The EMBO Journal vol.15 no.22 pp.6348-6361, 1996

The Tn7 transposase is a heteromeric complex inwhich DNA breakage and joining activities aredistributed between different gene products

Robert J.Sarnovsky, Earl W.May andNancy L.Craig1Howard Hughes Medical Institute, Department of Molecular Biologyand Genetics, Johns Hopkins University School of Medicine,Baltimore, MD 21205, USA

'Corresponding author

R.J.Sarnovsky and E.W.May contributed equally to this work

The bacterial transposon Tn7 translocates by a cutand paste mechanism: excision from the donor siteresults from double-strand breaks at each end of Tn7and target insertion results from joining of the exposed3' Tn7 tips to the target DNA. Through site-directedmutagenesis of the Tn7-encoded transposition proteinsTnsA and TnsB, we demonstrate that the Tn7 transpos-ase is a heteromeric complex of these proteins, eachprotein executing different DNA processing reactions.TnsA mediates DNA cleavage reactions at the 5' endsof Tn7, and TnsB mediates DNA breakage and joiningreactions at the 3' ends of Tn7. Thus the double-strand breaks that underlie Tn7 excision result froma collaboration between two active sites, one in TnsAand one in TnsB; the same (or a closely related) activesite in TnsB also mediates the subsequent joining ofthe 3' ends to the target. Both TnsA and TnsB appearto be members of the retroviral integrase superfamily:mutation of their putative DD(35)E motifs blockscatalytic activity. Recombinases of this class require adivalent metal cofactor that is thought to interact withthese acidic residues. Through analysis of the metalion specificity of a TnsA mutant containing a sulfur(cysteine) substitution, we provide evidence that adivalent metal actually interacts with these acidicamino acids.Keywords: active site/DD(35)E motif/DNAtransposition/protein-DNA complex/transposase

IntroductionThe movement of transposable elements between non-homologous insertion sites results from DNA breakageand joining reactions that disconnect the element fromflanking DNA at the donor site and then join the ends ofthe element to the target site. Central to the translocationof all elements examined in biochemical detail are DNAstrand cleavage reactions that expose the 3'. termini of thetransposon, and strand transfer reactions that join theseexposed 3' termini to staggered positions on the targetDNA (Mizuuchi, 1992a,b). Repair of the resulting gapsthat flank the newly inserted element generates the targetsite duplications characteristic of transposition. With someelements, the 5' transposon ends are also cleaved; whether

or not 5' end cleavage occurs has a profound impact onthe nature of the recombination product. When both the3' and 5' ends of the transposon are broken, the elementis excised from the donor site and inserted into the targetsite to form directly a simple insertion through a 'cut andpaste' mechanism. When only 3' end cleavage occurs, i.e.in the absence of cleavage at the 5' ends, the transposonremains attached to the donor site and is also attached tothe target site. Processing of this joint molecule, called aShapiro intermediate or strand transfer product (Arthurand Sherratt, 1979; Shapiro, 1979; Craigie and Mizuuchi,1985), by host replication functions (Craigie and Mizuuchi,1985; Kruklitis and Nakai, 1994) generates a structurecalled a co-integrate in which two copies of the transposonfuse the donor backbone and the target DNAs.The breakage and joining reactions that underlie trans-

position are mediated by transposases that bind specificallyto the ends of the transposon (Berg and Howe, 1989;Mizuuchi, 1992b; Craig, 1996a). The transposase is usuallyencoded by the mobile element itself, although hostproteins may also participate in recombination. In manycases, a single element-encoded gene product executesboth the breakage and joining steps. For example, atetramer of a single gene product forms the active transpos-ase of bacteriophage Mu (Lavoie and Chaconas, 1996).Analysis of transposase mutants defective in catalysisbut active in other recombination steps such as DNArecognition and assembly into higher order recombinationcomplexes has revealed that the individual breakage andjoining events at the two 3' ends of the transposon aredistributed among the transposase protomers, with thesame (or closely related) active site in each protomerbeing involved in a DNA breakage or joining reaction(Baker and Mizuuchi, 1992; Baker et al., 1994; Kim et al.,1995; Yang et al., 1995; Aldaz et al., 1996; Savilahtiand Mizuuchi, 1996). Similarly, a single gene product(integrase) executes the 3' end breakage and joiningreactions that underlie retroviral DNA integration (Katzand Skalka, 1994). In other cases, a single gene producttransposase can execute all the steps of cut and pastetransposition, i.e. both the 3' end breakage and joiningreactions and also 5' end breakage reactions. The bacterialtransposon TnJO is a particularly well studied element ofthis type (Kleckner et al., 1996). It has been shown thatthe same (or closely related) active sites in the single geneproduct TnJO transposase, which also functions as anoligomer, promote both the 3' and 5' transposon end DNAprocessing reactions (Bolland and Kleckner, 1996). Thetranslocation of many other cut and paste elements, forexample the Tc 1 and Tc3 elements of Caenorhabditiselegans and other members of the widespread marinerfamily of elements, is also probably mediated by a singlegene product transposase (van Luenen et al., 1994; Voset al., 1996).

6348© Oxford University Press

The Tn 7 transposase is a heteromeric complex

Although many transposition systems involve a singlegene product transposase, there are reactions in whichmultiple gene products are intimately involved in thebreakage and joining process. In the case of the P elementof Drosophila, the element-encoded recombinase binds tointernal sites which are not adjacent to the element terminiwhere breakage and joining will occur; rather, a hostprotein binds to this most terminal region (Kaufman andRio, 1992; Beall et al., 1994). Specific roles in DNAbreakage and joining have not yet been assigned to theseproteins. In V(D)J recombination, a reaction mechan-istically related to transposition (Craig, 1996b; van Gentet al., 1996), two proteins (Rag-I and Rag-2) collaborateto execute the double-strand breaks which initiaterecombination (McBlane et al., 1995). In a distinct reac-tion, conservative site-specific recombination, the highlyrelated XerC and XerD proteins execute related but distinctsteps in strand exchange (Blakely et al., 1993). In thecase of phage Mu, it is likely that a host-encoded activitydistinct from the MuA transposase itself can provokecleavage at the 5' ends of Mu following the MuA-mediated3' end breakage and joining reactions (Craigie andMizuuchi, 1985; Mizuuchi, 1992b). However, this 5'cleavage activity is not essential to recombination, sincethe 3' end breakage and joining reactions that result intransposition can occur efficiently in its absence.The bacterial transposon Tn7 (Barth et al., 1976; Craig,

1996c) moves through a cut and paste mechanism involv-ing double-strand breaks at both ends of Tn7 and joiningof the 3' ends to the target DNA (Bainton et al., 1991,1993) (Figure 1). Tn7 transposition involves multiple Tn7-encoded proteins: TnsA, TnsB, TnsC, TnsD and TnsE.Different subsets of Tns proteins promote insertion intodifferent target sites: TnsABC+D promote Tn7 insertioninto a specific site in the Escherichia coli chromosome,attTn7, and TnsABC+E promote Tn7 insertion into manynon-attTn7 sites (Rogers et al., 1986; Waddell and Craig,1988; Kubo and Craig, 1990). Molecular analysis of Tn7transposition into attTn7 revealed that all componentsof this reaction (TnsA, TnsB, TnsC, TnsD, the donortransposon DNA and the target attTn7 DNA) must bepresent for the initiation of recombination (Bainton et al.,1991, 1993). This requirement suggests that a prerequisitefor recombination is the formation of an elaborate nucleo-protein complex involving specific recognition of thesubstrate DNAs, and also involving synapsis of the trans-poson ends with each other and with the target DNA;multiple specific protein-DNA and protein-protein inter-actions are surely necessary to form this active complex.Which Tn7-encoded protein(s) in such a complex formsthe actual transposase and executes the DNA strandbreakage and joining reactions? We have been unable to

detect any breakage and joining activity by any Tnsprotein individually, probably reflecting the requirementfor proper complex assembly to 'activate' the breakageand joining activities. However, we have found thatTnsA+TnsB, in the absence of other Tns proteins andunder non-standard reaction conditions in vitro, can

together execute double-strand breakage and intramolecu-lar joining reactions that are related to but distinct fromstandard intermolecular transposition (M.Biery, M.Lopataand N.L.Craig, in preparation). This result focuses atten-tion on TnsA and TnsB as candidates for the Tn7 trans-

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Fig. 1. Tn7 transposition reactions. Tn7 transposition proceeds throuahtwo distinct steps. initiating with donor cleavage followed by targetjoining. Various substrates and products are shown. the substratesdiffering in whether or not they have already undergone donorcleavage (adapted from Gary et al., 1996). The cis-actingrecombination sequences at the Tn7 ends which include and extendbeyond Tn7's terminal 30 bp inverted repeats are designated by anopen triangle (Tn7L) and a closed triangle (Tn7R): the target DNA isshown as a dashed line. (A) An intact donor substrate in which thetransposon is flanked by donor backbone DNA. The position of Ndel(N) sites in the donor and target plasmids are indicated. DSBs resultfrom a double-strand break at either Tn7L (DSB.L) or Tn7R (DSB.R)3a simple insertion results from joining the two transposon ends of asingle Tn7 element to a target DNA; a single-end join (SEJ) resultsfrom the joining of a single Tn7 end to the target DNA. Both DSB-SEJs and ELT-SEJs can form. (B) An excised linear transposon (ELT)substrate which can form a simple insertion in which both Tn7 endsjoin to a single target. or a double-insertion single-end join[(DI) ELT.R-SEJ] when two Tn7R ends from two different ELTs jointo a single target.

posase. Other work has established that TnsB plays a keyrole in substrate recognition, being a sequence-specificDNA binding protein which interacts with multiple sites ateach Tn7 end (Arciszewska and Craig, 1991; Arciszewskaet al., 1991; Tang et al., 1991). TnsA has no detectableDNA binding activity (Bainton et al., 1993). The otherTns proteins, TnsC, TnsD and TnsE, are likely to playkey roles in selecting the element insertion site (Baintonet al., 1993; Craig, 1996c).

To probe the specific roles of TnsA and TnsB in theactual strand breakage and joining reactions, our strategyhere has been to generate and examine the properties ofsite-directed 'catalytic' mutants of TnsA and TnsB, i.e.mutants that are blocked in particular steps of strandbreakage and joining but which remain active in the manyother steps necessary for the formation of the multiproteinTn7 transposition complex. We have reported previouslythat a mutation in TnsA can block DNA breakage at the5' ends of Tn7 but does not alter the breakage and joiningreactions at the 3' ends of Tn7 (May and Craig, 1996).These experiments established that these 3' and 5' DNAprocessing reactions can be separated. Indeed, their separ-

6349

R.J.Sarnovsky, E.W.May and N.L.Craig

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Fig. 2. Working alignment of DD(35)E family members. Representative members of the DD(35)E superfamily of transposases and retroviralintegrases are aligned with TnsA and TnsB. The highly conserved DD(35)E residues are boxed; positions of identity between TnsA and TnsB areshaded. Mutagenized TnsA and TnsB residues are indicated except for E424A TnsB. Identical amino acids between TnsA (Flores et al., 1990; Orleand Craig, 1991) and TnsB (Flores et al., 1990) are shaded. The other proteins are: the p480 gene product of the Tn552 TnpA gene (Rowland andDyke, 1990); the IS3 transposase (Fayet et al., 1990); the RSV and HIV-1 integrases (Engelman and Craigie, 1992; Kulkosky et al., 1992); and thebacteriophage Mu transposase (Baker and Luo, 1994; Radstrom et al., 1994; Kim et al., 1995). Note that the spacing between the second and thirdconserved acidic residues of the Mu transposase is 55 amino acids, rather than 35. In this region, the alignment was directed by the 2° structuresimilarities of HIV integrase and the Mu transposase (Dyda et al., 1994; Rice and Mizuuchi, 1995).

ation has an interesting biological consequence in thatTn7 is converted, through specific blockage of only the5' end cleavage reactions, from a cut and paste element,like TnlO, into a replicative element, like phage Mu.These experiments did not, however, reveal which Tnsprotein is the transposase: are all the active sites for DNAprocessing reactions in one of the two candidate proteins,TnsA or TnsB, with the other protein acting as a 'modu-lator' of activity, or are the active sites for the DNAprocessing reactions disributed between TnsA and TnsB?The work presented here reveals that the Tn7 transposaseis a heteromeric complex in which TnsB and TnsAinterdependently contribute active sites for the DNAprocessing reactions; TnsA contains the active site for 5'end cleavage and TnsB contains the active site for 3' endbreakage and joining. This separation of active sitesbetween different gene products that act together to forma transposase has not been described previously, althoughit is not unreasonable to suggest that other recombinationreactions will also involve such collaborations.Our analyses also suggest that TnsA and TnsB are

likely to be members of the retroviral integrase superfamilyof recombinases. These Tns proteins, like the transposasesof retroviruses, retrotransposons, IS3 elements and

bacteriophage Mu, contain a limited homology to a highlyconserved array of acidic amino acids, the DD(35)E motif,in which the last D and E are generally separated by 35amino acids (Radstrom et al., 1994; Craig, 1996c; Polardand Chandler, 1995) (Figure 2). An attractive view, sup-ported by genetic, biochemical and structural studies, isthat these conserved amino acids lie at (or at least areintimately related to) the active sites which mediate thebreakage and strand transfer reactions during recombin-ation (reviewed in Craig, 1995; Grindley and Leschziner,1995). The DD(35)E motif is thought to provide a bindingsite for an essential metal ion, usually Mg2+, at or nearthese active sites (Baker and Luo, 1994; Dyda et al.,1994; Kim et al., 1995; Rice and Mizuuchi, 1995; Bujaczet al., 1996). We provide functional evidence here thatTnsA does indeed interact with a metal ion. We showthat when an aspartic acid, i.e. an oxygen-containingcarboxylate, within the proposed TnsA DD(35)E motif issubstituted with a cysteine, i.e. containing sulfur, the metalspecificity of the 5' processing reaction switches fromMg21 to Mn2+. The strategy of analyzing metal ionspecificity in the presence of sulfur-substituted reactioncomponents has been exploited previously to show aninteraction between an essential metal ion and the substrate

6350

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The Tn 7 transposase is a heteromeric complex

in RNA self-splicing (Dahm and Uhlenbeck, 1991;Piccirilli et al., 1993).A view that has emerged from the analysis of many

recombination systems is that these DNA breakage andjoining reactions are highly controlled and coordinated,ensuring that such potentially lethal reactions are limited toparticular chromosomal positions and occur at biologicallyappropriate times. A powerful strategy to impose suchcoordination is to link the ability to execute recombinationto the proper assembly of a nucleoprotein complex con-taining recombination proteins and DNA substrates, aprocess involving multiple protein-DNA and protein-protein interactions (Baker, 1993). The identification of amulti-gene product Tn7 transposase provides a new anddistinctive example of a 'division of labor' amongrecombination proteins and provides a new scenario forthe control of recombination reactions.

ResultsTnsA mutations block breakage at the 5' ends ofTn7 but do not alter 3' end processing reactionsWhich Tns protein(s) forms the Tn7 transposase? It hasnot been possible to detect any DNA breakage or joiningactivity with either TnsB or TnsA alone or when eitherof these proteins is simply omitted from recombinationreactions (Bainton et al., 1993; Gary et al., 1996); thus ithas not been possible to assign unique roles in DNAprocessing to either of these proteins. To dissect theirroles in transposition, we generated mutant versions ofthese proteins specifically defective in the DNA breakageand joining steps but still active in the many other stepsessential for recombination, such as specific recognitionof the substrate DNA and the ability to assemble into arecombination-competent nucleoprotein complex.We have reported previously that mutation of TnsA

D114 to alanine (DI14A) blocks cleavage at the 5' ends,but 3' end breakage and joining still proceed, forming afusion product (FP) in which the donor and target plasmidsare joined through a copy of the transposon, (May andCraig, 1996) (Figure 3A). Thus the Dl 14A mutant hasthe hallmarks of a 'catalytic' mutant, i.e. only 5' endbreakage is blocked and the other steps key to transposi-tion, such as recognition of DNA substrates and formationof an appropriate transposition complex, must still occur.This result does not, however, reveal the actual locationof the 5' cleavage active site; this active site could be inTnsA, or it might be in TnsB and regulated in some wayby TnsA. Our experiments here suggest that TnsA actuallycontains the active site for 5' end processing. A weakhomology is observable between a region of TnsA andthe active site region of other transposases, the conservedDD(35)E triad of amino acids, in which the last D and Eare generally separated by 35 amino acids (Figure 2). Thisputative DD(35)E motif in TnsA is not convincing. Indeed,a match was only possible after the identification ofhomology between TnsA and TnsB in this region of theprotein; the motif in TnsB is far more compelling (seebelow). Most notably, TnsA lacks an acidic amino acidthat obviously corresponds to the first D of the canonicalDD(35)E motif. However, the DI 14A mutation whichblocks 5' end processing is altered in the second D ofTnsA's version of the motif.

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Fig. 3. TnsA mutations block double-strand breaks and result intransposon-mediated fusions between the donor and target plasmids.(A) Mutation of TnsA can block 5' end cleavage and result in theformation of donor-target plasmid fusions. When 5' cleavage at bothtransposon ends does not occur, a fusion product (FP) is formed: FPOPP(not shown) containing Tn7 inserted in the opposite orientation inattTn7. can also form. When 5' end cleavage occurs at one Tn7 endbut not the other. two different fusions containing DSBs (FP.DSB) areformed. (B) In l'itro Tn7 transposition reactions were carried out withwild-type or mutant TnsA proteins using supercoiled donor plasmidDNA; the products were displayed on native agarose gels. Substratesand other products are labeled as in (A) and Figure 1. (C) Timecourses of recombination with wild-type and E149A TnsA. This timecourse was performed at 22°C instead of the standard 30°C. slowingthe reaction rate and allowing analysis of intermediates (Bainton et al.,1991).

To probe further the role of TnsA's putative DD(35)Emotif in recombination, we evaluated other mutants inin vitro reactions using purified Tns proteins and super-coiled plasmid donor substrates (Figure 3B). As previouslyreported, Dl 14A TnsA blocks cleavage at the 5' ends ofTn7 but does not affect breakage and joining at the 3'ends (May and Craig, 1996). E149A TnsA, altered in theE of TnsA's version of the DD(35)E motif, also results inreduction of 5' end cleavage (lane 12). In contrast to thecomplete block imposed by DI 14A TnsA, 5' cleavage isonly partially reduced with E149A TnsA; however, 5'cleavage with E149A TnsA in vivo is as reduced as

DI 14A TnsA (see below). Two species resulting fromreduced 5' end cleavage are observed, the FP and a partialfusion product, FP(double-strand break), in which 5'cleavage does not occur at one end of Tn7 but does occur

at the other end. Considerable amounts of simple insertionsand donor molecules broken by a double-strand break

6351

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R.J.Sarnovsky, E.W.May and N.L.Craig

(DSB) are also present. We note that in reactions withE149A TnsA, there is a disproportionate amount ofFP(DSB.L) compared with FP(DSB.R); thus E149A TnsAdisplays a bias in left versus right Tn7 end utilization,hinting that TnsA may also play a role in defining thefunctional identities of the transposon ends. This bias isin contrast to the equivalent production of FP(DSB.L) andFP(DSB.R) in reactions containing both wild-type andD114A TnsA (May and Craig, 1996). The partial 5'cleavage defect displayed with E149A reflects a decreasein cleavage rate, as revealed by a time course analysis ofthe E149A TnsA-mediated reaction (Figure 3C); at earlytime points, FPs and FP(DSB.L)s arise and, later in thetime course, further cleavage of the 5' ends occurs andthe amount of FP decreases.TnsA lacks an acidic amino acid corresponding to the

first D of the DD(35)E motif, although there are twoacidic residues in the region, D28 and E37. No alterationof recombination is observed with D28A (Figure 3B, lane3). With E37A TnsA, there is only a very modest decreasein 5' cleavage as evidenced by a slightly higher productionof FP(DSB.L) (lane 5). With an E37A/E149A doublemutant (lane 13), the ratio of fusion products to simpleinsertions is slightly greater than that of the singly mutatedE149A TnsA (lane 12). Other TnsA point mutations,T34A, D18A, E144A and E147A, had little effect upontransposition. Mutations at H57 and S60 dramaticallyreduced recombination; these proteins appear to have moreextensive defects, as addition of these proteins to reactionscontaining limiting wild-type TnsA had little effect (datanot shown).

These experiments reveal that TnsA has an intimaterole in 5' end cleavage: in certain TnsA mutants, 5' endcleavage is altered while 3' end activities persist. Itis notable that these TnsA mutants display a catalyticphenotype; although a specific chemical step fails to occur,i.e. 5' end cleavage, many other steps in recombinationdo occur to allow the formation of a nucleoprotein complexcompetent to execute the 3' end processing reactions.Assignment of the active site to TnsA on the basis ofthese results alone cannot be firmly made, in particularbecause of the limited homology between TnsA and theDD(35)E motif. However, we provide functional evidencebelow that TnsA interacts directly with the metal cofactor,supporting both the view that the active site for 5' endcleavage is in TnsA, and the hypothesis that TnsA is amember of the DD(35)E family.We have also evaluated the effects of these TnsA

mutations on Tn7 recombination in vivo (data not shown).We previously reported that with DI 14A TnsA, Tn7 makespredominantly (80%) co-integrate products rather than thesimple insertions obtained with wild-type Tn7 (May andCraig, 1996). Co-integrates are the in vivo consequenceof a failure in 5' end cleavage; the Shapiro intermediateformed by breakage and joining at exclusively the 3' endsof the transposon is the substrate for DNA replicationwhich generates the co-integrate. E149A TnsA also gener-ates a very high proportion of co-integrate moleculesin vivo (77%). The observation of co-integrate formationwith D114A and E149A TnsA supports the view thatthese mutant proteins are defective specifically in 5' endcleavage; with wild-type TnsA and with the other TnsAmutants examined, the recombination products are simple

inserts, consistent with double-strand breaks involvingboth 3' and 5' end cleavage. Moreover, all TnsA mutantsexamined supported overall transposition, i.e. joining ofthe 3' ends of the transposon to the target plasmid, at alevel comparable with that of wild-type (data not shown).We also note that while in vitro recombination usingH57A and S60A TnsA was reduced markedly, transposi-tion in vivo using these two mutants was unaffected(data not shown); the nature of this suppression is notunderstood.

Evidence for a direct interaction between metaland TnsAThe DD(35)E motif is thought to provide a binding sitefor a divalent metal at or near an active site(s) for DNAhydrolysis and strand transfer (Baker and Luo, 1994; Dydaet al., 1994; Kim et al., 1995; Polard and Chandler, 1995;Rice and Mizuuchi, 1995; Bujacz et al., 1996) Mg,+ isa critical cofactor in Tn7 recombination in vitro; norecombination is observed in the absence of divalent metal(Bainton et al., 1993). A reasonable hypothesis is that thismetal ion is a cofactor for TnsA (and/or TnsB, see below).However, the homology between TnsA and the DD(35)Emotif is limited; establishing that TnsA does contain ametal-dependent active site requires other evidence. Wehave used the differential chemistry of metal-sulfur andmetal-oxygen interactions (Pecoraro et al., 1984) to pro-vide evidence that an essential metal acts in close proximityto a specific aspartate in the DD(35)E motif of TnsA.Similar metal ion specificity experiments have been donewith ribozymes in which the metal requirements ofthiol-containing substrates were explored (Dahm andUhlenbeck, 1991; Piccirilli et al., 1993). The finding thatMn2+ was able to support the cleavage of the thiol-substituted RNA substrate far better than could Mg2+, i.e.that a change in metal ion specificity was observed,suggested an interaction between the metal and the sub-strate in the active sites of these enzymes.We have found that a cysteine substitution at the D114

position of TnsA changes the metal ion specificity ofthe 5' end cleavage reaction. To focus directly on theinvolvement of the divalent metal in the 5' end cleavageactivity, we temporally staged the reaction (Figure 4A).In the first step, we used DI 14 mutants of TnsA, eitherDI 14A or DI 14C, in a Mg2 -supported reaction to gener-ate high levels of FP, then added an additional metal andcontinued the incubation. Production ofFP(DSB) or simpleinsertions, in which double-strand breaks have been intro-duced at either or both transposon ends, are the signaturesof a suppression of the 5' end cleavage defect.

Strikingly, we observe a Mn2+-specific suppression ofthe DI 14C TnsA 5' cleavage defect (Figure 4B, lanes 10-12). In the presence of Mn2+ and D114C TnsA, specific5' end cleavage of the FP occurs. As the concentration ofMn2+ increases, more FP(DSB)s and simple insertionproducts are generated. It is interesting that 5' cleavageat Tn7L appears to occur preferentially, as indicated bythe disproportionately high level of FP(DSB.L). It shouldbe noted that Mn2+ can introduce a degree of permissive-ness to many in vitro reactions (Vermote and Halford,1992; Polard and Chandler, 1995). However, with DI 14ATnsA, we see no such permissive phenotype; no Mn>_supported 5' cleavage with DI 14A TnsA is observed (lanes

6352

The Tn 7 transposase is a heteromeric complex

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(cysteine) into TnsA changes the metal ion specificity of 5' cleavage

from Mgc to Mn>. In vitro Tn7 transposition reactions were staged

differently [(A) see text] using D114A or Dl 14C TnsA in the presence

of Mg> or Mn'; the products are displayed on native agarose gels.

Substrates and other products are labeled as in Figures 1 and 6.

4-6). Moreover, no 5' cleavage products are generated byany other combination of D114 mutations and Ca2+ or

Zn2+ (data not shown).The Mn2+-specific suppression of the sulfur-containing

Dl 14C mutant implies that this acidic residue in wild-type TnsA is, in fact, in a metal binding site, stronglyimplicating a metal-dependent active site in TnsA for thehydrolysis of the 5' end during Tn7 transposition. Thisnovel use of sulfur (cysteine) substitutions may prove

useful in the study of the role of metals in other protein-nucleic acid interactions. Indeed, a similar suppressionhas been observed recently within the Mu transposaseactive site (E.Krementsova and T.Baker, personal com-

munication).

TnsB mutations can block the joining of 3' Tn7ends to target DNA but do not alter 5' endprocessing reactionsThe results presented above provide evidence that theactive site for 5' end cleavage lies in TnsA. Where is theactive site(s) for the 3' end processing reactions ofbreakage and joining? Also in TnsA? In TnsB? A modesthomology to the DD(35)E motif is also present in TnsB(Figure 2). We also note the limited homologies betweenTnsA and TnsB other than the DD(35)E motif (Figure 2);indeed, this alignment of TnsA with TnsB was key to the

recognition of the degenerate DD(35)E motif of TnsA.We generated TnsB mutants and analyzed their activities in

recombination. As observed with TnsA DD(35)E mutants,these TnsB mutants also appear to display a catalyticdefect; 3' end breakage and joining reactions are blocked

specifically without dramatically affecting other aspectsof the reaction, such as the ability to enter into the

nucleoprotein complex and support the 5' end cleavageactivity of TnsA. Thus, the experiments described below

provide evidence that the 3' end processing reactions in

Tn7 transposition are mediated by active sites in TnsB.To evaluate directly the 3' end joining step in Tn7

transposition apart from DNA breakage activities, we used

an excised linear Tn7 element (ELT) as a substrate DNA.

i--lt s | t10 , 14 -1' '> | !wi-.t s

21 ! 1, 25 1' 25 73 Jm1uta t

FI:lT->

,INlIILLINSERI ION

24-

4 4sIE 4W -ELT

_ 1 4 -12 13 14 1; li' P;,

Fig. 5. TnsB mutations block strand transfer of exposed 3' Tn7 ends

to the target DNA. In vitro Tn7 transposition reactions were carried

out with either wild-type or mutant TnsB (A) or a mixture of wild-

type and mutant TnsB (B) using, ELTs as a donor DNA substrate-, the

products are displayed on native agarose gels. Substrates and products

are labeled as in Figure 1. In addition to a simple insertion, in which

both ends join to a single target. other recombination products are

particularly notable with ELT substrates: a singale-end join (SEJ) in

which only one transposon end is joined to one strand of the target.

and a double-insertion single-end join [(DI) ELT.R-SEJ] in which two

different ELTs join to a single attTnz7 (Bainton et al., 1991:- Gary

et al., 1996).

The ELT has been disconnected from the donor DNA by

restriction of specialized donor molecules (Bainton et al.,

199 1; Gary et al., 1996) (Figure 1). We note that TnsA is

required to support target joining with an ELT substrate,

although the chemical step executed by TnsA, 5' end

cleavage, is not actually required in this reaction (Gary

et al., 1996). This result reveals that the 3' end joininge

activity is dependent on the presence of TnsA in the

nucleoprotein complex.With an ELT substrate, no target joining can be detected

with D273N, D3O1A, D361N or E396Q TnsB (Figure

5A). D273, D361 and E396 are the signature amino acids

of the TnsB DD(35)E motif, suggesting, a critical role for

this motif in joining of the 3' Tn7 ends to the target DNA.

With E364A TnsB, target joining does occur but the major

products are single-end joins (SEJs), in which only one

end of the ELT has been joined successfully to the target.

Target joining using D278N, D283N, S304A, E371A or

E424A TnsB is not significantly different from that seen

with wild-type TnsB.

In ELT reactions using mixtures of wild-type and the

joining-defective TnsBs D273N, D36IN or E396Q TnsB,the addition of the mutant protein dramatically changes

6353

BU . (X),Q .'.

Z Z Z " --r" x r, -?'.. X :;

;, .1 "" -,X.\\ 1.\ i%I

R.J.Sarnovsky, E.W.May and N.L.Craig

/ /: /. .- /t / /

I. -

(.)\ ()II

K /I/

1., 1 \ 1 1.

*'\. ' P,B1.iv/\ '124

._ _ ..

N 4 - t- - S 11 1_ !-

AIN .N > IN I)

~;;, 'R _. 1 . 1.! 14. R; R;. 2 ;s zl, RZ , t,1

B- -* q-p

:1 1 LI,^

INI RI (IN

't alrn tt

%1 _ . 4 to - 11_ I" II 1| 1| I| u K I., I,

Fig. 6. TnsB mutations block the double-strand breaks that initiateTn7 transposition. In vitro Tn7 transposition reactions were carried outwith wild-type or mutant TnsB (A), or a mixture of wild-type andmutant TnsB (B), using supercoiled donor plasmid as substrate; theproducts are displayed on native agarose gels. Joining of the exposed3' end of a DSB to attTn7 results in a double-strand break-single-endjoin (DSB-SEJ). Substrates and products are labeled as in Figure 1.

the pattern of the ELT recombination products (Figure5B). Whereas the major products with wild-type TnsB aresimple insertions formed by the joining of both transposonends to the target, addition of these mutant proteins resultsin the accumulation of SEJs formed by the joining of asingle transposon end. We interpret these results to indicatethat strand transfer at both ends of Tn7 to form a simpleinsertion requires multiple TnsBs in the nucleoproteincomplex active in transposition, and that these TnsBmutants have catalytic defects; they do not promote endjoining but are active in other stages of recombination, suchas substrate recognition and formation of a multiproteincomplex. By contrast, addition of D301A TnsB hadlittle effect on wild-type recombination (data not shown),suggesting that this mutant has more than a simple catalyticdefect. No recombination was observed with pairwisecombinations of D273N, D361N and E396Q TnsB, norwith a mixture of all three mutants (data not shown).

TnsB mutations block breakage at the 3' ends ofTn7 but do not alter 5' end processing reactionsTo probe the effects of TnsB mutations on the initiatingdouble-strand break step, we examined the TnsB mutantsin reactions using supercoiled plasmid donor substrates(Figure 6A). Most notably, no DNA breakage or joiningreactions were detected with this assay using D273N,D301A, D361N or E396Q TnsB. Therefore, these TnsBmutant derivatives are not only altered in 3' end targetjoining activity, as shown above with ELT substrates, butare also altered in the preceding donor breakage reactions.

E364A TnsB is able to execute donor breakage, butappears altered in target joining, as indicated by theaccumulation of ELTs and ELT-SEJs. The patterns dis-played with other TnsB mutants (D278N, D283N, S304A,E371A, E424A) resemble that of wild-type.The examination of recombination products using this

non-denaturing electrophoresis assay does not reveal anysingle-strand nicking activity that may be present. Thedouble-strand breaks which cut Tn7 away from the donorbackbone actually involve two nicking reactions: cleavageto expose the 3' ends of the transposon and cleavage ofthe 5' ends (Gary et al., 1996). To analyze these twocleavage reactions individually, we examined DNAs fromrecombination reactions on denaturing gels using strand-specific probes for the top and bottom strands of Tn7Land Tn7R (Figure 7). With wild-type TnsB (lanes 1-3),3' end breaks (Tn7L = 172 nt, probe B; Tn7R = 204 nt,probe C) are observed only at low levels because most ofthe processed 3' ends are joined to the target DNA(Tn7L = 530 nt, probe B; Tn7R = 400 nt, probe C); themajor 5' strand products are cleavages at these ends(Tn7L = 171 nt, probe A; Tn7R = 203 nt, probe D). NoDNA cleavage is seen in the absence of TnsB (lane 14).Strikingly, wild-type levels of 5' end cleavage are observedwith D273N, D361N and E396Q TnsB but the 3' endreactions of breakage and joining are not detectable withthese mutants (lanes 4, 9 and 12). Thus, these DD(35)ETnsB mutants appear to be catalytic in nature; they areblocked in their own 3' end activities but are able to enterinto the transposase complex and support other aspects ofTn7 transposition. Neither 3' nor 5' end cleavages aredetectable with D301A (lane 7), suggesting again that theeffects of this mutation are more extensive than a simplecatalytic defect.When wild-type TnsB and the breakage-and-joining

defective TnsBs (D273N, D361N or E396Q) are mixedin reactions using supercoiled plasmid donor substrates,addition of the mutant protein results in the accumulationofDSBs and DSB-SEJs (Figure 6B). These results indicatethat the nucleoprotein complex which executes Tn7recombination contains multiple TnsB proteins, i.e. thatmultiple active sites* for 3' end processing are involved;similar results were obtained with ELT substrates (Figure5B). The patterns of recombination products obtained insuch mixing experiments can provide information aboutthe actual number of 3' end processing sites, i.e. thelikely number of transposase protomers, involved in arecombination reaction (Baker et al., 1994; Kleckner et al.,1996; Bolland and Kleckner, 1996). However, the mutantproteins may impose biases in the assembly and stabilityof recombination complexes (Baker et al., 1994; Bakerand Luo, 1994; Kim et al., 1995). Thus while theseTnsB mixing experiments do provide strong evidence thatmultiple TnsB active sites are involved in an activerecombination complex, a clear definition of the actualnumber of active sites in such a complex cannot beinferred easily from only these experiments.We have also examined the effects of TnsB on Tn7

transposition in vivo, analyzing TnsABC+D transpositionto attTn7 and also transposition mediated by a differentensemble of Tn7 transposition proteins, TnsABC+E, tonon-attTn7 targets (Rogers et al., 1986; Waddell andCraig, 1988; Kubo and Craig, 1990). TnsB DD(35)E

6354

'm M*4040,40 me.

40 sm 404* -am on NW as

.: 2:. 7" 7 .; , I .: .. :., .-:; 1! .: I :; :., ; ,-,

The Tn 7 transposase is a heteromeric complex

Z Zz . tor.x, el -?

I -

1]s* r r * c}~~*v3

DONOR

171--5'BREAKS

e46h * 1A 14

probe A

probe B1 * 3 *5 - C *11 '13

530- -3' JOINS

235- - - -

DONOR

172-3' BREAKS 2* a 4 * 5* S * l10 12 . 14

DONOROS 2x,94 X'

F7l 204I 1 4-tA b

probe A probe C

mTn7

H2eB7L )pmbe13prObe L)

-4 .. +172.....

D. ....-0--

20)3

-2-11

pr)be A probe C

nmTn -

proabe B~ probe L)

- -1113

s aRli S

SIMPLE INSERTION

z z Z. c:z t/

f44 ri C,

\\\'/ ///.1 . s.11'_

_ ___ _400

_ 3' JOINS

DONOR

-2043' BREAKS

. -' a 4 . h . S . 1{l.4

probe C

probe DI * 3 *;5 * 9 *11.13-----... - -294

DONOR

-2035' BREAKS

* *.4.*h * S*l* 1Z*lq 4

Tn7 Left Tn7 Right

Fig. 7. TnsB mutations block cleavage at the 3' ends of Tn7 but do not block 5' end cleavage. In vitro Tn7 transposition reactions were carried out

as in Figure 4, but the products are displayed on denaturing polyacrylamide gels; following transfer to nitrocellulose, the fates of the various strands

at the Tn7L and Tn7R ends were analyzed with strand-specific oligonucleotide probes as indicated (probes A. B, C and D). The diagram shows a

substrate donor DNA (top) and the simple insertion product (bottom). When cleavage of the 3' transposon ends occurs, these species are detected

principally as joins to the target DNA, whereas the products of 5' end cleavage remain as cleaved species. The hybridization positions of the probes

are shown and the sizes of the various substrate and product fragments are indicated. S = Sall, R = EcoRI. H = HiozdIll.

mutants are defective in both transposition pathways (datanot shown). Consistent with the view that alteration ofthe TnsB DD(35)E motif imposes a catalytic block onrecombination, these TnsB mutants act as dominant-negative inhibitors of transposition in vivo when bothwild-type and mutant TnsBs are present (data not shown).

Thus certain TnsB mutants, D273N, D361N and E396Q,are specifically defective in the chemical events thatinvolve the 3' ends of the transposon: the cleavagereactions which expose the 3' ends and, as also shownabove, the joining of exposed 3' ends to the target DNA.An attractive hypothesis is that these DD(35)E mutationsresult in catalytic defects and that the active site for thesereactions is contained in TnsB.

Mn2+ can suppress the breakage and joiningdefects imposed by TnsB mutationsTo challenge further the hypothesis that TnsB is a memberof the retroviral integrase family of metal-dependentrecombinases, we also examined Tn7 recombination in

the presence of Mn2+ (Figure 8). With wild-type TnsBand TnsA, DNA breakage and joining can occur withMn>, although Mn- -promoted recombination is notequivalent to that seen with Mg>+ (Bainton et al., 1993;M.Biery, M.Lopata and N.L.Craig, in preparation). Wealso analyzed the effects of Mn2+ on the DD(35)E mutantTnsBs that were shown above to be inactive in thepresence of Mg>. Mn- can partially suppress the blockimposed on double-strand break formation by D36 INTnsB (Figure 8A). This partial suppression is in contrastto the complete lack of suppression of the 5' end cleavagedefect of Dl 14A TnsA with Mn2+ (Figure 4B, lanes 4-6). The suppressive effects of Mn>+ on the 3' endjoining reactions are much more pronounced. With an

ELT substrate, considerable Mn2+-supported joiningoccurs with D273N and E396Q TnsB but virtually no

recombination is detectable with D361N TnsB (Figure8B). Thus, although Mn>+ can suppress some aspects ofthe recombination blocks imposed by the D273N, D361Nand E396Q TnsB mutants, the suppressive effects vary:

6355

R.J.Sarnovsky, E.W.May and N.L.Craig

1.

1 2I 15t I3 D B. i~ i|* |IS4h TN-I -|k! | - | i'Nl 1 2 i1

DLh-Ilii}o'.

13~~~~~~~~II

Iln

j XaX,I 1-I

" ,wr -.1..1,N 1 I§s ]()\

~dJd - I- L-1X--' 1E 13I l 14 J. 1v'1.V 1- 2

Fig. 8. Mn2+ can suppress the defects imposed on breakage andjoining by TnsB mutations. In vitro Tn7 transposition reactions werecarried out with wild-type and mutant TnsB using either a supercoileddonor plasmid (A) or an ELT donor (B) in the presence of Mg2+ orMn> as indicated; the products are displayed on native agarose gels.Recombination substrates and products are labeled as in Figure 1.

Mn2+ most effectively suppresses the breakage defect ofD361N but most profoundly suppresses the joining defectsof D273N and E396Q. No recombination is detectablewith Ca2+, Zn2+, Cd2+ or Co2+, with wild-type or mutantTnsB using either ELTs or intact supercoiled substrates(data not shown).The finding that the defects imposed on recombination

by TnsB DD(35)E mutations can be partially suppressedby the presence of the alternative metal cofactor Mn2+ isconsistent with the view that the DD(35)E motif in TnsBprovides a metal-dependent active site for the DNAbreakage and joining reactions at the 3' ends of Tn7.Furthermore, experiments presented above suggest that aDD(35)E motif in TnsA provides a metal-dependent activesite for 5' end processing. Thus the DNA breakage andjoining reactions that underlie the translocation of Tn7are mediated by collaborations between metal-dependentactive sites in two different gene products: TnsB andTnsA interdependently form the Tn7 transposase.

DiscussionA functional model for Tn7 transpositionTn7 transposition is initiated by double-strand breaks thatexcise the element from flanking DNA at the donor site;the newly exposed 3' ends of the excised transposon arejoined to the target DNA (Bainton et al., 1991, 1993).The experiments presented here provide evidence that Tn7uses a distinctive recombination mechanism. Two element-encoded proteins that appear to be members of theretroviral integrase superfamily, TnsA and TnsB, form aheteromeric Tn7 transposase; moreover, each gene product

provides different DNA processing activities which acton different strands at the transposon ends (Figure 9). Ouranalyses here of TnsA and TnsB catalytic mutants, i.e.protein mutants capable of entering the recombination-competent nucleoprotein complex but blocked at particularreaction steps, suggest that TnsB executes the 3' endbreakage and joining reactions, whereas TnsA cleaves the5' ends of the transposon. We note that it has also beenpossible to separate these 3' and 5' end activities throughDNA mutation in the ends of Tn7 (Gary et al., 1996).Because our experiments provide strong evidence thatparticular amino acids within each protein interact with adivalent metal important to catalysis, a reasonable hypo-thesis is that the designated active sites actually liewithin TnsB and TnsA. However, we cannot exclude thepossibility that amino acids from multiple polypeptidesmay actually contribute to a common active site (Jayaram,1994; Craigie, 1996).The Tn7 transposase thus consists of subunits of at

least two different proteins, TnsA and TnsB, and, throughexperiments involving the mixture of wild-type and mutantproteins, we can conclude that there are at least twoprotomers of each subunit in the active transposase. TnsBand TnsA are probably assembled onto the transposonends in different ways. TnsB mediates recognition of thedonor substrate, binding to specific sites at the ends ofthe element (Arciszewska et al., 1989; Arciszewska andCraig, 1991). No DNA binding activity by TnsA has beendetected (Bainton et al., 1993), therefore it is likely thatTnsA is recruited to the ends through interactions withTnsB. The other Tns proteins, TnsC, TnsD and TnsE,regulate the activities of TnsB+A transposase and modu-late target selection: TnsC serves as the connector betweenTnsB+A and the target protein TnsD and also probablythe alternative target-determining protein TnsE (Baintonet al., 1993; Craig, 1995a; M.Biery, M.Lopata andN.L.Craig, in preparation; A.Stellwagen and N.L.Craig,in preparation). TnsABC+D mediate insertion into Tn7'sspecific chromosomal site attTn7, and TnsABC+E medi-ate insertion into many different non-attTn7 sites (Rogerset al., 1986; Waddell and Craig, 1988; Kubo and Craig,1990; Bainton et al., 1993; Craig, 1996c).Tn7 uses a novel mechanism to catalyze double-strand

breaks: two proteins interdependently conduct cleavagereactions on different strands at the transposon ends. Thismechanism is distinctive not only among transpositionsystems (Haniford et al., 1989, 1991; Mizuuchi, 1992b;Chalmers and Kleckner, 1994; Kleckner et al., 1995), butalso among more general endonucleolytic processes (Linnet al., 1993). Although many DNA processing reactionsdo involve homo-oligomers in which different protomersact on different DNA strands, the strategy of using acollaboration between two different gene products toexecute a double-strand break in DNA is uncommon. Itis important to note that although TnsA and TnsB executedistinct chemical steps, they are dependent on each otherfor their own activities; no reaction is observed with eitherprotein alone (Bainton et al., 1993; Gary et al., 1996).Two different proteins also comprise the recombinase inthe Xer conservative site-specific recombination system(Blakely et al., 1993). Like TnsA and TnsB, XerC andXerD work interdependently to execute recombination;however, unlike TnsA and TnsB, the Xer proteins are

6356

The Tn 7 transposase is a heteromeric complex

Donor Cleavage Target Joining1II

TnsB v-nsA E==4TnsA ELI\

TnsBTnsAD114A TnsA

TnsBTnsA =

TnsBD114A TnsA

TnsB>TnsA ,.,

SimpleInsertion

Product

Fig. 9. A model of Tn7 transposition. TnsB and TnsA, two proteins of the retroviral integrase superfamily, execute the chemical steps in Tn7

recombination; the presence of both proteins is required for all chemical steps. Recombination initiates with donor cleavage in which double-strand

breaks disconnect the element from the donor backbone: TnsB executes the cleavages which expose the 3' ends of Tn7, and TnsA mediates the

5' end cleavage reactions. When both 3' and 5' end cleavage occur, an ELT intermediate is formed. When 3' end cleavage proceeds, but 5' end

cleavage is blocked through a mutation in TnsA, a nicked donor intermediate is formed. In the strand transfer step, TnsB joins the newly exposed

3' ends to the target DNA. Target joining occurs in two steps, each 3' transposon end joining to one target strand, forming a single-end join as a

recombination intermediate. A simple insertion is formed when the ELT joins to the target DNA; a fusion product is formed when the nicked donor

joins to the target DNA.

closely related and conduct parallel chemical steps

(Arciszewska and Sherratt, 1995; Colloms et al., 1996).TnsB is the protein which executes the critical breakage

and joining reactions at the 3' ends of Tn7 that actuallyunderlie transposition (Figure 9). We have demonstratedhere that changing single amino acids in TnsB can blockboth the cleavages that separate the 3' ends from flankingdonor DNA and the joining of these 3' ends to thetarget; these changes do not block 5' end cleavage. Thetransposition-defective TnsBs do retain the ability to

recognize other components of the recombinationmachinery, i.e. can enter into a nucleoprotein complex,and thus appear to be catalysis mutants. It is attractive to

suppose that in Tn7, as in other DD(35)E transposases,

the 3' end cleavage and target joining steps are profoundlyrelated (Engelman et al., 1991; Mizuuchi and Adzuma,1991; Vink et al., 1991; Mizuuchi, 1992a,b).Our results suggest that TnsB executes the DNA pro-

cessing reactions involving on/y the 3' ends of Tn7;however, Tn7 recombination also involves 5' end cleavage.We have reported elsewhere that a mutation in TnsA can

block the 5' end, but not 3' end processing reactions (Mayand Craig, 1996). The work presented here providesevidence that TnsA actually contains the active site for 5'

end cleavage and is thus distinct from the active site for

3' end processing in TnsB. In virtually all other systemsthat have been analyzed, the protein which binds specific-ally to the element ends, and thus mediates element

recognition, also executes the DNA breakage and joiningsteps (Mizuuchi, 1992b). Although TnsB can specificallyrecognize a Tn7 substrate and contains the active sites for

3' end processing, the presence of TnsA is required to

promote these TnsB activities, even when 5' end cleavagedoes not occur. Thus TnsA actually has at least two roles

in Tn7 recombination: executing the 5' end cleavageactivities and modulating TnsB 3' end processing.Similarly, the presence of TnsB is required for TnsA to

execute 5' end cleavage; TnsB probably positions TnsA

at the ends of Tn7 and also modulates the ability of TnsAto execute the cleavages. This interdependence of TnsAand TnsB is likely to play a central and key role in

controlling Tn7 transposition. Moreover, the requirementfor multiple TnsB protomers in recombination, as shownhere, and the requirement for multiple TnsA protomers

(May and Craig, 1996) is also consistent with the viewthat a critical step in Tn7 transposition, as in otherrecombination systems, is the assembly of higher ordercomplexes both to promote synapsis of the DNA substratesand to form an active recombinase. The requirement forsuch nucleoprotein complex assembly provides a criticalcheckpoint in the transposition mechanism, assuring thatall the critical components in transposition are present

before a potentially lethal double-strand break is intro-

duced into the bacterial chromosome or an inappropriatesingle-end join product is formed (Bainton et al., 1993).

Tn7's strategy of dividing the active sites for transposi-tion among multiple gene products has not been describedpreviously, but multiple gene products are known to be

intimately involved in several other recombination sys-

tems, for example P element transposition, Xer recombin-ation and V(D)J rearrangement. Indeed, there are several

resemblances between Tn7 transposition and V(D)Jrecombination (Craig, 1996b; van Gent et al., 1996). The

critical chemical steps in these reactions involve direct

transesterification reactions. Moreover, the key and initiat-

ing event in V(D)J recombination is the introduction of a

single-strand nick at the 5' end of the excisable DNA

segment containing the signal sequences located between

the immunoglobulin gene segments to be joined. In V(D)Jrearrangement, this single-strand nick is converted to a

double-strand break by an intramolecular attack of the

exposed 3' end. TnsA cleavage occurs with the same

chemical polarity and position, i.e. at the 5' end of an

excisable segment. It will be interesting to see if the 5'

nicks introduced by TnsA can be converted similarly into

double-strand breaks. It will also be interesting to see if

6357

TnsA TnsB

54 4,3'

3YT T5tTnsB TnsA

R.J.Sarnovsky, E.W.May and N.L.Craig

in the V(D)J system, as occurs with TnsA, the 5' cleavageactivity is positioned on the DNA substrate throughprotein-protein interactions with another protein thatspecifically binds to the signal sequences.

TnsB and TnsA probably belong to the DD(35)Etransposase superfamilyOur amino acid changes in TnsB and TnsA were guidedby comparison of their sequences with those of otherrecombinases containing the DD(35)E motif, a signaturecluster of acidic amino acids present in many otherrecombinases including retroviral integrases (reviewed inCraig, 1995; Grindley and Leschziner, 1995; Polard andChandler, 1995) Studies of other members of this super-family have provided strong evidence that these aminoacids play a critical role in the DNA strand cleavage andstrand transfer reactions underlying the translocation ofmany mobile elements (Engelman and Craigie, 1992;Kulkosky et al., 1992; van Gent et al., 1992; Leavitt et al.,1993; Baker and Luo, 1994; Vos and Plasterk, 1994; Kimet al., 1995). Indeed, alteration of this motif in many otherrecombinases imposes catalytic blocks, as we have alsoobserved with TnsB and TnsA mutants. The DD(35)Emotif is probably a site of action of the divalent metalcofactor essential in these recombination reactions (Bakerand Luo, 1994; Dyda et al., 1994; Kim et al., 1995; Riceand Mizuuchi, 1995; Bujacz et al., 1996). We havepresented two kinds of experiments, suppression by Mn2+of TnsB DD(35)E mutations and alteration of the metalion specificity with DI 14C TnsA, which support the viewthat metal does interact with the DD(35)E motif. Mostcritically, these TnsA metal ion specificity experimentsalso provide functional evidence that TnsA interacts witha divalent metal which is essential for execution of the5' processing reactions, probably acting at or near theactive site.

It is interesting that TnsA does not have a canonicalDD(35)E motif; it lacks an obvious candidate correspond-ing to the first D position. Although there are acidic aminoacids in an appropriate region of TnsA, only the E37ATnsA mutant shows at most a mild defect in catalysis,suggesting that this position is not critical in TnsAendonucleolytic cleavage. While it remains possible thatan acidic residue elsewhere in TnsA substitutes for thisposition in the motif, it is also possible that the TnsADD(35)E region has a structure that obviates the role ofthis particular residue in the motif function. Recall thatthe Tn7 3' strand transfer activity is supplied by TnsB,perhaps alleviating the necessity for an aspartate at thisposition. Indeed, the first acidic residue of other DD(35)Efamily members may not be as critical to the endonucleo-lytic step; mutation of this residue in the DD(35)E motifof HIV- 1 integrase can have a much stronger effecton target joining than on 3' end processing (Leavittet al., 1993).

The consequences of separation of the 3' and 5'end DNA processing activities of Tn7The separation of the 3' and 5' end cleavage activitiesinto different Tn7 proteins may allow their differentialexecution and result in different transposition lifestyles.Although direct formation of a simple insertion requiresboth 3' and 5' end DNA processing reactions, transposition

can also result when just 3' end reactions occur, forminga co-integrate product as in the replication of bacteriophageMu (Craigie and Mizuuchi, 1985; Mizuuchi, 1992b; Lavoieand Chaconas, 1996). Although co-integrates have notbeen detected with wild-type Tn7 transposition proteins(Waddell and Craig, 1988; Arciszewska et al., 1989), wehave shown elsewhere that Tn7 co-integration can occurin vivo when 5' end cleavage is eliminated by mutationsin TnsA (May and Craig, 1996). It is intriguing that theTnS52 element, which encodes a transposase containinglimited homology not only to the DD(35)E portion ofTnsB but across the entire protein, naturally generates co-integrate products (Rowland and Dyke, 1989). While thistransposon does not carry a TnsA homolog, it does encodeTn3-like co-integrate resolvase functions to generatesimple insertion through a replicative mechanism(Rowland and Dyke, 1990). It should be noted thatMu transposition can also involve both 3' and 5' endprocessing; 5' end cleavage of the strand transfer inter-mediate apparently requires host proteins, i.e. not just thetransposase itself, to form Mu simple insertions (Ohtsuboet al., 1981; Craigie and Mizuuchi, 1985; Mizuuchi,1992b; Lavoie and Chaconas, 1996). In contrast, Tn7transposition occurs by reactions mediated by Tns proteinsto form either simple insertions (Bainton et al., 1993) orco-integrates (May and Craig, 1996). The experimentshere show that the breakage and joining reactions aremediated by a Tn7 transposase containing multiple geneproducts. Other transposons which move by a cut and pastemechanism, notably TnJO, use a transposase containing asingle gene product both to execute the double-strandbreaks that result in excision from the donor, and toexecute target joining (Haniford et al., 1989; Chalmersand Kleckner, 1994; Kleckner et al., 1996; Bolland andKleckner, 1996). It will be interesting to compare in detailthe double-strand break reactions executed by singlegene product (TnJO) and multiple gene product (Tn7)transposases.

Concluding remarksWe have suggested previously that a key element inthe control of Tn7 transposition is the assembly of anucleoprotein complex containing the DNA substrates andTns proteins (Bainton et al., 1993), such a complexactually being required for the double-strand breaks whichinitiate recombination. A more detailed view of the Tn7transposase has now emerged. It is an oligomeric nucleo-protein complex consisting of multiple subunits of differentprotein protomers; TnsB specifically recognizes the endsof Tn7 and executes the breakage and joining events atthe 3' ends, and TnsA mediates the breakage reactions atthe 5' ends. Both TnsB and TnsA appear to be membersof the retroviral integrase superfamily in which a Mg2+_dependent phosphoryl transfer reaction can result in eitherDNA breakage or strand transfer, depending on the nucleo-phile: H20 in the strand breakage step or the 3' hydroxylof the transposon end in the strand transfer step. We havehere identified TnsB as the protein which executes themost central and fundamental of these breakage andjoining reactions and whose activity must, therefore, bedetermined carefully by this nucleoprotein assembly. Itwill be especially interesting to determine how the 3' endbreakage and joining activities of TnsB are coordinated

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The Tn 7 transposase is a heteromeric complex

interdependently with the TnsA 5' breakage activity toeffect the double-strand break and strand transfer reactionsunderlying Tn7 translocation.

Materials and methodsDNA substratesThe donor plasmid pEMA (5926 bp) contains a 1.6 kb miniTn7 elementcomprised of a 166 bp Tn7L segment and a 199 bp Tn7R segmentflanking a kanamycin resistance gene (Bainton et al., 1993). ELTs weregenerated by Mlul digestion of pMIM-L+R+ which exposes the terminal-CA-3' ends of Tn7 (Bainton et al., 1991); after restriction, ELTs wereisolated by TBE agarose gel electrophoresis and Qiaex extraction. TheminiTn7 in MIM-L+R+ is identical to that of pEMA except that theterminal 19 bp of Tn7L have been converted to the Tn7R sequence, a2 bp change. The 3290 bp target plasmid pRM2 (McKown et al., 1988)contains a 555 bp attTn7 segment (-342 to + 165) in the Accl siteof pUC 18.

TnsBIn Figures 3 and 4, TnsB-His6 (Gary et al., 1996) was used. In all otherexperiments, an alternative protein fusion with an epitope tag was used,TnsB-HSV-His6 which contains amino acids 1-694 of TnsB (702 aminoacids) fused to a 15 amino acid linker containing a herpes simplex virusepitope followed by a six residue histidine tag; its properties understandard reaction conditions are indistinguishable from those of authenticTnsB (data not shown). The tagged tnsB gene was created by introducingthe BamHI-AlwNI fragment from pET-25B (Novagen) into the BamHI-AlwiNI site of pCW12 (Waddell and Craig, 1988) which contains atranscriptional fusion of tnsB to the lac promoter. Mutant derivatives ofTnsB-HSV-His6 were generated by PCR with oligonucleotides containingappropriately altered tnsB sequences to generate fragments which werethen introduced into the TnsB-HSV-His6 plasmid by cloning; mutantsequences were verified by direct DNA sequencing. The resultingplasmids were introduced into CAG456 (E.coli K12 rpsL lacZam trpamphOam supCts htpRI65) which also contained a pSCIOI derivativecarrying laclq (Arciszewska et al., 1991). Cells were grown at 30°C toan OD600 = 0.4 in LB medium supplemented with 100 gg/ml carbenicillinand 5 jg/ml tetracycline, IPTG was added to 1 mM and growth continuedfor 1 h. The cells were harvested by centrifugation and resuspended at4°C in 40 mM imidazole, 20 mM Tris (pH 7.9) and 500 mM NaCl at0.2 g cells/ml. The cells were then lysed by sonication, centrifuged at30 000 g for 20 min, and the resulting supernatant passed through a0.45 gm filter. The filtrate was applied to a Ni2+ Sepharose B column(Pharmacia), the column washed with the same buffer and TnsB-HSV-His6 eluted with 150 mM imidazole, 20 mM Tris (pH 7.9) and 500 mMNaCl. Peak fractions were pooled and dialyzed against 25 mM Tris(pH 8.0), 500 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM EDTA and25% glycerol. About 300 ,ug of TnsB-HSV-His6 were obtained from 2 gof cells; the final fraction was >95% full-length TnsB. TnsB-HSV-His6derivatives were stored at -80°C in 25 mM HEPES (pH 8.0), 500 mMNaCl, 2 mM DTT, 1 mM EDTA and 25% glycerol.

Other Tns proteinsTnsA was expressed as a fusion with glutathione S-transferase, boundto glutathione-agarose beads, and then freed through a limited proteolytictreatment with thrombin (Bainton et al., 1993; May and Craig, 1996).Site-specific mutations were introduced into the tnsA gene using PCRas previously described (May and Craig, 1996). TnsA was stored at-80°C in 25 mM HEPES (pH 8.0), 1 mM EDTA, 150 mM NaCl, 1 mMDTT, 5% glycerol and 0.25 mM phenylmethylsulfonyl fluoride (PMSF).TnsC was fraction III (Gamas and Craig, 1992) and was stored at

-80°C in 25 mM HEPES, 1 M NaCl, 2.5 mM DTT, 1 mM ATP, 10 mMMgCl2, 0.1 mM EDTA, 10 mM CHAPS and 10% glycerol. TnsD was

TnsD-His, a derivative containing a C-terminal polyhistidine tag, andwas purified by Ni2+ affinity chromatography; its properties are indistin-guishable from wild-type TnsD in these assays (P.Sharpe and N.L.Craig,unpublished observation). TnsD-His was stored at -80°C in 50 mM Tris(pH 7.5), 2 mM DTT, 500 mM KCI, 1 mM EDTA and 25% glycerol.

In vitro transposition reactionsReactions were performed essentially as previously described (Baintonet al., 1993; Gary et al., 1996). Reaction mixtures (100 p.l final volume)contained 0.25 nM donor DNA (plasmid or ELT), 2.5 nM pRM2 attTn7target plasmid, 26 mM HEPES (pH 8.0), 2.1 mM DTT, 4 mM Tris

(pH 7.5), 100 pg/ml tRNA, 50 ,ug/ml bovine serum albumin (BSA),2.0 mM ATP, 0.05 mM EDTA, 0.1mM MgCl2, 0.1 mM CHAPS, 14 mMNaCl, 21 mM KCI, 1.2% glycerol, 4 pM PMSF and 15 mM MgAcunless otherwise indicated. Tns proteins were added as follows, unlessotherwise indicated. In Figures 3 and 4: 24 nM TnsA, 3 nM TnsB-His6,8 nM TnsC, 6 nM TnsD; in all other experiments: 24 nM TnsA, 3 nMTnsB-HSV-His6, 13 nM TnsC, 12 nM TnsD. Pre-incubation reactionmixtures containing all components except donor DNA, TnsA, TnsBand metal were assembled on ice; the concentrations of the componentsin this mixture varied by <15% from the stated final concentrations.The pre-incubation mixtures were then incubated for 20 min at 30°C,or 20 min at 22°C for time course experiments (Figures 3C ); donor DNA,TnsA, TnsB and metal were then added individually and sequentially, andthe incubation was continued for an additional 20 min, or for theindicated times (Figures 3C). Reactions were stopped by making them25 mM EDTA and 300 mM NaAc, extracted with phenol:chloroform(1:1): the DNA was ethanol precipitated, digested with restrictionenzymes and analyzed by 0.6% agarose gel electrophoresis. All reactionmixtures except those in Figure 7 were digested with NdeI which cutsonce in pEMA 1837 bp outside the right end, and once in pRM2 577 bpfrom the attTn7 insertion point. In Figure 7, the reaction products were

digested with Sall and HindlIl for hybridization with probes A and B,or with Sall and EcoRI for hybridization with probes C and D.

Reactions with Mn2+In Figure 8, pre-incubation mixtures containing all components exceptMn2+ were assembled on ice and then incubated for 20 min at 30°C;Mn2+ as indicated (final concentration 5-20 mM) was then added andincubation continued for an additional 20 min. In Figure 4, pre-incubationmixtures containing all components except metal were assembled on iceand then incubated for 20 min at 30°C; Mg2> was then added andincubation continued for another 20 min at 30°C; the second metal(Mg2+ or Mn2+) was then added and incubation continued for another2 h. Metal concentrations at the end of the reaction: 15 mM Mg>+ andeither 5 mM, 15 mM or 45 mM of the additional metal (either Mg2+or Mn2+).Gel electrophoresis and product analysisThe restricted DNAs of all reactions except Figure 7 were displayed byelectrophoresis on 0.6% agarose TBE gels. The gels were transferred to

Gene Screen Plus and analyzed by hybridization; the [35S]DNA ladderwas from Amersham. The restricted products of the reactions in Figure7 were displayed by electrophoresis on a 5% denaturing polyacrylamidegel, electrotransferred to Gene Screen Plus and hybridized with a miniTn7end-specific oligonucleotide. All blots were analyzed by autoradiographyusing Kodak X-OMAT-AR film or by Molecular Dynamics Phosphor-imager. The miniTn7-specific probe used for all blots except in Figure7 was the kanamycin gene segment between Tn7L and Tn7R. Therelevant gel-purified restriction fragment was labeled by random primingwith [R-32P]dCTP and the Klenow fragment of DNA polymerase I(BMB). Oligonucleotide probes were 5' end labeled with [y-32P]ATPand T4 polynucleotide kinase and purified by NICK Spin Columnchromatography (Pharmacia). Probe A: NLC 95 (5') ATAATCCTTAA-AAACTCCATTTCCACCCCT; probe B: NLC 97 (5') AGGGGTGGAA-ATGGAGTTTTTAAGGATTAT; probe C: NLC 163 (5') GTGAAAA-AGCATACTGGACTTTTGTTATGG; probe D: NLC 94 (5') AAAGT-CCAGTATGCTTTTTCACAGCATAAC.

AcknowledgementsThe authors wish to thank Tomonobu Kusano, Elena Krementsova andTania Baker for communication of information prior to publication; FredDyda and Phoebe Rice for HIV-1 integrase and Mu transposase crystalcoordinates prior to publication; Cynthia Wolberger for help withstructure interpretation; Melissa Moore for helpful discussions about themetal ion specificity experiments; Matt Biery for developing severalcritical methods; Patti Eckhoff for help in the preparation of thismanuscript, and also other members of the Craig laboratory for helpfulconversation during the work and the critical reading of this manuscript,especially Pat Gary, Pam Sharpe and Anne Stellwagen for supplyinguseful reagents; and Tania Baker and Howard Nash for comments on

earlier drafts of the manuscript. N.L.C. is an investigator of the HowardHughes Medical Institute.

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Received on June 11, 1996; revised on July 30, 1996

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