orthopho-sphate and puri®ed using a Qiagen column. Relaxedplasmid DNA was made by treating form I (supercoiled)DNA with E. coli topoisomerase I. All DNA concen-trations are expressed in moles of nucleotides.

Helicase substrates were prepared by annealing(50-32P)-labelled oligonucleotides (20, 30 or 66 nt inlength) to �X174 virion DNA essentially as described(Tsaneva et al., 1993). The 20-mer was complementary to�X174 DNA at nt 570-589, the 30-mer was complemen-tary to nt 570-599, and the 66-mer to nt 5357-36.

Annealed substrates were puri®ed by gel ®ltrationthrough a 3 ml Bio-Gel A-0.5 m (BioRad) column equili-brated with 10 mM Tris-HCl (pH 8.0), 1 mM EDTA,100 mM NaCl.

ATPase and DNA binding assays

ATPase assays, DNA binding assays and electronmicroscopy were carried out essentially as described byMeÂzard et al. (1997).

DNA unwinding assays

DNA helicase assays were carried out as described byMeÂzard et al. (1999), except that the reactions wereallowed to proceed for 25 minutes.

The unwinding of covalently closed DNA was carriedout as described (Adams & West, 1995) but with a fewmodi®cations. Reactions (20 ml) contained 15 mM relaxed32P-labelled pFB585 DNA in 25 mM Tris-HCl (pH 7.5),0.5 mM EDTA, 2 mM DTT, 100 mg/ml bovine serumalbumin, 15 mM MgCl2, 0.5 mM ATP and 0 to 5 mMATPgS as indicated. RuvA and RuvB/RuvBD113E wereadded as indicated. After 15 minutes at 37 �C, ®ve unitsof wheat germ topoisomerase I were added and incu-bation continued for two minutes, after which the reac-tions were stopped and deproteinised. DNA productswere puri®ed by extraction with phenol/chloroform fol-lowed by ethanol precipitation, and analysed by 0.7 %(w/v) agarose gel electrophoresis in 80 mM Tris-acetatebuffer (pH 7.5) containing 5 mM sodium acetate, 1 mMEDTA and 0.03 % (w/v) SDS at 1.67 V/cm for 36 hours.32P-labelled DNA was visualised by autoradiography.

Branch migration assays

Recombination intermediates were prepared from(30-32P)-labelled linear duplex (pAKE-7Z) and gDNA(pDEA-7Z) by RecA-mediated strand exchange and puri-®ed as described (Eggleston et al., 1997). The linearduplex is homologous to the gapped DNA but containsa 1668 bp heterologous insertion which blocks strandexchange. Branch migration reactions containing0.57 mM 32P-labelled recombination intermediates werecarried out as described (MeÂzard et al., 1997), using theindicated amounts of RuvB/RuvBD113E in the presenceand absence of RuvA. 32P-labelled DNA was visualisedby autoradiography and quanti®ed using a Phospho-rimager.

Acknowledgements

We are grateful to Fiona Benson, Robert Lloyd andHideo Shinagawa for providing strains, and to DavidAdams and Irina Tsaneva for proteins. We thank mem-bers of the recombination laboratory, both past and pre-sent, for helpful discussions, criticisms and carefulreading of the manuscript, and Jacques Dubochet forhis interest. This work was supported by the ImperialCancer Research Fund, the Human Frontiers ScienceProgram, the Swiss National Foundation and the Swiss-British Council Joint Research Program. H.G. is a recipi-ent of an ICRF studentship and C.M. was supported inpart by a fellowship from the European MolecularBiology Organisation.


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Edited by J. Karn

(Received 6 August 1999; received in revised form 17 September 1999; accepted 17 September 1999)

Helicase-defective RuvBD113E promotes RuvAB-mediated branch migration in Vitro

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