Proc. Nati. Acad. Sci. USAVol. 84, pp. 2189-2193, April 1987Biochemistry

Mutations in the 2-,pm circle site-specific recombinase that abolishrecombination without affecting substrate recognition

(strand cleavage/protein-DNA complexes/DNase "footprinting")

P. V. PRASAD, L.-J. YOUNG, AND M. JAYARAM*Department of Molecular Biology, Research Institute of Scripps Clinic, 10666 North Torrey Pines Road, La Jolla, CA 92037

Communicated by Ernest Beutler, December 16, 1986 (receivedfor review October 30, 1986)

ABSTRACT The site-specific recombinase encoded by theyeast plasmid 2-,um circle (FLP) forms a transient covalentlinkage with its substrate DNA via a tyrosine residue, whichappears to be located near its COOH terminus. The homologyof the COOH terminus of FLP with that of the Int family ofrecombinases suggests that tyrosine-343 of FLP could beinvolved in forming the DNA-protein bridge. We have mutatedtyrosine-343 to a phenylalanine or serine. We demonstrate thatthe binding of each of the two mutant proteins to its substrateis indistinguishable from that of wild-type FLP. However, bothmutant proteins are incapable of catalyzing strand cleavageand recombination.

Site-specific recombination, unlike generalized recombina-tion, is characterized by relatively short DNA substrates ofspecific sequences, by the requirement of rather limitedhomology of the recombining partners, and by strand ex-change mechanisms that require no DNA synthesis or exog-enous high-energy cofactor (1). The recombination reactioninvolves cleavage, swapping, and religation of the participantDNA strands. The energy of the phosphodiester bond isconserved during cleavage in the form of a phosphoproteinlinkage. This is reminiscent of the mode of strand exchangemediated by topoisomerases. The best-studied site-specificrecombination systems among prokaryotes are those medi-ated by the Int protein of phage X, the resolvase encoded bythe related transposons Tn3 and y8, and the Cre protein ofphage P1 (2-4). Recently, considerable information concern-ing the site-specific recombination mediated by the FLPprotein of the 2-,um yeast plasmid has become available(5-10). Like most topoisomerases, FLP and Int form aphosphotyrosine bridge with their DNA substrates duringcatalysis (refs. 11 and 12; C. Pargellis, S. Nunes-Duby, L.Vargas, and A. Landy, personal communication); in con-trast, the link between the resolvase and its substrate is aphosphoserine (13).The remarkable similarity in the chemistry of the reactions

catalyzed by the prokaryotic site-specific recombinases andthe FLP protein is contrasted by the unexpectedly largediversity among these proteins. However, in spite of thisglobal diversity, the site-specific recombinases encoded bybacteriophage X, 080, P22, 186, P4, and P1 can be aligned intheir COOH-terminal halves (14). A 40-residue region nearthe COOH terminus is well-conserved in all these proteinsand shares a conspicuous degree of homology with a regionnear the COOH terminus of the FLP protein. This family ofrecombinases (the integrase family) does not appear to berelated to other site-specific recombinases, such as theprokaryotic invertases or the yS resolvase (3, 15-18). Withinthe integrase family, three residues (histidine, arginine, andtyrosine) located at alignment positions 396, 399, and 433,

respectively, are perfectly conserved. It has been speculatedthat these residues form part of the active site of this familyof recombinases and that tyrosine-433 forms the transientcovalent linkage to DNA during strand breakage and reunion(14).

In this communication, we describe the conversion oftyrosine-343 (family alignment position 433) of the FLPprotein to serine or phenylalanine. We demonstrate that boththe mutant proteins are inactive in recombination in vivo inEscherichia coli. They are also incapable of catalyzingrecombination or strand cleavage in vitro. However, likewild-type FLP, both mutant proteins bind to the DNAsubstrate to give three hierarchical DNA-protein complexes.Furthermore, the DNase "footprints" left by the mutantproteins on the substrate are indistinguishable from those leftby the wild-type FLP.

MATERIALS AND METHODSPlasmids. The construction of plasmids pMI and pJK (Fig.

1) has been described (5). The structure of pMI is almostidentical to that of pMJ, which was extensively used as thesource of FLP in recombination assays in vivo (5). The pMIhomologs, pMS and pMP (which contain serine-343 andphenylalanine-343, respectively) were obtained by directedmutagenesis using synthetic oligodeoxynucleotides.

Mutagenesis. The tyrosine-343 of FLP was mutated toserine or phenylalanine by the double-primer method (19).The oligodeoxynucleotides were synthesized in an A-380Applied Biosystems (Foster City, CA) nucleic acid synthe-sizer by the phosphoramidite method (21). Mutagenesis wascarried out on an Sph I/Xba I segment of 2-,um circle thatencompasses FLP, cloned into M13mpl8. The mutationswere then transferred to pMI by replacing the EcoRV/Xba Ifragment of pMI encoding the COOH-terminal two-thirds ofFLP with the corresponding mutant fragments.

Purification of FLP. The wild-type and mutant proteinswere purified to 25-40% homogeneity by modifications ofpublished procedures (22, 23). . The crude extracts preparedas described (23) were subjected to streptomycin sulfateprecipitation (4%), followed by ammonium sulfate precipita-tion of the resulting supernatant (40%). The pellet wasdissolved in 50 mM Tris HCl, pH 7.5/20 mM 2-mercaptoeth-anol/200 mM NaCl/1 mM EDTA, desalted with SephadexG-25, and chromatographed on Accell CM cation exchanger(Waters Associates) and on double-stranded DNA-cellulose(Pharmacia). On NaDodSO4 polyacrylamide gels, FLP mi-grated at -43 kDa. Transfer of gel-fractionated proteins tonitrocellulose and probing with FLP antibodies were done bypublished procedures (24).Recombination Assays. In vivo recombination assays were

done as described by Jayaram (5). In vitro recombinationswere done according to Prasad et al. (6).

*To whom reprint requests should be addressed.

2189

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 84 (1987)

A.

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Second Primer

C.EcoRI

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FIG. 1. Plasmids that express wild-type or altered FLP in E. coli. (A) In pMI, the FLP gene is expressed from the XPR promoter; the plasmidalso harbors the temperature-sensitive repressor c1857. At 42°C, the repressor is inactivated and FLP expression is turned on. Plasmids pMSand pMP are derivatives of pMI in which tyrosine-343 of FLP has been mutated to serine and to phenylalanine, respectively. Mutations wereproduced by the double-primer method on a single-stranded FLP template cloned in M13mp18 (19). The sequences of the mutagenic primersand the second primer are shown. The coordinates are according to the 2-gm circle numbering scheme of Hartley and Donelson (20). The 2-,mcircle portion of pMI is represented as a thick line. The wavy line stands for the FLP coding region. (B) After mutagenesis, the accuracy of themutations was verified by DNA sequencing. The altered bases in pMS and in pMP are indicated by the arrows. (C) The pJK plasmid (5), whichis the substrate for FLP recombination in E. coli, is a pACYC derivative. This plasmid is compatible with the ColEl plasmid pMI or its mutanthomologs pMS and pMP. The consequence of recombination between the directly repeated FLP sites is the loss of the kanamycin-resistance(KanR) marker. CamR, chloramphenicol resistance; AmpR, ampicillin resistance; ORI, origin.

Strand Cleavage, DNA Binding, and DNase Footprinting.All three assays were carried out using an 100-base-pair(bp) HindIII/EcoRI fragment that includes the complete FLPrecombination site. The fragment was labeled at the HindIIIend on the bottom strand by the Klenow polymerase reactionin presence of [a-32P]dCTP and the other three unlabeleddeoxynucleoside triphosphates.Approximately 0.1-0.2 pmol of the labeled fragment was

incubated at 30°C for 30 min with 0.5-1.0 ,ug of partiallypurified FLP in 50 mM Tris-HCl, pH 7.5/50 mM NaCl/5 mMMgCl2/1 mM dithiothreitol/75 ,ug of sonicated and phenol-extracted calfthymus DNA per ml, in a total vol of 50 1,u. Thereaction was stopped by the addition of 1 ,ul of 20%NaDodSO4. After phenol extraction and ethanol precipita-tion, the DNA was dissolved in a small vol of 80% forma-mide/10mM NaOH/0.1% xylene cyanol/0.1% bromophenolblue, heated at 90°C for 3 min, and run on 10% denaturingpolyacrylamide gels (19:1 cross-linking). Since cleavage ofthe DNA by FLP results in a free 5'-OH and a protein-bound3'-phosphate, no proteolysis was required to detect thelabeled breakage product resulting from the 3'-end-labeledsubstrate.Binding reactions were done by slight modifications of the

procedure of Prasad et al. (6). Approximately 0.1-0.2 pmolof the end-labeled fragment was incubated with 0.5-1 ,g ofprotein in 50 Al containing 50 mM Tris HCl (pH 7.5), 50 mMNaCl, 200 ,tg of bovine serum albumin per ml, 100 ug ofdenatured calf thymus DNA per ml, and 1 mM dithiothreitolfor 30 min at 30°C. After addition of heparin (5 ,g/ml) and

incubation for 5 min at room temperature, 5 ,ul of a solutioncontaining 10 mM Tris-HCl (pH 7.5), 30% (vol/vol) glycerol,and 0.1% bromophenol blue was added to the reactionmixture. Aliquots ofthe samples were electrophoresed on 5%polyacrylamide gels (30:1 cross-linking; 16 x 14 x 0.3 cm) at4°C in 1 x TBE (25 mM Tris HCl, pH 7.4/25 mM boric acid/1mM EDTA).For footprinting, the binding reactions were carried out

essentially as described above, except that the incubationmixtures included 10 mM MgCl2. The subsequent steps wereas described by Andrews et al. (25).

General Methods. Bacterial transformations, isolation ofplasmid DNA, restriction enzyme digestions, and othermiscellaneous methods followed published procedures (26).DNA sequences were determined by the method of Maxamand Gilbert (27) or Sanger et al. (28).

RESULTSIn Vivo and in Vitro Recombination Activity of Serine-343

FLP and Phenylalanine-343 FLP. Replacement of tyrosine-343 of FLP by serine and phenylalanine was achieved byusing mutagenic oligodeoxynucleotides as described (seeFig. 1A). Mutagenesis was carried out on an Sph I/Xba Isegment from the 2-,m circle genome (20) spanning the FLPcoding region that had been cloned into M13mpl8. Aftermutagenesis, the EcoRV/Xba I FLP fragment that includesthe mutation was excised and used to replace the correspond-ing wild-type FLP fragment in plasmid pMI (5). In plasmid

2190 Biochemistry: Prasad et al.

Proc. Natl. Acad. Sci. USA 84 (1987) 2191

Table 1. In vivo recombination assays for FLP, serine-343 FLP,and phenylalanine-343 FLP

Number of transformants

Source of FLP AmpR CamR AmpR CamR KanRpMI (FLP) 216 48pMS (serine-343 FLP) 362 362pMP (phenylalanine-343 FLP) 412 412

Each of the FLP plasmids (pMI, pMS, or pMP) was introducedinto a recA E. coli strain harboring the pJK plasmid. The resultantAmp)R Came transformants were replica-plated on kanamycin platesat 420C. The presence of functional FLP is manifest as kanamycinsensitivity. AmpR, ampicillin resistance; CamR, chloramphenicolresistance; KanR, kanamycin resistance.

pMI, FLP expression is driven by the XPR promoter, whichis controlled by the temperature-sensitive repressor cI857.Unlike plasmid pMJ used in earlier in vivo recombinationassays (5), pMI contains an intact FLP site within the 2-,umcircle repeat. The derivatives of pMI containing the FLPmutations, pMS (tyrosine-343 to serine-343) and pMP (tyro-sine-343 to phenylalanine-343), were used for in vivo assaysfor FLP recombination as described (5). The rationale of theassay is that recombination between two directly repeatedFLP sites bordering the TnS-derived kanamycin-resistance

A.FLP SER-343 FLP PHE-343 FLP

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gene results in the loss of this marker on a circular piece ofDNA that lacks a replication origin. The recombination eventis thus manifest as kanamycin-sensitive colonies of a recA E.coli host harboring the substrate plasmid pJK (see Fig. IC).Both serine-343 FLP and phenylalanine-343 FLP were inac-tive in this recombination assay (Table 1).To confirm these in vivo results, partially purified wild-type

and mutant FLP proteins were used in in vitro recombinationassays. The assay is based on the fact that recombinationbetween the FLP sites on two suitably chosen DNA frag-ments (P1 and P2; Fig. 2A) results in two new fragments (R1and R2) whose sizes are different from those of the parents(6). The results of the assay are shown in Fig. 2A. While thewild-type protein catalyzed efficient recombination, both themutants failed to do so.The Serine-343 FLP and Phenylalanine-343 FLP Cannot

Mediate Strand Cleavage. The results of the in vivo and invitro recombination assays demonstrate that replacement oftyrosine-343 ofFLP by either serine or phenylalanine resultsin the loss of the recombination potential of FLP. It waspossible that these proteins catalyzed cleavage of the DNAsubstrate but failed to complete the reaction by strandexchange and ligation. We tested this possibility by using thesynthetic FLP substrate J3 (Fig. 3A; ref, 5). This substratenot only includes the minimal FLP recombination site con-

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FiG. 2. In vitro recombination and strand cleavage. (A) The substrate for the in vitro recombination reactions was an equimolar mixture ofPvu II- or EcoRI-digested pJR1 (6). Plasmid pJR1 contains a 900-bp Hindll fragment from the A form of the 2-,m circle (20), which includesa copy of the repeated segment cloned into the HindIII site of pBR322. An FLP-mediated cross-over between the two types of molecules resultsin two recombinant linear molecules. The parental molecules are labeled PI and P2; the recombinant products are labeled R, and R2. The reactionconditions were those used by Prasad et al. (6). Protein concentrations were 0, 0.5, and 1.0 yg in lanes 1, 2, and 3, respectively. (B) The substrateused in the strand cleavage assay (shown in Fig. 3A) was labeled close to its 3' end on the bottom strand. The position of the label is indicatedby the C with the asterisk in Fig. 3A. Details of the assay are described in the text. The labeled fragment resulting from cleavage by FLP (F)was visualized by electrophoresis on a denaturing polyacrylamide gel followed by autoradiography. An Xba I digest of the labeled substrate(X) was run alongside for reference. The positions of strand cleavage caused by FLP and by Xba I are marked F and X, respectively, in Fig.3A. Protein concentrations were 0 ,ug (lanes 1), 0.5 /Lg (lanes 2), and 1.0 1jtg (lanes 3). (C) Approximately 40-50 ,ug of the wild-type and mutantFLP preparations used in the assays described above were electrophoresed on a 10o NaDodSO4/polyacrylamide gel (30:1 cross-linking),transferred to nitrocellulose (24), and probed first with anti-FLP antibodies and subsequently with peroxidase-conjugated anti-rabbit IgG. Theblot was developed with 4-chloro-1-naphthol (Sigma) and H202 (Baker). A protein preparation from an E. coli host containing the vector'withoutthe FLP gene (purified by the same protocol used for FLP and the FLP mutants) shows the absence of the 43-kDa protein that reacts withanti-FLP antibodies. (Left) Coomassie blue-stained gel. (Right) Immunoblot probed with FLP antibodies.

Biochemistry: Prasad et al.

Proc. Natl. Acad. Sci. USA 84 (1987)

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FIG. 3. Substrate binding and DNase foot-printing of FLP, serine-343 FLP, and phenylal-anine-343 FLP. (A) The substrate for footprint-ing was an 4100-bp HindIII/EcoRI fragmentthat includes the complete FLP site consisting ofthe three symmetry elements la, 1'a, and l'bplus the 8-bp core of the la-l'a dyad symmetry.Wavy line indicates sequences derived frompUC19. The bottom strand was labeled at theHindIII end with [a-32P]dCTP using the Klenowreaction. The deoxynucleotides incorporated bythe Klenow enzyme are italicized and the posi-tion of the label is marked by an asterisk. (B)Binding reactions were carried out as describedin the text. Samples were run on 5% polyacryl-amide gels at 40C in Tris borate buffer andautoradiographed. The FLP-DNA complexesare called I, II, and III. The input substrate isindicated by S. Protein concentrations were 0 ug(lanes 1), 0.5 ,g (lanes 2), 0.75 ,g (lanes 3), and1.0 ,.ug (lanes 4). (C) The footprinted sampleswere run on 12% sequencing gels. The sequenceladder obtained by chemical cleavage of thesubstrate at G, G+A, C, and C+T is alignedwith the DNase protection pattern. The bandresulting from the FLP-induced cleavage of thesubstrate (F) is absent in the footprints ofserine-343 FLP and phenylalanine-343 FLP.The region of the substrate strongly protectedby FLP is shown as the solid bar below it (seeA); the weakly protected region is symbolizedby the hatched bar. Protein concentrations were0 ,ug (lanes 1), 0.5 ,ug (lanes 2), 1.0 ,ug (lanes 3),1.5 ug (lanes 4), and 2.0 jpg (lanes 5).

sisting of the nearly perfect dyad symmetry (la-l'a) plus the8-bp. core, but also a second copy of the symmetry element(1'b), which is not essential for recombination. Since J3 isbordered by HindIII and BamHI 'sites, a stock of thissubstrate was. made in pUC19 digested with the above.enzymes. However, during propagation of this plasmidstock, the BamHI site was lost. due 'to the deletion of a single

* CG. Therefore, the FLP site. was excised from this plasmidby cutting.with EcoRI andHindIlIand was gel-purified. Thebottom strand was labeled at the HindIlI end by the Klenow

. polymerase reaction using [a-32P]dCTP and the other. threeunlabeled deoxynucleotides.'The cleavage.assay was done asdescribed. While 2-4%.of the input substrate was cleaved bywild-type FLP, no detectable cleavage could be observedwith the mutant proteins (Fig. 2B). We have verified that theprotein preparations used in the assay contained approxi-mately the same concentrations of the wild-type and themutant proteins by using immunoblots probed with anti-FLPantibodies (ref. 24; Fig. 2C).

Binding of FLP, Serine-343 FLP, and Phenylalanine-343FLP to Its Substrate. The experiments described aboveestablish that the two mutant FLP proteins are unable tocatalyze not only complete recombination but also strandbreakage, which is assumed to be the intermediate step in thereaction. Can these mutant proteins recognize and bind to theFLP substrate in a manner similar to the wild-type protein?To test this question, we used the same labeled FLP substrateas the one used for the strand cleavage assay. After incuba-

tion of this substrate with protein, the reaction mixtures wereanalyzed on polyacrylamide gels (29) to detect DNA-proteincomplexes. The wild-type FLP gave rise to three distinctcomplexes (I, II, and III) (see Fig. 3B). Kinetic experimentshave shown that complexes I, II, and III are formed in asequential order (data not shown). Furthermore, in theabsence of the third symmetry element, I'b, complex III was'not formed. Thus, these complexes appear to be trulyrepresentative of hierarchical binding ofprotomers ofFLP toits substrate. Similar results have been obtained by B. J.Andrews, L. G. Beatty, and P. D. Sadowski (personal com-munication). In addition, they have shown that complex Irepresents loose FLP binding, whereas complexes II and IIIrepresent tighter FLP binding and are recombination profi-cient. In our experiments, both serine-343 FLP and phenyl-alanine-343 FLP gave rise to the same three complexes as didFLP, as judged by the gel migration of these complexes (Fig.3B).

Finally, we performed DNase footprinting experiments(30) to determine whether substrate binding by the mutantproteins is qualitatively different from that of the wild-typeFLP. The footprints left by the three proteins on the bottomstrand of the substrate (end-labeled as described in previousassays) are shown in Fig. 3C. The region normally protectedby FLP consists of an -50-bp segment that spans the threesymmetry elements (la, l'a, l'b) as well as the core thatconstitutes the FLP site (Fig. 3A; ref. 25). The patterns ofprotection obtained with serine-343 FLP and phenylalanine-

B.

2192 Biochemistry: Prasad et A

Proc. Natl. Acad. Sci. USA 84 (1987) 2193

343 FLP contained no obvious differences from that obtainedwith FLP. However, as expected, the band corresponding tostrand cleavage by FLP was absent in the footprints of themutant proteins.

DISCUSSIONHomology alignments of prokaryotic site-specific recombin-ases assign them to two families: the Int-related and theHin-related families. The Hin family is composed of thehighly homologous invertases ofSalmonella (Hin), phage Mu(Gin), phage P1 (Cin), and E. coli (Pin) (15-18). The resolvaseproteins of the transposons Tn3 and y8 (31, 32) also belong tothis class of recombinases. The Int family consists of thesite-specific recombinases encoded by bacteriophage X andthe lambdoid phage 080 and P22, the related phage P2 and186, and the phage P4 and P1. The Int family differs from theHin family in encompassing an unexpectedly high degree ofsequence diversity (14). Nevertheless, there is a relativelyhigh level of conservation within a 40-amino acid sequencelocated near the COOH-terminal portion of these proteins.This homology appears to extend to the COOH-terminalregion of the 2-gm circle FLP protein as well (14). We maytherefore look upon FLP as a distant member ofthe Int familyof recombinases. The most remarkable feature shared by themembers of this family is the perfectly conserved trinity ofhistidine-396, arginine-399, and tyrosine-433. These familyalignment positions correspond to residues 305, 308, and 343,respectively, of the FLP protein.

In spite of the individual differences among the Int-relatedrecombinases, the reactions they catalyze share the samebasic features, especially in regard to the chemistry of strandcleavage and exchange. The three proteins Int, Cre, and FLPnick their substrates at specific positions to create 3' protein-bound termini and 5' overhangs with a free hydroxyl group.The 5' protruding ends are 6 bp long for Cre, 7 bp for Int, and8 bp for FLP (25, 33, 34). Presumably, the phosphoproteinbridge provides the energy required for the strand resealingstep of recombination.The fact that only three amino acid residues are invariant

in a family of proteins that catalyzes reactions whose chem-istry is almost identical implies that these residues areinvolved in recognition of specific DNA sequences and/orform part of the active sites of these proteins. We concen-trated on one of these conserved residues, tyrosine-343 of theFLP protein, for the following reasons. First, the aromaticportion of the tyrosine residue could be involved in interac-tions with DNA. Second, for FLP as well as Int, thephosphoprotein linkage has been shown to occur via atyrosine residue (ref. 12; C. Pargellis, S. Nunes-Duby, L.Vargas, and A. Landy, personal communication). Third, it isalso known that, in FLP, this residue is in the COOH-terminal portion of the protein, although its precise locationis undetermined (12). Based on these premises, one mayexpect the following properties for phenylalanine-343 FLPand serine-343 FLP. The change from tyrosine to phenylal-anine would likely eliminate DNA cleavage and protein-DNA linking. The effects of the mutation on DNA binding,however, could be minimal. On the other hand, the tyrosineto serine mutations could have large effects on DNA binding;nevertheless, if substrate binding is not altered by this aminoacid change, the mutant protein has the potential to form aphosphoprotein linkage. Indeed, in the case of the resolvaseof Tn3 or of yc the DNA-protein linkage is establishedthrough a serine (13, 35). We have shown here that neither thetyrosine to phenylalanine nor the tyrosine to serine mutationsignificantly affects the binding of FLP to its substrate. Bothmutations, however, abolish cleavage of the substrate andformation of the covalent protein-DNA complex and recom-

bination. The fact that the mutant proteins are not affected instability or substrate binding rules out a general distortion intheir secondary and tertiary structures. Thus, the resultsstrongly suggest that tyrosine-343 forms part of the FLPactive site but has minimal, if any, role in DNA binding. Ourdata are completely consistent with but do not prove the ideathat tyrosine-343 is the residue that becomes linked to DNAduring recombination.Accepting that tyrosine-343 is part of the FLP active site,

what is the role of the invariant histidine and arginine of theInt family in recombination? How do mutations of histidine-305 and arginine-308 of FLP (family alignment positions 396and 399) affect DNA binding and or catalysis by FLP?Experiments to answer this question using mutant FLPproteins (histidine to glutamine, arginine or leucine, arginineto isoleucine or glycine) are currently under way.

This work was supported by a research grant from the NationalInstitutes of Health.

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