The EMBO Journal vol.12 no.12 pp.4577-4584, 1993

Complementation of bacteriophage lambda integrasemutants: evidence for an intersubunit active site

Yiping W.Han1, Richard I.Gumport2 andJeffrey F.Gardner' 3

'Department of Microbiology and 2Department of Biochemistry andCollege of Medicine, University of Illinois at Urbana-Champaign,Urbana, IL 61801, USA3Corresponding author

Communicated by R.Kahmann

Site-specific recombination of bacteriophage lambdastarts with the formation of higher-order protein-DNAcomplexes, called 'intasomes', and is followed by a seriesof steps, including the initial DNA cleavage, top-strandexchange, branch migration and bottom-strandexchange, to produce recombinant products. One of theintasomes formed during excisive recombination (the attLcomplex) is composed of the phage-encoded integrase(Int), integration host factor (1HfF) and one of therecombination substrates, attL DNA. Int is the catalyticrecombinase and has two different DNA bindingdomains. When IHF is present, Int binds to two typesof sites in attL DNA, the three arm-type sites (P'123) andthe core-type sites (B and C') where the reciprocal strandexchange takes place. The Tyr342 residue of Int servesas a nucleophile during strand cleavage and covalentlyattaches to the DNA through a phosphotyrosyl bond. Invitro complementation assays have been performed forstrand cleavage using attL suicide substrates and mutantproteins containing amino acid substitutions at residuesconserved in the integrase family of recombinases. Wedemonstrate that at least two Int monomers are requiredto form the catalytically-competent species that performscleavage at the B site. It is likely that the active site isformed by two Int monomers.Key words: attL complex/complementation/intasome/integrase mutants/trans-cleavage

IntroductionThe bacteriophage lambda int gene encodes a site-specificrecombinase (Int) that catalyzes the strand exchangesrequired for both the integrative and excisive reactions. Intbinds to specific sites on the attachment (att) site DNA inconjunction with accessory proteins to form higher-orderprotein-DNA complexes, called 'intasomes', that are

required for recombination. Once the intasome is formedand the two recombination partners undergo synapsis, thereaction proceeds through an ordered series of stepsinvolving (i) initial DNA cleavage and exchange of the topstrands to form a Holliday junction, (ii) branch migration,and (iii) cleavage and exchange of the bottom strands togenerate the recombinant products (for reviews, see Landy,1989; Thompson and Landy, 1989).

Int is a member of the integrase family of the site-specificrecombinases, which includes the Cre protein of phage P1

©O Oxford University Press

and the Flp recombinase of the 2 / circle (Argos et al., 1986;Abremski and Hoess, 1992). This family shares a group offour invariant residues that are equivalent to Arg212, His3O8,Arg3 11 and Tyr342 of the Int protein and Arg 191, His3O5,Arg3O8 and Tyr343 of the Flp protein. These sequencesimilarities, along with the common characteristics of thereactions they catalyze, indicate that the chemistry of strandexchange is likely to be identical among these recombinases.The hydroxyl group of the conserved Tyr residue serves asa nucleophile to attack the phosphodiester bond at the strandexchange point and to covalently attach to the 3' end of thecleaved strand through the formation of a phosphotyrosylbond. The newly exposed 5'-OH group of the DNA thenserves as a nucleophile to attack the phosphotyrosylInt-DNA bond of the other recombination partner to finishstrand transfer (Gronostajski and Sadowski, 1985; Hoess andAbremski, 1985; Pargellis et al., 1988; Evans et al., 1990).By testing for complementation between the Flp(Y343F) andFlp(R191S) or Flp(R308Q) mutants in cleaving artificial half-site substrates, Chen et al. (1992) showed that the Tyr343residue that cleaves a half-site is part of a Flp monomerbound to a partner half-site, rather than to the site that iscleaved. This mode of reaction is termed trans-cleavage. Incontrast, Kim et al. (1990) inferred from experiments in thelambda system that, in the attL complex, an Int moleculecleaves at the core site to which it is bound by a cis-cleavagemechanism.

In this study, complementation was tested betweenInt-Y342F and mutants of Int containing substitutions ofthe conserved Arg212 and Arg3l1 residues (R212Q, R31 1Hand R31 IC) in cleaving the core-type sites in attL complexes.With respect to the cleavage of the B site in the attL complex,it is demonstrated that cleavage (i) requires at least two Intmonomers to form one catalytic site and (ii) is intramolecular- the B site is cleaved by Int bound in the same complex.The implications of these findings for the mechanism of thestrand cleavage are discussed.

ResultsThe cleavage activities of wild-type and mutant Int proteinswere measured using attL suicide substrates (see Figure 1)in the presence of integration host factor (IHF). Thesesubstrates contain a nick three nucleotides away from theInt cleavage site on either the top or bottom strand. Uponcleavage, the Int protein covalently attaches to the 3' endof the DNA strand and the trinucleotide cleavage productdissociates. These events prevent both the progress andreversal of the reaction, and covalent protein-DNAcomplexes accumulate (Nunes-Duby et al., 1987; Pargeiliset al., 1988). The protein-DNA complexes can be resolvedfrom unreacted substrates by electrophoresis on SDS-polyacrylamide gels and, if the substrate contains 32p,quantification of the radioactivity is a direct measure ofcleavage by Int (Kim et al., 1990).

4577

Y.W.Han, R.i.Gumport and J.F.Gardner

B

Xba I Sal IO* C' Po c' 7

CIA I Sal I*~~~ *

B 0 C' PI

CIa I Xba I Sai I

B O C P

CIa I Xba I BamH I* %.ntt$

B O C P

B O C' P1) tac *

CIa I Xba I

Fig. 1. Structure of the suicide substrates and the DNA fragmentsused to generate them. The names and sizes are indicated on the left.All suicide substrates are labeled at the 5' end (marked with '*') of thestrand which carries the nick. The restriction sites used to generatethese substrates are marked with arrows above or below the DNAstrands. B and C': the core-type binding sites for Int; P': the P' arm

of attL which contains the IHF binding site, H', and the arm-typebinding sites for Int, P'123. The nicks were all generated at the XbaIsites. The XbaI site used to generate the top-strand nick is located atthe + 1 position in the overlap region (0), while the XbaI site used togenerate the bottom-strand nick is at the -2 position. The curvedarrows indicate where the cleavage of the B or C' sites takes place,which are three bases away from the nick ('t' in the top strand, 'cat'in the bottom strand). After cleavage, Int becomes covalently attachedto the 3' end of the cleaved strand through its Tyr342 residue, whichforms a phosphodiester bond with the DNA. The trinucleotides ('ttt' or

'cat') diffuse into the solution and prevent both the progress and thereversal of the reaction. Therefore the protein-DNA complexaccumulates.

Complementation between mutants R212Q, R31 1C,R31 1H and Y342F in cleavage of the B site in attLcomplexesTo test complementation between mutants R212Q, R31 IC,R31 1H and Y342F in cleavage of the B site, the top-strandnicked suicide substrate CS was used (see Figure 1 andMaterials and methods). The results are shown in Figure 2.When wild-type Int was added to the reaction mixture andincubated at room temperature for 2-3 h, - 80% of theCS substrate was cleaved and converted into a radiolabeledcomplex that is sensitive to proteinase K, indicative of a

protein-DNA complex (compare lanes 2 and 3). The CXDNA, which contains only the B site, was also cleaved andformed covalent protein-DNA complexes in assays usingthe wild-type protein (lanes 2 and 3). No cleavage occurredwith any of the four mutant proteins when tested individually(lanes 4-7) or when the R212Q and the R311C or R311Hmutants were mixed (lanes 8 and 9). However, when theY342F mutant was mixed with any of the Arg-substitutedmutants, - 15-20% of the CS DNA was cleaved andconverted into protein-DNA complexes. Thiscomplementation was - 15-25 % as efficient as the reactionwith the wild-type protein. No CX -Int complexes wereformed when mutant proteins were used (lanes 10-12).Complementation between Int-Y342F and the Arg-

substituted mutants in cleaving the CS DNA suggests thatthe Arg212 or Arg311 residues on the Y342F mutant andthe Tyr342 residue on the Arg-substituted mutants form one

4578

catalytic site that is competent to cleave the B site. Inaddition, because mutant R212Q failed to complement eitherR311C or R311H, the Arg212 and Arg311 residues arecontributed by the same monomer.The complementation between these Int mutant proteins

differs from that of the mutant Flp proteins in that both thecore and arm of attL are required. No complementation wasseen with the CX half-site substrate, which contains the Bcore-type site but lacks arm-type sites (see Figure 1). In thecase of Flp, however, complementation with half-sitesoccurred. This difference is probably caused by the relativebinding affinities of Int and Flp for their respective core-type sites. Flp binds to its half-sites strongly andrecombination does not require accessory proteins or flankingarms (Amin et al., 1991; Qian et al., 1992). Int, on the otherhand, needs arm-type binding sites and other accessoryproteins to enable binding to the core-type sites (Ross andLandy, 1982, 1983; Moitoso de Vargas et al., 1989; Kimet al., 1990; Kim and Landy, 1992; MacWilliams, 1992).The Int mutants may have an impaired affinity for the core-type sites and thus are unable to complement in cleavageof the CX half-site.

DNA cleavage in order-of-addition experimentsFigure 3 shows the time course of an order-of-additionexperiment using mutant proteins R212Q and Y342F. Whenthe R212Q protein was added to the reaction mixturecontaining the CS DNA 10 min before the Y342F protein,a significant amount (10-15%) of cleavage at the B siteoccurred (lanes 1-6 in panel A, solid square curve in panelB). When the order of addition was reversed, cleavage wasweak (< 5%; lanes 7-12 in panel A, open triangle curvein panel B). The other two Arg-substituted mutants, R31 ICand R311H, behaved similarly (Figure 4). If Y342F waspreincubated with the substrates, weak cleavage was detected(lanes 5 and 7). These results suggest that when Y342F isprebound to the attL site, a complex is formed that inhibitscleavage of the B site when an Arg-substituted mutant proteinis added. In contrast, preincubation with an Arg-substitutedmutant, which presumably forms a complex similar to theone formed by Y342F, allows efficient complementationwhen the Y342F mutant is added. We interpret these resultsto indicate that preincubation of attL DNA with a mutantInt protein leads to the formation of a complex that containsboth tightly- and weakly-bound Int monomers. At least oneweakly-bound Int monomer can exchange readily with othermonomers in solution. According to this interpretation,cleavage occurs when an Arg-substituted protein forms astable attL complex and a Y342F monomer from solutionassociates with it to facilitate strand cleavage at the B site.

Differentiating intermolecular from intramolecularcleavage of the B siteTo test whether the B site in an attL complex is cleaved bya Tyr342 residue from an Int monomer bound in the samecomplex or from one bound to a different DNA molecule,a mixing experiment was performed. The Y342F and Arg-substituted mutants were separately incubated with twodifferent suicide substrates (CS and CB) for 10 min beforethe two reactions were combined and incubated at roomtemperature for 2-3 h. Both substrates were nicked in thetop strand, but were of different lengths so that the covalent

ChIA Xba I

"CX" (582 bp) * _ _ _

Xba I-Sal I fragment (474 bp)

Cla I-Sal Ifagment(1056 bp)

"CS" (1056 bp)

"CB" (780 bp)

"SC" (1098 bp,

Complementation of integrase mutants

_a a m m__

S

__ r:

Fig. 2. Complementation between Int-Y342F and Int-R212Q, Int-R311C or Int-R311H. CS, top-strand nicked attL suicide substrate; CS-Int,protein-DNA complexes formed after the B site is cleaved; CX, DNA with only the B site; CX-Int, protein-DNA complexes formed after the Bsite is cleaved. Lane 1, a reaction mixture with no Int protein added. Lane 2, wild-type Int was added to the reaction mixture and incubated for 2-3h before loading onto the gel. Lane 3, same as lane 2 except that the sample was treated with proteinase K prior to loading onto the gel. Lanes4-7, Int-Y342F or the indicated Arg-substituted mutants alone was added to the reaction mixture. Lane 8, R212Q and R311C were mixed togetherbefore addition to the reaction mixture. Lane 9, R212Q and R31 1H were mixed together before addition to the reaction mixture. Lanes 10-12,Int-Y342F was mixed with R212Q, R31 IC or R31 lH, respectively, before addition to the reaction mixture, as labeled on top of the lanes.

A .: ..-; 9-

a - _ a - a" as am

B

Lj1

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Fig. 3. A time course order-of-addition experiment with the Y342F and R212Q mutant proteins. (A) In lanes 1-6, the R212Q mutant waspreincubated for 10 min with the suicide substrate, CS, before the Y342F mutant was added. The reaction was then stopped at different times asindicated on top of each lane. Lane 1 represents the time point when the Y342F mutant was added to the reaction mixture. Lanes 7-12 are thesame as lanes 1-6 except that the Y342F mutant was preincubated with the substrates and the R212Q protein was added 10 min later. CS, top-strand nicked attL suicide substrate; CS-Int, protein-DNA complexes formed after the B site is cleaved; CX, DNA with only the B site. (B) Theresults shown in (A) plotted as cleavage curves. The percent suicide substrate that is cleaved is presented as a function of the reaction time. Thecleavage shown in lanes 1-6 in (A) is represented by solid squares (U), while the cleavage shown in lanes 7-12 in (A) is represented by opentriangles (A). The arrow indicates the point when the second protein was added.

protein-DNA complexes formed with either could bedetected by electrophoresis.

If an Arg-substituted mutant (R212Q, R31 IC or R3 1 1H)was preincubated with the CS DNA, and Y342F with CB,the Int binding sites in CS would be occupied with the Arg-

substituted mutant, while the same sites in CB would bebound by Y342F. Upon mixing, there would be a mixtureof four predominant attL complexes after exchange of Intmonomers at the weakly-bound site, as illustrated in Table I.If the cleavage takes place intramolecularly, it is predicted

4579

ioa-,

a'-s A

Fig. 4. Results of the order-of-addition experiments with the R212Q, R311C, R311H and Y342F mutants. All reactions were incubated at room

temperature for 2-3 h. Lane 1: no protein was added to the reaction mixture. Lane 2: wild-type Int was added to the reaction mixture. Lanes 3, 5

and 7: the Y342F mutant was added to the reaction mixture 10 min before the second protein, R212Q, R311C or R311H, respectively, was added.

Lanes 4, 6 and 8: cell extracts of mutants R212Q, R31 IC or R31 lH, respectively, were preincubated with the reaction mixtures for 10 min before

Y342F was added. CS, top-strand nicked attL suicide substrate; CS-Int, protein-DNA complexes formed after the B site is cleaved; CX, DNA

with only the B site; CX-Int, protein-DNA complexes formed after the B site is cleaved.

that only Complex II would result in strong cleavage.Complexes I, HI and IV would be incapable of cleavage.If this hypothesis is correct, CS DNA, the substratepreincubated with the Arg mutant, would be cleavedpredominantly. However, if the cleavage takes placeintermolecularly, and two Int monomers on each substrateare required for cleavage, one would expect both the CSand CB DNA to be cleaved similarly because the Arg-substituted mutant bound to CS DNA could complement theY342F mutant bound to the weak site on the CB DNA. Asshown in Figure 5, a strong cleavage bias toward thesubstrate that was preincubated with an Arg-substitutedmutant occurred (compare lanes 5 and 6, 7 and 8, 9 and10). The simplest interpretation of these results is that thecleavage of the B site in an attL complex is an intramolecularevent involving only one nucleoprotein complex and doesnot require interactions between different attL complexes.

Cleavage of the C' site in an attL complexThe cleavage of the bottom strand in the C' site of attLcomplexes was also tested with the Y342F and Arg-substituted mutants using the bottom-strand nicked attLsuicide substrate SC (see Figure 1). The results aresummarized in Table II. Surprisingly, 6-9% cleavage ofthe DNA was detected repeatedly when only the Y342Fprotein was present, unlike the case using the top-strandnicked suicide substrates. Crude extracts of the Y342Fmutant behaved similarly (data not shown). The product ofY342F cleavage is sensitive to proteinase K (data notshown), indicative of a covalent protein-DNA complex.We speculate that, although the active nucleophilic hydroxylof Tyr342 has been replaced by hydrogen in the non-activePhe residue, another nucleophile in proximity to the scissilephosphodiester bond may promote the observed cleavage.

When complementation between the Y342F and Arg-substituted mutants was tested, however, <4-5% of thesubstrate was cleaved. These activities are not only muchlower than those with the top-strand nicked suicide substrates(15-20%), but are also lower than those observed whenthe Y342F mutant was incubated with SC alone (6-9%).

DiscussionTo study the mechanism of Int-mediated strand cleavage,attL suicide substrates were used in complementation studieswith mutant proteins. The Int mutants contained substitutionsof three of the four residues that are completely conservedin the Int family of recombinases. The mutants containedsubstitutions of Tyr342 (Tyr342 to Phe), Arg212 (Arg212to Gln) and Arg311 (Arg311 to Cys or His). Using an attLsuicide substrate nicked in the top strand of the B site, wefound that the Y342F mutant complemented all of the Arg-substituted mutants. In contrast, mutants with substitutionsat Arg212 and Arg311 failed to complement one another.There are two mechanisms that can explain the

complementation patterns of the mutant Int proteins. Onepossibility is that the mutants show structural comple-mentation. Each mutant has a slightly disrupted structure sothat they cannot function by themselves. When they arepresent together in the presence of DNA they are able tointeract and adapt to a structure that allows function. Analternative mechanism is one in which mutant monomersform a catalytic site. In this mechanism, the Arg212 andArg311 residues in Y342F and the Tyr342 residue in theArg-substituted mutants form a shared intersubunit activesite. Although the two mechanisms are difficult todistinguish, we prefer the latter model for three reasons.First, previous work has suggested that Arg212, Arg3l1 and

4580

Complementation of integrase mutants

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Fig. 5. Results of mixing experiments. The Y342F and Arg-substituted mutants (R212Q, R31 IC or R31 1H) were separately incubated with the twodifferent suicide substrates (CS or CB) for 10 min before the two halves were mixed together, followed by incubation at room temperature for 2-3h. The CS and CB substrates are of different lengths as indicated in Figure 1. CS-Int, CB-Int: protein-DNA complexes after the B site iscleaved; CX: DNA with only the B site, which is cleaved by wild-type Int to form CX-Int. CX-Int comigrates with CB. Lane 1, CB DNA only;lane 2, CB DNA incubated with the wild-type protein; lane 3, CS DNA only; lane 4, CS DNA incubated with the wild-type protein; lanes 5, 7 and9, Y342F preincubated with CB DNA and the R212Q, R311C or R311H proteins, respectively, preincubated with CS DNA before mixing; lanes 6,8 and 10, Y342F preincubated with CS DNA and the R212Q, R311C or R311H proteins, respectively, preincubated with CB DNA before mixing.The proteins and substrates are indicated at the top of the figure.

Tyr342 are part of the catalytic site (Pargellis et al., 1988;Burgin and Nash, 1992; Han et al., 1993). Second, theY342F mutant complements both Arg212- and Arg3 11-substituted mutants, but the Arg-substituted mutantsdo not complement each other. Third, evidence from theFlp system, where the same complementation patterns wereobserved for mutants with substitutions at Argl91, Arg3O8and Tyr343, indicates that the active site is formed by twomonomers (M.Jayaram, personal communication).Kim et al. (1990) showed Int cleavage of the B site of

an attL suicide substrate when all the Int binding sites,including the B site, were occupied. Our results, inconjunction with the titration experiments reported by Kimet al. (1990), indicate that strand cleavage requires one Intbound to the B site and another bound elsewhere on attL.However, it is a formal possibility that a simple monomerof the wild-type protein can function by itself.

Cleavage of DNA by the Tyr342 residue could occur byeither a cis or trans mechanism (Chen et al., 1992). A cismechanism involving two Int monomers is one in whichcleavage is mediated by an Int monomer at the site to whichit is bound. In the case of the attL suicide substrate containinga nick in the B site, the B-bound monomer would donatethe active Tyr342 residue and a different Int monomer wouldprovide the Arg residues. Trans-cleavage would involvecleavage by the Tyr342 of an Int monomer presumablybound to a core-type site different from the one that isactually cleaved. Trans mechanisms can be placed into twoclasses depending on the source of the Tyr342 donor usedin the cleavage. Trans-horizontal cleavage would involvecontribution of the active Tyr342 by an Int monomer boundto the C' site. In this case, the C'-bound Int would bridgethe overlap region to cut the B site. A second type of trans-

Table I. Composition of the four predominant anL complexes formedin one of the mixing experiments a

Substrate CS Substrate CB

tightly-bound weakly-bound tightly-bound weakly-bound

Complex I R RComplex II R YComplex Im y yComplex IV Y R

a This table shows only the complexes formed when an Arg-substitutedmutant is preincubated with the CS DNA and Y342F preincubatedwith CB (see text for details). R, Arg-substituted mutants; Y, Y342F;tightly-bound = arm-bound; weakly-bound = core-bound.

cleavage would involve an Int monomer whose C-domainwas presumably bound to one of the core-type sites of apartner DNA substrate. Depending on which core-type siteof the partner substrate was bound by Int and the alignmentof the substrates during synapsis (parallel or antiparallel) thecleavage could be trans-vertical or trans-diagonal. In the caseof Flp, Chen et al. (1992) proposed a trans-diagonalcleavage mechanism if the substrates align in a parallelconfiguration during synapsis.How well can our complementation results and previous

information about the structure of the attL intasome be usedto support either a cis-cleavage or trans-cleavagemechanism? Kim et al. (1990) have proposed a model forthe structure of the attL complex (Figure 6). In their model,IHF bound to the H' site induces a bend in the DNA bringingthe core-type and arm-type sites close to each other andthereby facilitating the simultaneous binding of an Int

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Y.W.Han, R.i.Gumport and J.F.Gardner

Table IH. Cleavage of the C' site in the bottom-strand nicked suicidesubstrate

Samplesa % Cleavage of the C' sitein suicide attL DNA

wild-type Int 70-80Y342F 6-9

R212Q _bR311C _bR311H _b

Y342F/SC 10 min R212Q 3-4R212Q/SC 10 min Y342F 1-2

Y342F/SC 0min R311C 3.8-4R311C/SC lO min Y342F 1.8-2.6

Y342F/SC 10 min R31 IH 3-5R31 IH/SC 10 mm Y342F 0.5-1.5

a The suicide substrate, SC (see Figure 1), and the reaction conditionsare described in Materials and methods. In the order-of-additionexperiments (the bottom six samples) testing the complementationbetween the Y342F and the Arg-substituted mutant proteins, eitherY342F or an Arg mutant protein was preincubated with the substratesfor 10 min before the addition of the second protein. The reaction wasthen incubated at room temperature for 2-3 h.b No detectable cleavage was observed.

Fig. 6. A model for the assembly of the attL complex from Kim et al.(1990). The circles represent the large C-domains of the Int monomersthat bind to the core-type sites (B and C'). The ovals represent the N-terminal domains that bind to the arm-type sites (P'123). When IHF(the stippled horseshoe) binds to the H' site, it causes a bend in theDNA and facilitates bridging of one Int monomer between the core-and arm-type sites with its C-domain bound to the C' site and the N-terminal domain bound to the P'1 site. A second and third Int arebound to the P'2 and P'3 sites, respectively. These three Ints arestably bound in this complex and are darkly shaded in this figure. TheB site-bound Int (lightly shaded) is brought into the attL complexthrough protein-protein interactions with the P'-bound Ints. This Intreadily exchanges with the Int molecules in solution.

monomer to the C' core-type site and the P' 1 arm-type sitevia its C- and N-terminal domains, respectively. A secondInt monomer binds to the P'2 site via its N-terminal DNAbinding domain leaving its C-domain unbound. A third Intmonomer binds to the P'3 site, although it is not requiredfor excisive recombination. A fourth Int monomer isrecruited from solution and binds to the B site. The Intmonomers bound to P' 1 and P'2 stabilize binding of the latterInt through protein-protein interactions. The recruitedmonomer exchanges more readily with Int monomers insolution than can those bound to the arm-type sites.The results from our order-of-addition and mixing

experiments are consistent with this model. If the model isapplied to our results, when the Y342F mutant occupies theP'123 and C' sites, it inhibits cleavage of the B site by an

Arg-substituted mutant so that the active Tyr342 residue ofthe latter protein cannot form a covalent link with the DNA.If an Arg-substituted mutant occupies the P' 123 and C' sites,one of the Tyr342 residues can form an active site, inconjunction with the Arg residues from Y342F bound to theB site. The model implies that Int acts by a mechanismsimilar to that of Flp (Chen et al., 1992). The Arg 212 andArg311 residues are provided in cis to the B site, whereasthe Tyr342 residue is provided in trans by an Int monomerbound to the P'l or P'2 site.Our results do not establish which of the P'-bound Int

monomers provides the Tyr342. Because the P'3 site isdispensable for excision (Bauer et al., 1986; Winoto et al.,1986; Kim et al., 1990; Numrych et al., 1990), either theP' 1-bound or P'2-bound Int could donate the active Tyr342hydroxyl group. If the Tyr342 residue is provided by an Intmonomer bound simultaneously to the P' 1 and C' sites, themechanism would be classified as trans-horizontal in thenomenclature proposed by Chen et al. (1992) (see above)because Int monomers bound to the adjacent B and C' core-type sites would form the catalytic site. On the other hand,if the active Tyr342 residue is provided by an Int monomerbound to the P'2 site, the mechanism would be similar tothe trans-diagonal mechanism proposed for Flp. Althoughthe P'2-bound Int is bound to the same attL molecule as theB-bound Int, its association with the attL DNA is throughits N-terminal domain, while the C-domain that is responsiblefor binding to the core-type site is vacant. During excisiverecombination with the partner substrate present, thisP'2-bound Int would presumably be bound to the C core-type site of the partner attR DNA through its C-domain aswell (Kim and Landy, 1992). Therefore, the active Tyr342of this Int monomer can be considered as coming from attR.If so, the catalytic site is formed by a B-bound monomerfrom attL and a C-bound monomer from attR. Dependingon whether synapsis occurs with the DNA in a parallel oran antiparallel configuration, this would be classified astrans-vertical or trans-diagonal in the nomenclature of Chenetal. (1992).

Considering these two mechanisms, the second modelseems more attractive for two reasons. First, Flp and Intare members of the same family of recombinases. In bothcases, amino acid residues from more than one monomerform the catalytic site. Chen et al. (1992) provided evidencethat the active tyrosine residue of a Flp monomer cleavesthe DNA in a partner substrate. Second, the mechanismsuggests a means for coupling synapsis and strand cleavageduring excisive recombination. Formation of the intersubunitcatalytic site for cleavage of the B site of attL would requiresynapsis of the attL and attR substrates.Both mechanisms are consistent with the footprinting and

gel shift assays of Kim et al. (1990). Mutations in the P' 1and P'2 sites decreased protection of the core-type sites inattL in footprinting experiments. Multiple mutations in P' 1decreased binding to both the B and C' sites, while mutationsin the P'2 site disrupted binding to the B site. Gel shift assays,that monitored both the stoichiometry of Int binding to attLand the formation of the covalent protein-DNA complex,indicated that four monomers are required for cleavage ofthe suicide substrate containing a top-strand nick in the B site.There are, however, some differences in the interpretation

of our results and those of Kim et al. (1990). In oneexperiment, they used the same attL suicide substrates that

4582

i...

II

'v .

Complementation of integrase mutants

we used with nicks in either the top (B site) or bottom (C'site) strand. The substrates were preincubated with theY342F mutant and the wild-type protein was then added ata molar ratio of 0.04 relative to the Y342F mutant. Theyfound that the substrate containing the nick in the top strandwas cleaved 5-fold faster than the substrate with the nickin the bottom strand. Because models under considerationat the time assumed that the C-domain of a single Intmonomer was responsible for cleavage, they interpreted theresults to indicate that a wild-type Int from solutionexchanged readily with the Y342F mutant bound to the Bsite and thus cleaved in a cis fashion. Cleavage of the C'site was slower because a cis-cleavage mechanism wouldrequire dissociation of the complex and formation of a newone with wild-type Int bound to the P'1 and C' sites.Our results using the attL substrate with a nick in the C'

site of the bottom strand did not show a straightforwardcomplementation pattern. In contrast to the results with anattL suicide complex with a nick in the B site, we foundthat the Y342F mutant itself could cleave the C' site andform a covalent protein-DNA complex. Furthermore,addition of the Arg-substituted mutants failed to increase thefrequency of this aberrant cleavage of the C' site. The onlysimple explanation we can offer for these differences in thecomplementation patterns for cleavages of the B and C' sitesis differing mechanisms of cleavage for each substrate.Recombination between attL and attR proceeds with adefined order of strand exchanges (Nunes-Duby et al., 1987;Kitts and Nash, 1988a,b). In the attL complex, the initialstrand exchange is almost exclusively at the B site, and theC' site is cleaved only after the Holliday junction is formed.Because the Holliday junction and the attL complexes differin topology and probably in the spatial arrangement of Intmonomers, it is possible that Int interacts with each complexdifferently. It may be more appropriate to test complementa-tion of the C' site using suicide Holliday junctions ratherthan atL.We think a trans-cleavage model is attractive because it

makes the mechanisms of Int and Flp cleavage similar. Moredefinitive experiments will be required to determine thepositions of the Int monomers that form the active site todetermine whether the trans-cleavage mechanism is correct.Our finding that the active site is formed by at least two Intmonomers complicates the architecture of the atL complex,making it risky to use cleavage patterns of suicide complexesto assign positions of Int monomers in the complex.

Materials and methodsConstruction of attL suicide substratesPlasmids containing attL [pSK3 (Kim et al., 1990) or pBF32 (Nunes-Dubyet al., 1987)] were used to construct attL suicide substrates nicked on thetop or bottom strand. The nicks in these substrates are generated at the XbaIsites in the overlap region and are located three bases 3' from the Int cleavagesites (Nunes-Duby et al., 1987; Figure 1). The suicide substrates wereprepared as described by Nunes-Duby et al. (1987) with a slight modification.To prepare top-strand nicked suicide substrates, pSK3 was digested withClaI, treated with bacterial alkaline phosphatase and the 5' ends were labeledwith [-y-32P]ATP and T4 DNA polynucleotide kinase. The sample wasdigested by XbaI and the fragment containing the 32p label in the top strand,designated CX (-580 bp) in Figure 1, was purified from a 5%polyacrylamide gel after electrophoresis. The unlabeled XbaI-SalI (-480bp) and ClaI-SalI (- 1060 bp) fragments (see Figure 1) were purifiedfrom 1% low melting point agarose gels after endonuclease digestion andelectrophoresis. The DNA concentrations of the purified fragments weredetermined by ethidium bromide staining as described by Sambrook et al.

(1989). The labeled CX and unlabeled XbaI-Sall and ClaI-Sall fragmentswere mixed at ratios of 1:5:5 to 1: 10:10, denatured on ice in 0.2 N NaOH,10 mM EDTA for 5-10 min, followed by neutralization with 10 M aceticacid. The DNA was annealed at concentrations of 10-15 Ag/ml in a solutionof 0.3-0.5 M sodium acetate and 10mM Tris-HC1 (pH 7.9) after heatingat 65'C for 30 min followed by slow cooling. After the annealing, the DNAproducts (including the suicide substrate CS, - 1060 bp) were purified bycentrifugation through a G-50 spun column. A portion of this DNA wastreated with BamHI to generate a shorter substrate CB ( - 780 bp). BothCS and CB are top-strand nicked attL suicide substrates, labeled at the 5'ends of their top strands (see Figure 1) and were used to monitor Int cleavageat the B site in the attL complexes. The bottom-strand nicked attL suicidesubstrate, SC (- 1020 bp), was prepared like the CS substrate except thatthe DNA was labeled at the 5' end of the bottom strand (see Figure 1).This substrate was used to detect Int cleavage of the C' site in the attLcomplexes.

Preparation of cell extracts, Western blotting and quantitationof the proteinsThe cell extracts containing wild-type and mutant Int proteins were preparedas described by Y.W.Han, R.I.Gumport and J.F.Gardner (manuscriptsubmitted). Cells bearing int plasmids were grown at 37'C to an OD65oof 0.1 in 300 ml of LB-ampicillin medium, followed by induction withIPTG (1 mM) for 60 min. Cell pellets were collected after centrifugationat 4°C, resuspended in 300 A1 of 50 mM Tris-HCI, pH 7.4, 10% sucrose,and frozen on dry ice before being stored at -70'C. To lyse the cells,the pellets were thawed on ice prior to the addition of 25 Al of freshly-made solution of lysozyme (10 mg/mil) in 250 mM Tris-HCI, pH 7.4,followed by a 40 min incubation on ice. After 30 min of centrifugationin a microcentrifuge at 4°C, the supernatant was carefully removed andaliquots were frozen in liquid N2 and stored at -700C. The amount ofInt protein in crude extracts was determined by Western blotting as describedby Sambrook et al. (1989). One unit of wild-type Int (pure or crude) isdefined as the minimum amount of protein needed to yield the maximumamount of products in the in vitro recombination assays. One unit of purifiedY342F protein, a gift from Dr A.Burgin Jr, is defined as the amount ofprotein needed to retard the same amount of attL DNA as does 1 unit wild-type Int in the in vitro gel retardation assays (A.Burgin Jr, personalcommunication). One unit of the Arg-substituted Int mutants (cell extractsof R212Q, R311C or R311H) was defined by Western blot analysis as anamount comparable with 1 unit of the wild-type Int protein.

DNA cleavage assaysDNA cleavage assays (20 Al) contained 25 mM Tris-HCI, pH 7.4, 50mM KCI, 5 mM spermidine, 250 jig/ml BSA and -0.2 nM of theradiolabeled suicide substrates. In addition, 1-2 nM intact (non-nicked)unlabeled atL DNA was present as a result of the method used to preparethe substrates (see above). The lengths of these substrates are indicated inthe figures. The concentrations of the proteins were: 1 unit IHF, 1 unitY342F and -1 unit of the R212Q, R31 IC or R311H mutants. The reactionswere carried out at room temperature for 2-3 h, unless otherwise indicated,before loading onto 5% polyacrylamide gels. The gels and the running bufferwere made with 1 x TBE containing 0.1% SDS. After electrophoresis,the gels were dried and subjected to autoradiography and the radioactivitywas quantified on a PhosphorImager (Molecular Dynamics).

Chemicals and enzymesRestriction endonucleases, bacterial alkaline phosphatase and T4polynucleotide kinase were purchased from Bethesda Research Laboratories.Proteinase K (Boehringer Mannheim) was used at a final concentration of1 tg4ll. [-y-32P]ATP was obtained from DuPont.

AcknowledgementsWe are grateful to Drs A.Landy and S.Nunes-Duby for providing plasmids.We thank Dr A.Burgin Jr for purified Y342F protein and appreciate thesuggestions from Drs A.Landy and S.Nunes-Duby regarding the preparationof the suicide substrates. We thank Drs A.Burgin Jr, N.Craig, H.Nash andA.Segall for comments on this manuscript. This work was supported byNIH grant GM28717.

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Received on June 23, 1993; revised on August 23, 1993

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