JOURNAL OF BACTERIOLOGY,0021-9193/97/$04.0010

Feb. 1997, p. 1117–1125 Vol. 179, No. 4

Copyright q 1997, American Society for Microbiology

Specific DNA Cleavage Mediated by the Integrase ofConjugative Transposon Tn916

KATHRYN L. TAYLOR AND GORDON CHURCHWARD*

Department of Microbiology and Immunology, Emory UniversitySchool of Medicine, Atlanta, Georgia 30322

Received 28 May 1996/Accepted 30 November 1996

The conjugative transposon Tn916 encodes a protein called INTTn916 which, based on DNA sequencecomparisons, is a member of the integrase family of site-specific recombinases. Integrase proteins such asINTl, FLP, and XERC/D that promote site-specific recombination use characteristic, conserved amino acidresidues to catalyze the cleavage and ligation of DNA substrates during recombination. The reaction proceedsby a two-step transesterification reaction requiring the formation of a covalent protein-DNA intermediate.Different requirements for homology between recombining DNA sites during integrase-mediated site-specificrecombination and Tn916 transposition suggest that INTTn916 may use a reaction mechanism different fromthat used by other integrase recombinases. We show that purified INTTn916mediates specific cleavage of duplexDNA substrates containing the Tn916 transposon ends and adjacent bacterial sequences. Staggered cleavagesoccur at both ends of the transposon, resulting in 5* hydroxyl protruding ends containing coupling sequences.These are sequences that are transferred with the transposon from donor to recipient during conjugativetransposition. The nature of the cleavage products suggests that a covalent protein-DNA linkage occurs via aresidue of INTTn916 and the 3*-phosphate group of the DNA. INTTn916 alone is capable of executing the strandcleavage step required for recombination during Tn916 transposition, and this reaction probably occurs by amechanism similar to that of other integrase family site-specific recombinases.

Conjugative transposons are genetic elements that are capa-ble of transferring themselves from the chromosome of a do-nor bacterial cell to a new site in the genome of a recipient cellthrough a process that requires intercellular contact. Theseelements are remarkably promiscuous, with conjugation occur-ring between members of different species and genera of gram-positive bacteria. Since most conjugative transposons isolatedto date carry genes encoding resistance to antibiotics, they areimportant in the spread of antibiotic resistance among gram-positive bacterial pathogens. One of the most extensively stud-ied conjugative transposons is Tn916, an 18-kb element carry-ing the tetM gene, which specifies resistance to tetracycline(22). This transposon was first identified in the chromosome ofEnterococcus faecalis and is closely related to Tn1545, whichwas isolated from Streptococcus pneumoniae (9, 12, 17, 23). Theproperties of these conjugative transposons have recently beenreviewed in detail (14, 16, 45, 46, 48).Conjugative transposition differs from transposition of other

bacterial elements because insertion of the transposon doesnot result in duplication of the target DNA. Instead, a shortregion (6 bp) of adjacent DNA from the donor, termed “cou-pling sequence,” is transferred with the transposon (11, 15, 38).This coupling sequence can then be found at either junction ofthe transposon with target DNA following insertion into a newtarget site (11). Subsequent excision of the transposon mayoccur precisely to restore the original target sequence or resultin replacement of target DNA nucleotides by the couplingsequence originally brought in with the transposon (11, 39).During transposition, Tn916 and related elements first ex-

cise and form a nonreplicative circular intermediate (11, 38, 41,49). Excision apparently occurs by staggered endonucleolyticcleavage at the transposon termini, and ligation of the resulting

noncohesive ends produces a short stretch of heteroduplexDNA, composed of the coupling sequences, between juxta-posed transposon ends (11). The existence of this heteroduplexregion in the circular excised transposon has been shown byobserving the inheritance of the two sequences during trans-position, nucleotide sequence analysis of circular transposonmolecules, and the resistance of junction regions in the circularintermediate to restriction enzyme digestion (11, 33).The isolated circular intermediate has been shown to inte-

grate into different chromosomal sites upon transformationinto Bacillus subtilis protoplasts (49). To insert at a new site,staggered cleavage of the circular intermediate as well as thetarget DNA presumably occurs to produce protruding ends onrecombining strands. Since the coupling sequences of the in-coming transposon are usually not the same as that of thetarget site, strand ligation results in the formation of hetero-duplex regions, consisting of coupling and target sequences atthe transposon termini, which are subsequently resolved byreplication. When circular transposon DNA is used to trans-form a recipient, both coupling sequences of the intermediateappear in the transformants at about the same frequency (11,28). However, during normal conjugation, only the 59 couplingsequence is recovered in transconjugants, suggesting that onlyone strand of the intermediate is transferred between donorand recipient cells (47). An origin of conjugal transfer has beenidentified in Tn916 and is similar to those found in plasmids ofgram-negative bacteria (29).Genetic analysis has shown that the int gene of Tn916 and

Tn1545 is important for transposition in gram-positive bacteriaand is required for excision of the transposon in gram-negativebacteria (8, 38, 51, 53). The int genes of these two transposonsdiffer by a single nucleotide which causes a conservative aminoacid substitution (38, 53). The ends of the two transposons areidentical for at least 186 bp at the left end and 108 bp at theright end (10, 15), and the int genes complement each othergenetically (8, 31). The int gene encodes a protein that shares

* Corresponding author. Phone: (404) 727-2538. Fax: (404) 727-3659. E-mail: ggchurc@microbio.emory.edu.

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local sequence homology with members of the integrase familyof site-specific recombinases, including those encoded by bac-teriophage lambda (INTl) and the yeast 2mm plasmid (FLP)(3, 38, 39). Although the ;30 members of this family exhibit alarge diversity in their sequences, the family is defined by adistinctive structural feature. Four amino acid residues (Arg,His, Arg, and Tyr) found in two regions of the protein (desig-nated domains I and II) are conserved (1, 3). Some familymembers such as INTl and FLP cleave DNA via the transientformation of a covalent protein-DNA linkage with the con-served Tyr residue (26, 36), and mutation of this Tyr residue,as well as the other conserved residues, inhibits recombination(21, 27, 36, 37, 40). In contrast, IS1 transposase and the EcoRIIrestriction endonuclease have similar conserved residues thatare required for enzymatic activity, yet these types of enzymesdo not form covalent bonds with their DNA substrates (52, 54).In vivo, the polarity of strand cleavage during Tn916 transpo-sition is the same as that of other integrase family members(33). However, given the unique characteristics of Tn916 trans-position, it is possible that DNA strand cleavage and ligationby INTTn916 occur by a mechanism that differs from that of thesite-specific recombinases of the integrase family.Unlike most other members of the integrase family of re-

combinases, INTTn916 contains two DNA binding domains(31). The N-terminal domain of INTTn916 binds specifically toregions at each end of Tn916 that contain repeated DR-2elements sharing the consensus sequence AGTAGTAAATT(31). The C-terminal domain of INTTn916 (residues 82 to 405),which contains the conserved amino acid residues characteris-tic of integrase proteins, binds independently to the transpo-son–bacterial-DNA junction, protecting the intervening cou-pling sequence as well as ;20 bp on each side from DNase Idigestion. The C-terminal domain of INTTn916 has also beenfound to bind to bacterial sequences known to be target sitesfor Tn916 insertion (32). In gram-positive bacteria, these tar-get sites are used with widely differing frequencies (47). Acharacteristic feature of these target sites is the presence of anA- or T-rich region flanking the coupling sequence (47). It isalso likely that, in addition to the primary nucleotide sequence,other structural features of the target site are important forrecognition and INTTn916 binding. Analysis of three target sitesby circular permutation and gel electrophoresis has shown thatthese sites behave in a manner consistent with the presence ofa static bend centered within the region protected by INTTn916

(32).Recombination during Tn916 transposition requires the ini-

tial cleavage of parental DNA strands. INTTn916 is the onlyTn916-encoded protein required for excision in Escherichiacoli, and it binds to DNA sequences which encompass thecoupling sequences at the target site and transposon-targetjunctions, suggesting that INTTn916 catalyzes the cleavage andreunion of DNA strands involved in the Tn916 excision/inser-tion cycle. To investigate the recombination mechanisms in-volved in conjugative transposition, we used purified protein todetermine whether INTTn916 has the ability to cleave DNAsubstrates containing one end of the transposon and adjacentbacterial target sequences. We found that INTTn916 alone iscapable of cleaving these substrates and that cleavage occurs atspecific sites adjacent to the coupling sequence to produce 59hydroxyl protruding ends, as predicted from experiments invivo (33). In addition, our results provide evidence that DNAstrand cleavage occurs via a transient covalent DNA-proteinlinkage between INTTn916 and the 39-phosphate of the DNA atthe site of cleavage. These observations suggest that the mech-anism of DNA strand cleavage by INTTn916 is similar to that ofother integrase family members.

MATERIALS AND METHODS

Reagents. Restriction enzymes and exonuclease III were purchased from NewEngland Biolabs and Gibco/BRL. Radioisotopes were purchased from Dupont,and T4 polynucleotide kinase and proteinase K were from Boehringer Mann-heim. MBPINTTn916 and MBPINT78 were prepared as previously described (31).Cloning and expression of INTTn916 using the baculovirus system. The gene

(orf2) encoding the integrase of Tn916 was amplified from pAM120 (24) by PCRusing the primer pair 59-CGTGGATCCATGTCAGAAAAAAGACGTG-39 and59-GATGAATTCCTAAGCAACAAGACGCTCC-39. Amplification was per-formed by using a 35-cycle program of 948C for 1 min, 458C for 1 min, and 728Cfor 2 min with an MgCl2 concentration of 2.5 mM. The resulting product wasrestricted with BamHI and EcoRI, and ligated into the multicloning site ofpVL1393 (Invitrogen). The ligation product was transformed into E. coli (DH5a)by electroporation, and recombinant plasmid was prepared from a single colonywith the Qiagen Maxiprep kit. The presence of the complete int gene with thecorrect DNA sequence was confirmed in this construct by nucleotide sequencing.Insect cells (Sf9 from Spodoptera frugiperda) were transfected with the recombi-nant vector and linearized Autographa californica nuclear polyhedrosis virusDNA in accordance with the manufacturer’s instructions. Polyhedrin-negativeplaques were selected for amplification and screened by PCR for recombinantbaculovirus. Expression of the Tn916 integrase was assessed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting ofSf9 and High Five (Trichoplusia ni) insect cells for 0 to 6 days postinfection.Cloning and expression of INTTn916 in E. coli. The int gene was PCR amplified

as described above by using the primer pair 59-CAAATGCCATGGCAGAAAAAAGACG-39 and 59-GATGAATTCCTAAGCAACAAGACGCTCC-39. Theresulting product was restricted with NcoI and EcoRI and ligated into apTrcHisB plasmid (Invitrogen) from which the NcoI-EcoRI fragment encodingthe histidine tag had been removed. The resulting nonfusion construct,pTrcINT7, encoded a Ser3Ala change at the second amino acid residue ofINTTn916. The rest of the sequence was as predicted. Following electroporationof pTrcINT7 into SG22094 cells (del lon clpP1 cat resA166 Km) (25), selectedcolonies were screened for recombinant plasmid by PCR and restriction enzymedigestion of minipreps. Expression of INTTn916 was assessed by SDS-PAGE andWestern blotting of whole-cell samples harvested 0 to 4 h after induction with 0.8mM isopropyl thio-b-D-galactoside (IPTG).Purification of INTTn916 from High Five insect cells. High Five insect cells

grown in Excell 401 media (Gibco/BRL) in 150-cm2 flasks were harvested bycentrifugation 5 days postinfection with recombinant baculovirus. The cell pelletwas suspended in 15 ml of extraction buffer containing 25 mM Tris (pH 7.4), 1.5M NaCl, 1 mM EDTA, 1 mM dithiothreitol (DTT), 2 mM phenylmethylsulfonylfluoride, and 4 ml of a protease inhibitor mix per ml (625 mg each of chymostatin,pepstatin A, leupeptin, elastinal, antipain, and aprotinin per ml) and immediatelytreated with 1 ml diisopropylfluorophosphate per ml before being frozen at2808C overnight or until use. All subsequent procedures were performed at 48C.After defrosting, the extract was diluted to 30 ml, sonicated for four pulses of 40 seach, and centrifuged at 20,000 rpm for 30 min in a Sorvall SS-34 rotor. Theresulting supernatant was dialyzed overnight against 2.5 liters of buffer A (25 mMTris [pH 7.4], 0.50 M NaCl, 1 mM EDTA, and 1 mM DTT) and centrifuged asbefore to remove insoluble material. The extract was loaded at 0.8 ml/min ontoa 1-ml heparin HiTrap cartridge (Pharmacia) previously equilibrated in buffer Aby using a Pharmacia fast protein liquid chromatography system. The cartridgewas washed with buffer A until the absorbance at 280 nm was#0.05, and then theintegrase was eluted with a 50-ml linear gradient of 0 to 100% buffer B (same asbuffer A but with 1.5 M NaCl). Two-milliliter fractions were collected andanalyzed by SDS-PAGE and Western blotting. Appropriate fractions were com-bined, concentrated by using a Centricon 30 microconcentrator (Amicon), andaliquoted for storage at 2808C.Purification of INTTn916 from E. coli by heparin affinity chromatography. E.

coli containing pTrcInt7 was grown in medium containing 100 mg of ampicillinand 30 mg of chloramphenicol per ml to an absorbance at 600 nm of 0.5 and theninduced with 0.8 mM IPTG for 3 h. The cells (1 liter) were harvested bycentrifugation, resuspended in 30 ml of ice-cold extraction buffer (describedabove), and frozen at 2808C. The integrase was extracted and purified by hep-arin affinity chromatography in a manner identical to that described above exceptthat the column gradient was run over a 40-ml volume.Western blotting and N-terminal sequencing. Blotting from SDS–15% poly-

acrylamide gels onto nitrocellulose or Immobilon PSQ (Millipore) membraneswas performed in 15 mM Na2CO3 (pH 9.9) containing 10% methanol for 16 to20 h at 20 V. The nitrocellulose membranes were probed with polyclonal anti-bodies prepared against the C-terminal maltose-binding protein–Tn916 integrase(MBPINTTn916) construct previously described (31), followed by an alkalinephosphatase-conjugated second antibody. Immobilon membranes were stainedwith Fast Green (0.03% in a 5/4/1 ratio of methanol-water-acetic acid), and theband corresponding to INTTn916 was excised. After treatment with 0.6 M HCl in10%methanol overnight at room temperature, the membranes were subjected toN-terminal sequencing at the Emory University Microchemical Facility.Preparation of DNA substrates. PCR products containing the sequence from

one end of Tn1545 and adjacent streptococcal sequences were cloned intopUC19 as previously described (31). The excised fragments containing the left(408 bp) or the right (180 bp) end of Tn1545 were purified by agarose gel

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electrophoresis and electroelution from gel slices. The fragments were selectivelylabeled at one end by using DNA polymerase I Klenow-exo2 fragment (Pro-mega) and the appropriate a-P32-labeled deoxynucleoside triphosphate (specificactivity, ;3,000 Ci/mmol).Cleavage reactions. Incubations were performed in siliconized 1.5-ml Eppen-

dorf tubes in a final volume of 100 ml containing 50 mM Tris (pH 7.4), 70 mMKCl, 1 mM EDTA, 1 mM DTT, 10 mMMgCl2, 200 mg of bovine serum albuminper ml, 10% glycerol, and labeled DNA substrate (;0.4 pmol). Integrase wasadded at a 100-fold molar excess to the DNA substrate just prior to incubationfor 30 min at 378C. Cleavage reactions were stopped by the addition of 100 ml ofphenol-chloroform-isoamyl alcohol (25/24/1 ratio) and rapid vortexing to pre-cipitate the protein. One hundred microliters of Tris-EDTA (TE) containing 4mg of calf thymus DNA was then added, and the sample was vortexed andcentrifuged at 16,000 3 g for 5 min in a microcentrifuge. The aqueous phase wascarefully transferred to a clean tube, avoiding the removal of precipitated pro-tein, and the organic phase was extracted twice with 200 ml of TE to removeunbound DNA. The resulting organic phase containing the precipitated proteinwas mixed with 300 ml of isopropanol and centrifuged as above. The proteinpellet was washed with 0.5 ml of 70% ethanol and dried in vacuo. The pellet wassuspended in 100 ml of TE containing 0.2% SDS, 4 mg of calf thymus DNA, and2 mg of proteinase K. The samples were digested for 2 h at 378C, diluted with 100ml of TE, and extracted three times with 100 ml of phenol-chloroform-isoamylalcohol and once with 100 ml of chloroform. The liberated DNA in the aqueousphase was then precipitated by the addition of a 0.5 volume of 7.5 M ammoniumacetate (pH 7.6) and 3 volumes of 100% ethanol, and the samples were stored at2808C for at least 1 h. The DNA samples were pelleted by centrifugation,washed twice with 0.5 ml of 70% ethanol, and dried in vacuo. The samples weresuspended in 10 to 20 ml of loading buffer (80% formamide, 10 mM EDTA, 0.1mg of xylene cyanol per ml, and 0.1 mg of bromphenol blue per ml) and heatedat 908C for 3 min. Samples (10 ml/lane) were loaded on a 6.5 to 7.5% denaturingacrylamide sequencing gel and electrophoresed at 65 to 75 W for 2.5 to 6.0 hfollowed by autoradiographic analysis of the dried gel.Miscellaneous procedures. DNA pellets were solubilized in 100 ml of the

supplied buffer and treated with bacterial alkaline phosphatase (100 U; Gibco/BRL) for 1 h at 658C or 20 U of T4 polynucleotide kinase at 378C for 30 min inthe presence of 0.3 mM ATP. Samples were precipitated, washed, and dried asdescribed above. DNA substrates were 59 radiolabeled with T4 polynucleotidekinase as previously described (31) and subjected to cleavage reactions as de-scribed above. The resulting DNA pellets from proteinase K digestions wereresuspended in 29 ml of TE, and 1 ml of 6 M NaOH was added. Samples wereincubated at 378C for 2 h, and the DNA was renatured by the addition of anequivalent volume of 6 M HCl, 15 ml of TE, and 5 ml of the supplied 103exonuclease III buffer. The samples were then treated with exonuclease III (100U; Promega) for 1 h at 378C and precipitated as described above. The rapidmethod of Maxam-Gilbert sequencing was performed on DNA substrates aspreviously described (35).

RESULTS

Purification of INTTn916 from insect cells. High Five cellswere infected with recombinant baculovirus containing the intgene and shown to express maximal levels of INTTn916 5 daysafter infection (Fig. 1, lanes 1 and 2). In order to solubilizeINTTn916, it was necessary to extract the cell pellet with 1.5 MNaCl in the presence of protease inhibitors. Prior to chroma-tography, the concentration of NaCl was reduced to 0.5 M bydialysis. Lowering the salt concentration further resulted inloss of activity due to precipitation of the integrase. Loss ofactivity during rapid dialysis against buffers of low ionicstrength has also been reported for INTl and FLP (4, 30).INTTn916 species in the crude extract were then absorbed to aheparin column, unbound material was removed by washingwith loading buffer, and INTTn916 was eluted from the columnwith a linear gradient of increasing salt concentration. Themajority of the immunoreactive protein was eluted between0.75 and 0.85 M NaCl. SDS-PAGE and Western blot analysisof the eluted fractions corresponding to this peak revealed thepresence of INTTn916 (;45 kDa) as the major protein species(Fig. 1, right panels). The immunoreactive bands of lowermolecular weight (MW) may have arisen from translation ini-tiating at sites within the int coding sequence or proteolyticdegradation. Approximately 1.5 mg of purified INTTn916 wasobtained from infecting ;6 3 108 cells in a monolayer culture.Purification of INTTn916 from E. coli. Because of the cloning

strategy used to construct a plasmid that would produce

INTTn916, the protein purified from E. coli contained aSer3Ala substitution at position 2. By varying both the in-ducer concentration and the time of induction, we found thatE. coli cells containing pTrcINT7 showed maximum expressionof INTTn916 3 h after induction with 0.8 M IPTG (Fig. 2, lanes1 and 2). Lower-MW INTTn916 species similar to those foundin insect cells were observed. Extraction of the cell pellet withhigh-concentration salt and purification of INTTn916 by hepa-rin affinity chromatography were accomplished by essentiallythe same procedure as that found effective for INTTn916 puri-fication from insect cells. INTTn916 from E. coli was elutedfrom the column at approximately the same NaCl concentra-tion as INTTn916 from insect cells (Fig. 2, left panel), andINTTn916 bands from the two sources appeared to migrateidentically during SDS-PAGE. With E. coli, approximately 1.5mg of purified INTTn916 was obtained per liter of cell culture.N-terminal sequencing of INTTn916 from E. coli. INTTn916

was transferred to Immobilon membranes and subjected toN-terminal sequencing. Without prior acid treatment of theimmobilized protein, no sequence was obtained from INTTn916

from either E. coli or insect cells. Since these results suggestedthat the amino terminus of the Tn916 integrase was blocked,the immobilized INTTn916 from E. coli was subjected to acidhydrolysis prior to sequencing. The sequence AEKRRDNRGRIL was then obtained, corresponding to amino acids 2 to 13of INTTn916. It is not clear why the terminal methionine wasnot detected, although the FLP protein expressed in E. colialso appears to lack an N-terminal methionine (4). It is possi-ble that the extended acid treatment prior to sequencing mayhave caused hydrolysis of this residue.Cleavage of DNA substrates by MBPINTTn916. The con-

struction and purification of MBPINTTn916 has been previ-ously described (31). Since this fused protein has been shownto bind to specific DNA fragments containing one end of the

FIG. 1. Purification of INTTn916 expressed in insect cells. Cell extracts wereprepared and chromatographed on a heparin column as described in Materialsand Methods. The column elution profile is shown on the left, and collectedcolumn fractions 1 to 4 are indicated by the double arrow. SDS-PAGE analysisof protein samples is shown at the upper right. Lane 1, uninfected cells; lane 2,cells 5 days postinfection; lane 3, soluble cell extract; lane 4, soluble extractfollowing dialysis; lane 5, column flowthrough; lanes 6 to 9, fractions 1 to 4,respectively, collected from the column and concentrated. An identical gel (low-er right) was blotted onto nitrocellulose and probed with antibodies preparedagainst INTTn916.

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transposon and adjacent bacterial sequences, we used smallDNA fragments containing transposon junctions as substratesto determine if the integrase of Tn916 could catalyze DNAstrand cleavage. Following incubation of MBPINTTn916 withradiolabeled DNA substrates, analysis of the sample by dena-turing PAGE did not reveal a cleavage product. However,when the sample was treated with a phenol-chloroform-iso-amyl alcohol mixture to precipitate the protein, a small amountof labeled DNA was coprecipitated. Subsequent washing of theorganic phase containing the precipitated protein was per-formed to remove unbound DNA, and the isolated proteinpellet was digested with proteinase K in 0.2% SDS. This re-sulted in the release of the labeled DNA from the protein,which was then isolated from the aqueous phase by ethanolprecipitation. As shown in Fig. 3, analysis of the DNA obtainedin this manner revealed that, when the incubation was per-formed in the presence of MBPINTTn916, a smaller DNA spe-cies was present in addition to the original substrate (lane 4).This species was not observed in samples from incubationsperformed in the absence of INTTn916 (lane 2) or when MBPfused to the amino-terminal portion (residues 3 to 78) of theTn916 integrase (MBPINT78) (31) was included in the incu-bation (lane 3). It therefore appears that the C-terminal do-main of INTTn916 mediated the cleavage of the DNA substrateto produce a product that remained associated with the en-zyme. No product could be detected in the aqueous phase ofthe incubation mixture following removal of the precipitatedprotein (lanes 9 to 11).Cleavage by INTTn916 produces a 5*-OH terminus. Integrase

family recombinases cleave DNA substrates by covalent link-age of the protein to the 39-phosphate group of the DNA,leaving a free 59-OH end at the cleavage site. To determinewhether INTTn916 cleavage resulted in a product with a 59-OHterminus, the cleavage product liberated by proteinase K di-gestion was treated with T4 polynucleotide kinase and ATP or

with bacterial alkaline phosphatase (Fig. 3, lanes 5 and 6,respectively). Whereas treatment with phosphatase did notaffect the mobility of this band, treatment with kinase resultedin a small increase in the mobility of the cleavage product suchthat it aligned directly to a band in the sequencing laddergenerated by Maxam-Gilbert chemistry (compare lanes 4 to 6with lane 7). Since single-stranded DNA with a 59-OH isknown to migrate more slowly under these conditions than thesame DNA strand with a 59-phosphate, these results indicatedthat the cleavage product produced by INTTn916 had a free59-OH group and suggested that the covalent DNA-proteinlinkage occurred via the 39-phosphate of the DNA at the site ofcleavage.INTTn916 forms a covalent linkage with the 3*-phosphate

group of the DNA at the site of cleavage. To investigate wheth-er INTTn916-mediated cleavage produced a covalent protein-DNA linkage with the 39-phosphate of the DNA, MBPINTTn916

was incubated with a substrate that had been radiolabeled atthe 59 end (Fig. 4). The DNA that was coprecipitated with theprotein was isolated following proteinase K digestion and sub-jected to either exonuclease III digestion (lane 3), alkalineconditions, and renaturation (lane 4) or alkaline conditionsFIG. 2. Purification of INTTn916 expressed in E. coli. Cell extracts were pre-

pared and chromatographed on a heparin column as described in Materials andMethods. The column elution profile is shown on the left, and collected columnfractions 1 to 4 are indicated by the double arrow. SDS-PAGE analysis of proteinsamples is shown at the upper right. Lane 1, uninduced cells; lane 2, cells 3 hpostinduction; lane 3, soluble cell extract; lane 4, soluble extract following dialy-sis; lane 5, column flowthrough; lanes 6 to 9, fractions 1 to 4, respectively, col-lected from the column and concentrated. An identical gel (lower right) was blottedonto nitrocellulose and probed with antibodies prepared against INTTn916.

FIG. 3. INTTn916-mediated cleavage of a 39-end-labeled bottom strand DNAsubstrate containing the right end of Tn916 and adjacent bacterial sequences.Lane 1, substrate only. DNA was isolated as described in Materials and Methodsfrom incubations performed with no additions (lane 2), MBPINT78 (lane 3), andMBPINTTn916 (lanes 4 to 6). Samples were subsequently treated with T4 polynu-cleotide kinase plus ATP (lane 5) or alkaline phosphatase (lane 6). Lanes 7 and8, T and A1G Maxam-Gilbert sequence reaction products, respectively: lanes 9to 11, samples of the aqueous phase following removal of precipitated proteinfrom incubations corresponding to lanes 2 to 4.

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followed by renaturation and exonuclease III digestion (lane5). Whereas exonuclease III digestion of the uncleaved DNAsubstrate was apparent, the cleavage product was resistant todigestion prior to, as well as following, alkaline treatment andrenaturation. These observations suggested that the exonucle-ase III resistance of the cleavage product was due to the block-age of its 39 end via chemical linkage with another moleculeduring cleavage. Furthermore, this 39 linkage was stable underalkaline conditions, since the relative mobility of the cleavageproduct did not change, and exonuclease sensitivity was notapparent following alkaline treatment.INTTn916 cleaves both strands in DNA substrates containing

transposon ends. The results shown in Fig. 3 were obtained byusing a bottom-strand-labeled DNA substrate containing theright end of Tn916 and adjacent bacterial sequences. To de-termine whether INTTn916 cleaves both strands of this sub-strate, the top strand was labeled and incubated with INTTn916

species under the same conditions (Fig. 5). Protein-associatedcleavage products were obtained when incubations includedMBPINTTn916 (lanes 4 and 9), INTTn916 from E. coli (lane 5),or INTTn916 from insect cells (lanes 6 and 10) but not whenMBPINT78 was added (lane 3) or when INTTn916 was omitted(lane 2). In addition to a major product produced by cleavage,two minor products which flanked the major band were visible.These products were easily detected from incubations withMBPINTTn916 but were present at lower levels in samplesincubated with INTTn916 from E. coli or insect cells. The lowerlevels of product produced by INTTn916 purified from E. colimight be due to an effect of the mutation affecting residue 2 of

this protein. Since the activities of INTl and FLP are stabilizedin the presence of high salt concentrations (4, 30), incubationswith INTTn916 were also performed at a higher NaCl concen-tration in the absence of glycerol. Under these conditions, theamount of cleavage product obtained with MBPINTTn916 wasnot significantly altered (compare lane 4 to lane 9) whereas theamount of cleavage product obtained with INTTn916 from in-sect cells was increased (compare lane 6 to lane 10). Theseobservations suggested that the higher yield of cleavage prod-uct obtained with MBPINTTn916 was due to its greater solu-bility under the incubation conditions used for this study. Thepresence of Mg21 ions also increased the amount of cleavageproduct isolated, although cleavage could still be detected inthe absence of Mg21 (data not shown). Other changes in theincubation conditions with respect to metal ions, pH, reactiontemperature, and incubation time did not increase the amountof cleavage product obtained (data not shown).Incubations were also performed to determine whether

INTTn916 cleaved a DNA substrate containing the left end ofTn916 and adjacent bacterial sequences. This substrate waslabeled on either the bottom strand (Fig. 6) or the top strand(Fig. 7) and incubated with INTTn916 proteins under the same

FIG. 4. INTTn916-mediated cleavage results in 59 cleavage products that areresistant to exonuclease III degradation. The DNA substrate (lane 1) containingthe left end of Tn916 and adjacent bacterial sequences was labeled at the 59 endon the bottom strand. DNA was isolated from incubations performed withMBPINTTn916 as described in Materials and Methods and subjected to nofurther treatment (lane 2), exonuclease III digestion (lane 3), alkaline treatment(lane 4), and alkaline treatment followed by exonuclease digestion (lane 5).Lanes 6 and 7, T and A1G Maxam-Gilbert sequence reaction products, respec-tively.

FIG. 5. Site-specific cleavage of a 39-labeled top strand DNA substrate con-taining the right end of Tn916 and adjacent bacterial sequences. Lane 1, sub-strate only. DNA was isolated as described in Materials and Methods from incu-bations performed with no additions (lane 2), MBPINT78 (lane 3), MBPINTTn916

(lane 4), INTTn916 from E. coli (lane 5), and INTTn916 from insect cells (lane 6).Lanes 7 and 8, T and A1G Maxam-Gilbert sequence reaction products, respec-tively; lanes 9 and 10, products from incubations performed as for lanes 4 and 6,respectively, except that 0.2 M NaCl was included and glycerol was excludedfrom the incubation buffer.

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reaction conditions. As shown in Fig. 6, MBPINTTn916 (lane 3)as well as INTTn916 from insect cells (lane 6) and E. coli (lane7) produced a single cleavage product in the bottom strand ofthis DNA substrate. A single cleavage product was also ob-served when the top strand was labeled (Fig. 7). However, theamount of cleavage product produced by top strand cleavageappeared to be significantly less than that of bottom strandcleavage (lanes 3 and 6).INTTn916 produces specific staggered cleavages near the

ends of Tn916. The sites on the DNA substrate at whichINTTn916 cleavage occurred were determined by comparingthe positions of the cleavage products on a denaturing poly-acrylamide gel with that of Maxam-Gilbert sequencing reac-tions on the appropriate substrate (Fig. 3 and 5 to 7). With theDNA substrate containing the right end of Tn916, bottomstrand cleavage by INTTn916 was observed between the firsttwo nucleotides of the bacterial sequence adjacent to the cou-pling sequence (Fig. 8). The major site of top strand cleavagewas at the end of the transposon adjacent to the couplingsequence with minor cleavage to each side of the major cleav-age site (Fig. 8). With the bottom-strand-labeled DNA sub-strate containing the left end of Tn916, a single product wasdetected which resulted from cleavage at the first nucleotide ofthe transposon sequence (Fig. 8). However, it was not possibleto unambiguously determine whether cleavage took place 59 or39 to this residue because of the complication in aligning mol-

ecules with 59-OH groups generated by INTTn916 with mole-cules with 59-P groups generated in the sequencing reactions.This was also the case for top strand cleavage which mapped tothe first nucleotide of the bacterial sequence preceding thecoupling sequence. Thus, INTTn916 appears to mediate thestaggered cleavage of these substrates in close proximity toeach side of the coupling sequence to generate 59 protrudingends.

FIG. 6. Site-specific cleavage of a 39-labeled bottom strand DNA substratecontaining the left end of Tn916 and adjacent bacterial sequences. Incubationswere performed with no additions (lane 1), MBPINT78 (lane 2), MBPINTTn916

(lane 3), INTTn916 from insect cells (lane 6), and INTTn916 from E. coli (lane 7).Lanes 4 and 5, T and A1G Maxam-Gilbert sequence reaction products, respec-tively.

FIG. 7. INTTn916-mediated cleavage of a 39-labeled top strand DNA sub-strate containing the left end of Tn916 and adjacent bacterial sequences. Lane5, substrate only. Incubations were performed with no additions (lane 1),MBPINT78 (lane 2), MBPINTTn916 (lane 3), and INTTn916 from insect cells (lane6). Lane 4, Maxam-Gilbert sequence reaction product for T.

FIG. 8. INTTn916-mediated cleavage occurs at specific sites to produce 59protruding ends. A partial sequence of the DNA substrates utilized in this studyis shown. The sequences corresponding to the ends of Tn916 are boldfaced, andadjacent bacterial sequences are italicized. The 6-bp coupling sequence is boxed.The positions of the major cleavage sites are indicated by large arrows above (topstrand cleavage) or below (bottom strand cleavage) the sequence. Sites wherecleavage was found to occur to a lesser extent are indicated by smaller arrows.

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DISCUSSION

The integrase of Tn916 has been expressed and purifiedfused to MBP. Although fused proteins have been used for thestudy of other recombinases (5, 20), the possibility that thepresence of MBP may alter the properties of the Tn916 inte-grase led us to utilize the baculovirus expression system forproduction of the native form of INTTn916 in insect cells.INTTn916 containing a single amino acid substitution at posi-tion 2 was also purified from a protease-deficient strain of E.coli. Although this conservative Ser3Ala change was not ex-pected to alter the properties of the integrase, since it was notwithin the conserved domains thought to comprise the activesite of integrase family members, it may have affected theactivity of the protein used in these experiments. Heparinaffinity chromatography was used to partially purify INTTn916

from both sources, and these protein species were indistin-guishable by SDS-PAGE. INTTn916 migrated with a slightlylower apparent MW (;45,000) than expected (;47,000), al-though anomalously fast migration of FLP has also been ob-served (4). Western blot analysis and N-terminal sequencingconfirmed the identity of the purified protein as INTTn916.Western blot analysis also revealed the presence of bands oflower MW in heparin column fractions containing INTTn916

which appeared to be truncated forms of the integrase pro-duced either from alternate translational start sites or by pro-teolysis. These shorter forms of INTTn916 have been observedpreviously during in vitro synthesis (48) and are presumablycapable of binding to the transposon ends. It has been sug-gested that these INTTn916 species may have a regulatory rolebecause in E. coli excision of Tn916 from a plasmid is inhibit-ed by a second plasmid encoding a C-terminal segment ofINTTn916 (16).We have shown that INTTn916 mediates the site-specific

cleavage of DNA substrates that contain transposon ends andadjacent bacterial target sequences. Site-specific cleavage ofsimilar duplex substrates by other integrase recombinases oc-curs by a mechanism similar to that used by topoisomerases.Cleavage by these enzymes is accompanied by covalent attach-ment of the protein to the 59-phosphate group of the DNA atthe site of cleavage via the hydroxyl group of an active-sitetyrosine residue (13, 42). This linkage conserves the energy ofthe phosphodiester backbone for the subsequent strand rejoin-ing reaction. Another feature of topoisomerase activity is thatthe amount of cleavage product does not increase with incu-bation time, reflecting the capacity of these enzymes to resealthe cleaved DNA. Transient protein-DNA complexes havebeen detected in the INTl and FLP systems by SDS-PAGE,and treatment with proteinase K releases the cleaved DNAspecies (18, 50). When we incubated MBPINTTn916 orINTTn916 with a radiolabeled DNA substrate, a cleavage prod-uct was detected only following protein precipitation and re-lease of the protein-associated DNA by proteolytic digestion,suggesting that INTTn916 also forms a covalent protein-DNAlinkage at the site of cleavage. The paucity of cleavage productsuggests that religation of cleaved DNA by INTTn916 is veryrapid under these conditions. The cleavage product obtainedwas the same regardless of whether MBPINTTn916 or INTTn916

from either insect cells or E. coli was used, indicating thatneither the MBP portion of the fused protein nor the Ser3Alachange of the E. coli protein alters the specificity of cleavage.To investigate the nature of the 59 ends produced by

INTTn916-mediated cleavage, the cleavage product was sub-jected to kinase or alkaline phosphatase treatment and com-pared to Maxam-Gilbert (35) sequencing products. DNAstrands which terminate in a 59-OH group migrate more slowly

in a denaturing gel than the corresponding sequencing prod-ucts which are 59 phosphorylated (18, 44a, 50). The smallincrease in mobility of the 39-labeled cleavage product follow-ing treatment with kinase resulted in the close alignment ofthis species with a band in the corresponding sequencing lanes.Phosphatase treatment had no effect. These observations sug-gest that the 39 cleavage product produced by INTTn916 has a59-OH group and that INTTn916 becomes covalently linked tothe corresponding 59 cleavage product via its 39-phosphategroup, as found for INTl and FLP (18, 26). The 59 cleavageproduct was resistant to exonuclease III digestion and mi-grated more slowly (2 to 3 bp) than expected from cleavage atthe site identified by using the same 39-labeled substrate.Therefore, exonuclease resistance is probably due to the pres-ence of an amino acid(s) which remains linked via the 39-phosphate group of the DNA at the site of cleavage followingproteolytic digestion. Restoration of exonuclease sensitivitywas not observed under alkaline hydrolysis conditions that areknown to cleave phosphodiester linkages with serine or threo-nine (34). Since phosphodiester linkages with tyrosine aremore stable, these experiments provide indirect evidence thatthe covalent linkage between INTTn916 and the 59 cleavageproduct occurs via a tyrosine residue.By comparing the size of isolated cleavage products to ap-

propriate DNA sequencing ladders, the sites of INTl and FLPcleavage have been identified (2, 18, 50). We have now simi-larly mapped the sites of cleavage by INTTn916 on both ends ofthe transposon using DNA substrates containing a couplingsequence flanked on one side by terminal transposon se-quences and on the other side by bacterial target sequences.The results indicate that INTTn916 causes staggered cleavagenear each end of the transposon which, if double-stranded,would produce 59 protruding ends containing the couplingsequence. This type of cleavage is expected because excisionalrecombination leads to the formation of a circular intermedi-ate of Tn916 which contains a heteroduplex coupling sequencebetween transposon ends (11) and is consistent with the po-larity of cleavage found in vivo (33). However, our studiesindicate that cleavage in vitro by INTTn916 does not occur at theends of a 6-bp coupling sequence as predicted from the anal-ysis of excision and integration events in vivo. Rather, at theright end of Tn916 and possibly at the left end as well, the siteof cleavage may vary to produce staggered cuts as far as 8 bpapart.There is evidence for cleavage at different sites at the right

end of Tn916 in vivo leading to two classes of excised trans-poson (11, 44). Class I excision appears to occur via cleavage atthe right transposon terminus, and the transposon excises with5 T residues, whereas cleavage 1 bp within the right end of thetransposon generates class II excisants that carry 4 T residues.It has been suggested that class II excision results from lack ofcleavage precision due to the slippage of protein factors thatbind to the stretch of 5 or 6 T’s at the right end of Tn916 (11).We have found that INTTn916 cleaves to a different extent ateach of the three adjacent T’s at the right end of the transpo-son.In naturally occurring transposition it has been difficult to

determine the length of the coupling sequence unambiguouslybecause of the particular nucleotide sequences at the ends ofan integrated transposon or in a specific target. Estimates ofthe length of the coupling sequence have been discussed indetail (48). Experiments specifically designed to measure thelength of the coupling sequences in vivo have shown that in oneinstance it was 6 bp but not 5 bp and in another case it was 6bp and not 7 bp (44). The available data are consistent with a6-bp coupling sequence but do not rule out the possibility of

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variability in the distance between the sites of INTTn916 cleav-age. Other recombinases of the integrase family cleave withdifferent distances between the sites of cleavage. For example,INTl normally cleaves at sites 7 bp apart (18), and FLP cleavesat sites 8 bp apart (2, 50). In some cases a single recombinasecan cleave at different distances. INTl can catalyze recombi-nation between mutant substrates where an extra nucleotidehas been inserted between the sites of cleavage (19, 43). TheXERC/D recombinase acting alone on chromosomal substratesequences cleaves 6 bp apart, but acting with accessory pro-teins on plasmid substrates, it cleaves 8 bp apart (6, 7). It is notknown whether the conditions used here are optimal forINTTn916 binding and cleavage or whether accessory proteinssuch as Xis (38, 53) are necessary for certain protein-proteinand/or protein-DNA interactions which would enhance eitherthe activity or specificity of INTTn916 cleavage. The short linearDNA fragments we used as substrates presumably differ topo-logically from natural substrates involved in Tn916 recombina-tion in vivo, and no additional proteins that might modify DNAstructure were present. Therefore, interactions that may nor-mally position INTTn916 correctly at the transposon ends topromote cleavage immediately adjacent to the 6-bp couplingsequence could not occur, resulting in cleavage 1 bp removedfrom the coupling sequence.Since MBPINT78 did not mediate substrate cleavage and

cleavage was not detected near sequences shown to bindMBPINT78, it appears that the N-terminal domain of INTTn916

directs binding but not cleavage, as has also been observed forINTl. This conclusion is supported by the observation that theMBP fusion containing the C-terminal portion of INTTn916

(residues 82 to 405) mediates DNA strand cleavage at thesame site as the full-length protein (data not shown). Thus,cleavage by INTTn916 is promoted by recognition of sequencesat the ends of the transposon and adjacent bacterial target sitesby the C-terminal DNA binding domain. This portion of theTn916 integrase contains the conserved active-site tyrosine ofdomain II like other members of the integrase family. Mutantsof INTl and FLP in which this tyrosine residue has beenchanged to Phe are not capable of strand cleavage, and similarstudies indicate that the histidine and arginine residues of thisconserved domain II triad are also important for recombina-tion (21, 27, 36, 37, 40). It is therefore likely that the cleavageactivity observed in our studies is due to the participation ofthese residues at the active site of INTTn916 and that covalentlinkage to the DNA at the site of cleavage occurs via theconserved tyrosine of INTTn916. Further studies are necessaryto confirm this prediction, which, if substantiated, indicatesthat strand cleavage and recombination in the Tn916 systemoccur by the same mechanism as that of other integrase familymembers.

ACKNOWLEDGMENTS

We are indebted to Fang Lu for the cloning and preparation ofMBPINT and DNA substrates used in this work and to Jan Pohl of theEmory University Microchemical Facility for help in sequencing andanalysis of the recombinant Tn916 integrase.This work was supported by grant GM50376 from NIH.

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