INTRODUCTION

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Bacteriophage lambda integrase (Int) is a recombinase which inserts and excises the phage genome into and out of the Escherichia coli chromosome. It belongs to a large family of tyrosine recombinases whose members carry out site-specific recombination of phage, plasmid, and chromosomal sequences (6, 29). Int mediates recombination via four distinct pathways (summarized in Table 1), in which different protein-DNA complexes are assembled by Int and its accessory factors on four types of recombination target sites, known as attachment or att sites (the structure of attL is shown in Fig. 3). In addition to the catalytic domain, Int has two DNA binding domains: a relatively high-affinity binding domain contacts the "arm" binding sites, which are distal to the loci of strand exchange, while a low-affinity binding domain contacts the "core" binding sites, which directly flank the loci of strand exchange (32, 41).

View this table: [in this window] [in a new window]   TABLE 1.   Summary of the four pathways of bacteriophage  site-specific recombination

Int cleaves and rejoins four phosphodiester bonds in the host and phage DNAs via transient covalent intermediates between itself and DNA. Presumably because abortive reactions would be detrimental both to the phage and to the host, the recombinases have evolved to be very efficient, and very few abortive products are seen (30). At worst, aborted recombination events would generate breaks in both the bacterial and phage genomes; at best, integration or excision would be less efficient due to unproductive rounds of strand cleavage and joining and the lysogeny versus lysis decision of the phage would be impeded. Abortive recombination may be minimized by carrying out strand exchange in the context of specific synaptic complexes containing both of the DNA substrates and all of the protein subunits necessary for the reaction. This indeed appears to be the case (1, 31, 35, 36). Moreover, the DNA strand cleavage and ligation events in integrative and excisive recombination are highly concerted, for which interactions between Int monomers are presumed to be important. Protein-protein interactions appear to be a central feature of site-specific recombination reactions, including phase variation of flagellar antigens in Salmonella enterica (12, 22), resolution of transposon-generated cointegrate structures (14, 25), and 2µ plasmid inversion mediated by Flp (reference 21 and references therein).

The DNA binding and catalytic properties of Int, as well as those of many protein-DNA intermediates assembled by Int in conjunction with its accessory factors, have been studied extensively (reviewed in references 20 and 27). However, no direct information is available on the interactions between Int monomers occurring during recombination. Two major observations suggest that protein-protein contacts between Int monomers are important. First, binding of Int to the att sites is highly cooperative (16, 36). Second, different catalytic mutants of the Int protein complement each other for strand cleavage activity (10), suggesting intimate interactions between at least two Int monomers. More recently, further information has become available from several crystal structures of Int family proteins. Four structures have been solved, namely, those of the catalytic domain of Int (19), the catalytic domain of the Haemophilus influenzae phage HP1 Int (13), the E. coli XerD protein (39), and the cocrystal of the bacteriophage P1 Cre protein bound to a Holliday junction (7, 9). The Int structure and the XerD structure were solved as monomers and thus provide information on protein-protein contacts only by homology alignments with the catalytic domains of HP1 Int, solved as a dimer, and with the Cre structure, which was solved as a pseudosymmetric tetramer (9). However, Int and the other bacteriophage integrases share little or no recognizable homology in the noncatalytic domains of their relatives (6). Therefore, while the Cre and HP1 Int structures give insight into protein-protein contacts lying within the catalytic domain, none of these structures is very informative about interactions occurring in the N-terminal half of the Int protein.

In order to obtain more information about protein-protein interactions necessary for Int's function, we have investigated interactions between Int molecules using several approaches. First, we have used a genetic assay to localize the regions where protein-protein contacts occur. Second, we have investigated cooperative interactions among Int monomers binding to DNA. Third, we have used protein-protein cross-linking as a physical assay to determine the predominant multimeric species assembled by Int. We find that residues which contribute to stable protein-protein contacts are spread throughout most of the Int primary sequence. The genetic and physical assays agree that Int's N-terminal domain, previously thought to comprise only the DNA binding domain necessary for contacting Int's high-affinity arm binding sites, also provides an important protein-protein interface between Int monomers. We show that cooperative interactions at the N terminus of Int affect the stable binding of Int to its core binding sites, the loci of strand exchange. Last, we show that Int forms several types of multimers and we test the effect on recombination of modifying cysteines and tethering Int protomers to each other.

	MATERIALS AND METHODS

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Strains. The tester strain for examining the transcription-inducing activities of Int-AraC fusion proteins was M8834, F (araOC leu)1109 rpsL150 (Str^r) (generously provided by Malcom Casadaban). It was constructed such that araC was deleted but a functional araBAD operon remained. Transcription of araBAD depends on appropriate dimerization of the AraC DNA binding domains (AraC_D). An hsdR deletion was introduced by P1 transduction to facilitate direct cloning of PCR-generated ligation products into the M8834 strain. Mutations in lon and clpP were introduced by transducing M8834 with clpP::Tn5 linked to a lon allele (donor strain was kindly provided by S. Gottesman). Phage P1 transductions were performed according to standard protocols, and mucous Kan^r transductants were used as recipients of the cloned fusion proteins (lon mutations confer a mucous phenotype). The clone expressing the His-tagged Int gene was the generous gift of J. Gardner, while the clone expressing the IntC25S gene was the generous gift of R. Tirumalai and A. Landy.

Generation of LacZ::Int::AraC chimeric genes. Fusion proteins were constructed by introducing portions of the Int gene, generated by PCR, upstream of and in frame with the AraC_D. Plasmid pKM19-C170 (from N. Lee via M. Casadaban) is a modified pUC19 plasmid containing the AraC_D, consisting of amino acid residues 170 to 292 (23). Upstream of the truncated AraC protein are the translation start sites and the first five amino acids of LacZ, all of which are under the control of a ptac promoter. PCR primers were designed to introduce restriction enzyme sites upstream (HindIII) and downstream (BamHI) of appropriate Int sequences to allow cloning upstream of and in frame with the AraC_D in pKM19-C170. The sequences of the primers used are summarized in Table 2. The PCR products, generated with VENT polymerase (New England Biolabs [NEB]) were purified with a Wizard PCR Preps kit (Promega), digested overnight with BamHI and HindIII (NEB), gel purified, and ligated to gel-purified, digested vector DNA. The ligated plasmids were recovered in DH5 cells and electroporated into M8834 or its relatives. Screens for araBAD activity were carried out on MacConkey agar (Difco) containing 1% L-arabinose (Sigma).

Cell extracts. Extracts of cells expressing the fusion proteins were made by a freeze-thaw lysozyme procedure, as follows. Overnight cultures of the tester strain containing the fusion proteins were subcultured 1:10 into 40 ml of 1× NCE (4) supplemented with 1% casein digest (Sigma) and 1% arabinose with 100 µg of ampicillin (Sigma) per ml, grown at 37°C for 2.5 to 3 h to an optical density at 650 nm of 0.7 to 0.8, and pelleted. Cell pellets were resuspended in a solution containing 200 µl of 0.5 M Tris HCl (pH 7.4) and 10% sucrose (Sigma) and frozen at 80°C. Pellets were thawed on ice, and the following concentrations of protease inhibitors were added: 0.5 µl of leupeptin (5 mg/ml in H₂O; Sigma), 0.25 µl of pepstatin (10 mg/ml in H₂O; Sigma), 0.5 µl of soybean trypsin inhibitor (100 mg/ml in H₂O; Sigma), and 5 µl of phenylmethylsulfonyl fluoride (100 mM in ethanol; Sigma). The cells were then lysed by incubating them on ice for 30 min with a 1/20 volume of 10-mg/ml lysozyme (in 10 mM Tris HCl [pH 7.4]). The cellular debris was removed by centrifugation at 15,000 rpm in a Sorvall RCSC centrifuge with an SA 600 rotor for 20 min. Total protein present in the extract was measured by a modified Bradford protein assay (Bio-Rad). The extracts were then frozen in a dry ice-ethyl alcohol bath and stored at 80°C.

Western blot analysis. Approximately 120 ng of cell extracts was mixed 1:1 with Laemmli loading buffer (Bio-Rad), boiled for 10 min, and electrophoresed on 10 to 20% polyacrylamide gradient minigels (Novex). Electrophoresis was carried out for 2.5 h at 120 V. The gels were transferred to nitrocellulose (Bio-Rad) in a Novex Western Blot Module according to the manufacturer's instructions at 30 V for 2 h in transfer buffer. The nitrocellulose was probed with antibody, washed, and treated with 10 ml of SuperSignal peroxide substrate (Pierce) for 10 min before exposure to Kodak XAR film. For reprobing with a different primary antibody, each blot was gently shaken in stripping solution (2% sodium dodecyl sulfate [SDS; Sigma], 62.5 mM Tris-HCl [pH 6.8], 0.1 M -mercaptoethanol [Sigma]) at 65°C for 30 min and was rinsed several times before the Western blot procedure described above was repeated.

Arabinose isomerase activity assay. In order to quantitate the activity of the arabinose operon induced by the fusion proteins, arabinose isomerase (AraA) activity assays were performed using published methods (3, 33). The colorimetric assay measures the activity of the AraA gene at time zero and at several time points thereafter. The zero time point served as the reference, and activities were calculated from the linear part of the curve using the following equation: (2 × 10¹⁰) (optical density at 550 nm)/(minutes of incubation)(number of cells in assay).

Purification of His-tagged Int. E. coli BL21 (DE3 pLysS) cells (250 ml) carrying the Int gene cloned into the pET28a vector (Novagen) were grown in Luria broth supplemented with neomycin sulfate (100 µg/ml; Sigma) at 37°C to mid-log phase. Int expression was induced with 0.5 mM IPTG (isopropyl--D-thiogalactopyranoside; Bachem) for 3 to 4 h; during induction, cultures were shaken at room temperature. Cell pellets were resuspended in extraction buffer (50 mM TrisCl [pH 8], 0.8 M KCl, 10% sucrose) after one freeze-thaw cycle. Extracts were made by homogenization on ice in the presence of phenylmethylsulfonyl fluoride, pepstatin A, leupeptin, and soybean trypsin inhibitor (concentrations of protease inhibitors were as for the previous cell extracts). The supernatant was frozen at 80°C, and the pellet was reextracted in 400 µl of extraction buffer. The supernatants were combined and mixed with 200 to 300 µl of Talon metal-affinity resin (Clontech) which had been washed in binding buffer (50 mM Tris-Cl [pH 8], 300 mM KCl, 10% glycerol, 10 mM -mercaptoethanol). The beads were rotated with the protein extracts for 2 h at 4°C and then washed five times (30 min each) with 500 µl of extraction buffer. Two sequential elution steps were performed, each with 150 µl of elution buffer (50 mM Tris-Cl [pH 8], 600 mM KCl, 10% glycerol, 0.6 M imidazole or histidine, 10 mM -mercaptoethanol). The resin was stripped with elution buffer containing 1 M imidazole. Fractions showing the greatest activity were quantitated by Western blot analysis.

Purification of non-His-tagged Int. E. coli BL21 (DE3 pLysS) or HN1463 (IHFHU) cells (500 ml) carrying the Int gene cloned into a pT7 vector (41) or in a pLex 5B vector (5) were grown in Luria broth supplemented with ampicillin (100 µg/ml; Sigma) at 37°C to mid-log phase. Int expression was induced with 0.5 mM IPTG (Bachem), and cultures were shaken at room temperature for 4 h. Cell extracts were prepared as described above for His-tagged Int purification. Int was isolated from these extracts by mixing 200 µl of phosphocellulose equilibrated in buffer X (50 mM Tris-Cl [pH 8], 400 mM KCl, 1 mM EDTA, 10% glycerol, 10 mM -mercaptoethanol) with 800 µl of cell extract. After being rotated for 2 h at 4°C, the resin was pelleted gently, the supernatant containing unbound proteins was removed, and the resin was washed with 400 µl of buffer X for 30 min. Two sequential elution steps were performed with 100 µl of buffer X. The first elution step was performed with buffer containing 600 mM KCl, while the second elution step was performed with buffer containing 1 M KCl.

Gel mobility shift and in vitro recombination assays. Linear substrates containing the attL or attL tenP'1 (27) sequence were end labeled using T4 polynucleotide kinase (NEB) and [-³²P]ATP (NEN). All recombination and binding reactions were performed in 20-µl total volumes. Recombination reaction mixtures contained 1 nM labeled att site (179 bp long) and 4 nM unlabeled att site (496 bp long), while binding reaction mixtures generally contained 1 to 4 nM labeled att site, as specified in figure legends. Each reaction mixture contained 0.1 to 1 µg of sonicated salmon sperm DNA, 44 mM Tris-Cl (pH 8), 60 mM KCl, 0.05 mg of bovine serum albumin per ml, 11 mM Tris-borate (pH 8.9), 1 mM EDTA, 13.6% (vol/vol) glycerol, and 5 mM spermidine. Int and integration host factor (IHF) were present at 50 and 35 nM, respectively. Recombination reactions were stopped by the addition of 5 µl of 2% SDS-containing bromophenol blue and heated at 65°C for 5 min. These samples were loaded onto 5% polyacrylamide Tris-SDS gels and electrophoresed in Tris-Tricine-SDS buffer at a 100-mA constant current. For the bent-L pathway, reaction mixtures were incubated at 30°C for 90 min. For the straight-L pathway, reaction mixtures were incubated at room temperature for 90 min. Gel mobility shift reactions were assembled as described above and were layered without loading dye directly onto 5% native polyacrylamide-0.5× Tris-borate-EDTA gels (29:1 acrylamide-bisacrylamide). Gels were run at 165 V at 4°C. Band shift assays using the oligomeric substrate coding for the individual P'1 arm site were loaded onto 10% native polyacrylamide gels, and reactions were carried out as described above but without nonspecific DNA competitor. All gels were dried and analyzed with a Molecular Dynamics PhosphorImager.

Protein-protein cross-linking assays. Reaction mixtures for gel mobility shift assays were assembled as described above except that the concentration of the att site was 4 nM. After 0 to 30 min of incubation at room temperature, bismaleimidohexane (BMH; Pierce) was added to 0.3 mg/ml, disuccinimidyl glutarate (DSG; Pierce) was added to 0.25 mg/ml, or dithio-bis-maleimidoethane (DTME; Pierce) was added to 0.3 mg/ml. All cross-linkers were resuspended in dimethyl sulfoxide (Sigma). The reaction mixtures were incubated for an additional 10 to 30 min at room temperature (no difference in extents of cross-linking was seen over this time period). To quench the cross-linkers, dithiothreitol (DTT; Sigma) was added to a final concentration of 20 mM for BMH-containing reaction mixtures or Tris (pH 8) was added to a final concentration of 150 mM for DSG reactions. The DTME tether was cleaved by adding 20 mM DTT. Laemmli's sample buffer (Bio-Rad) was added to the samples, which were then boiled for 10 min (Int multimers are very stable and are dependent on cross-linker only when reaction mixtures are boiled for 10 min in the presence of a reducing agent [data not shown]). Cross-linking products were separated on a 4 to 12% Tris-glycine gel (Novex) at 125 V for approximately 2 h. Proteins were electroblotted to nitrocellulose (Bio-Rad) using a Novex blot module, and Int was detected with polyclonal Int antibody and horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Jackson Laboratories). SuperSignal or Super Signal West Dura substrates (Pierce) were used to develop the blot.

Assaying the effect of cross-linking on recombination and protein-DNA intermediates. Reaction mixtures were assembled as for the in vitro recombination assays described above. Cross-linker was added either when the reaction mixtures were assembled, after 45 min of incubation at room temperature, or as specified in the figure legends. Following a total incubation time of 90 min, reaction mixtures were loaded onto a 5% polyacrylamide-Tris-Tricine-SDS gel to assay for recombination or a 5% native polyacrylamide gel to assay the formation of protein-DNA complexes. Samples assayed for recombination received 5 µl of 2% SDS plus dye and were heated at 65°C prior to electrophoresis.

	RESULTS

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Fusion assay for localizing intermonomer contacts among Int molecules. The fusion assay, developed by Malcolm Casadaban (unpublished results), takes advantage of properties of the transcriptional activator AraC (Fig. 1). This activator must bind two sites, araI₁ and araI₂, upstream of the paraBAD promoter in order to activate transcription from this promoter (reviewed in reference 34). The AraC activator consists of two independent domains, a dimerization domain and a DNA binding domain, connected by a hinge. The DNA binding domain by itself is sufficient to bind the araI sites and weakly activate paraBAD in the absence of arabinose (23). However, efficient activation of the natural promoter occurs only if a dimer of an AraC_D contacts araI. This is accomplished in the wild-type AraC protein via protein-protein contacts mediated by the dimerization domain. The natural dimerization domain has been successfully replaced by heterologous multimerization domains such as leucine zippers (2) or by entire proteins which can dimerize (M. Casadaban, unpublished data).

View larger version (29K): [in this window] [in a new window]   FIG. 1.   Identification of Int regions involved in protein-protein contacts using the activation of paraBAD as an assay. The araC gene together with the upstream araC binding sites are deleted in the M8834 strain, and thus the repression loop cannot form. (A) Efficient transcription initiation of paraBAD requires the presence of an AraC dimer bound at the I1 and I2 sites. AraC protein consists of two domains linked by a flexible tether; one binds to DNA, and the other mediates protein-protein contacts between two AraC monomers. (B) The AraCD cannot bind to DNA stably in the absence of the multimerization domain, resulting in poor activation of the operon. (C) Fusion of heterologous multimerization domains to the AraCD results in efficient activation of the operon. (D) Fusion of proteins or protein fragments which mediate weak or inappropriate interactions between two monomers do not activate the operon.

View larger version (20K): [in this window] [in a new window]   FIG. 2.   (A) Domain organization of the bacteriophage  Int protein. The figure depicts the regions of the protein responsible for contacting the arm-type sites of the att recombination targets, for contacting the core-type sites which flank the points of strand exchange, or for catalytic activity (refer to Fig. 3A for the structure of the attL site). The numbers above the diagram refer to the amino acids in the protein. (B) Structures of eight fusion proteins between fragments of Int (rounded rectangles) and the AraCD (oval). The numbers above the proteins refer to the Int amino acids included in the fusion protein. The darker the Int portion, the darker the color on MacConkey agar plates of colonies carrying the fusion protein: the fusion represented by white hatching on a black background turned colonies bright red, the fusion represented by black hatching on a white background conferred a light pink color, while the fusions represented in white conferred no color. On the right is listed the activity of the AraA protein in units per cell (see Materials and Methods); the averages of triplicate measurements from one representative experiment are shown (all fusions were tested at least three independent times). In this assay, the activity of the wild-type AraC protein was 923 U. "none" refers to AraA activity in a strain which did not contain any part of the AraC protein. nd, not determined. (C) Western blot of selected strains with anti-Int polyclonal antibody. Lane 1 shows a purified Int protein control. Lane 2 shows an extract of the tester strain M8834, containing the araBAD operon and the araC deletion (see Materials and Methods), which was used to express all the fusion proteins. Lane 3 shows M8834 with the pKM19-C170 plasmid, which expresses only the AraCD on a multicopy plasmid. Lanes 4, 5, and 6 show extracts of M8834 expressing Int-AraCD fusion proteins containing the Int amino acids depicted above the figure.

An N-terminal six-His tag confers a cooperativity defect. Because we wanted to examine the properties of many Int mutant proteins and to perform pull-down assays, we investigated in detail the recombination and DNA binding activities of an N-terminal six-His-tagged Int protein. The N terminus of Int contacts the arm binding sites present outside the core region of three out of four att sites (32), and thus we were interested in how the His tag affects interactions of Int with DNA. Landy and colleagues have shown, using DNA footprinting, that the three arm binding sites on the attL site (Fig. 3A) are filled cooperatively and that Int forms a protein-DNA intermediate called an intasome consisting of IHF and several Int protomers (16). Comparison of the His-tagged wild-type Int protein with the non-His-tagged protein showed that the His tag itself decreased cooperative interactions of Int with the wild-type attL site in the presence of IHF (Fig. 3B). We tested the cooperativity of the two proteins in a more stringent assay. In bandshift assays with a DNA substrate comprising only the arm sites, wild-type Int formed three complexes, corresponding to the filling of each arm site (Fig. 3C, lane 1). This binding was cooperative. We compared the cooperativities of the two proteins using a mutant DNA substrate in which one arm site, P'1, was wild type and the two neighboring arm sites were defective for Int binding due to triple substitutions, known as ten mutations, within the arm site consensus sequence (the ten mutations were shown to prevent Int binding [28]). The wild-type Int protein made a very prominent complex corresponding to the filling of two arm sites rather than one, and even showed a small amount of a complex containing three filled arm sites (Fig. 3C, lane 2). In contrast, the His-tagged Int shifted at least 50% of the substrate, mostly to the position corresponding to a single Int protomer bound to the DNA substrate (Fig. 3C, lane 3). Thus, the His-tagged Int binds DNA but is very defective in cooperative interactions. In order to more definitively separate the ability of the His-tagged Int to bind DNA from its ability to mediate cooperative interactions, we compared the extents of DNA binding of the wild-type and His-tagged Int proteins to a short DNA duplex containing a single arm binding site, P'1, and found that the two proteins shifted this DNA substrate to comparable extents (Fig. 3D, lane 4 versus lane 5). This binding was very sensitive to nonspecific DNA competitor in the cases of both proteins, and the reactions in panel D were performed without any competitor. We thus tested the specificity of Int interactions with the short probe by comparing the levels of binding of IHF and HU (the latter is a well-known nonspecific DNA binding protein) to the same fragment. While both these proteins clearly bound to the fragment, they did so to a lesser extent and formed different complexes than either of the two Int proteins (compare Fig. 3D, lanes 2 and 3 with lanes 4 and 5). We conclude that the His tag has little or no effect on DNA interactions but that it predominantly affects cooperative binding to the arm sites. This conclusion supports our genetic evidence that the N-terminal domain of the Int protein is involved in protein-protein interactions in addition to protein-DNA interactions.

View larger version (22K): [in this window] [in a new window]   FIG. 3.   Comparison of cooperative interactions and intermediate assembly by His-tagged and non-His-tagged wild-type Int. (A) Structure of the attL site. Triangles represent the low-affinity core-type binding sites for Int which flank the points of strand exchange. The diamond labeled H denotes the IHF binding site. The black arrows denote the higher-affinity arm-type Int binding sites. (B) Percentages of total counts found as intasome complexes at different Int concentrations. These complexes contain one to several Int monomers as well as a bending protein (IHF or HU). (C) Ability of His-tagged and non-His-tagged Int proteins to bind to the arm sites of attL. The attL fragments comprised coordinates +12 to +110 on attL, with either wild-type (wt) arm binding sites or a wild-type P'1 arm site and mutated P'2 and P'3 sites. Reaction mixtures contained 3 nM labeled DNA fragment, 100 ng of salmon sperm DNA, and 120 nM Int. Bands representing arm sites occupied by one, two, or three Int monomers are depicted in the cartoons shown to the left (the cartoons refer strictly to the stoichiometry rather than the actual position occupied by Int monomers). (D) Ability of His-tagged and non-His-tagged Int proteins to bind to an individual arm site, the P'1 sequence (coordinates +54 to +63 of attL plus five flanking residues). All reaction mixtures contained 1 nM labeled DNA (nonspecific DNA was omitted). Lanes 2 and 3 show control reaction mixtures containing 37 nM IHF or HU, respectively. Lanes 4 and 5 show reaction mixtures containing a 7.5 nM concentration of the specified Int protein. (E) Formation of intasome and BMC complexes by wild-type or His-tagged Int. All reaction mixtures contained 1 nM labeled full-length attL (coordinates 69 to +110) and 1 µg of salmon sperm DNA. Lane 1 contains labeled attL and IHF only. Lanes 2 to 4 contain non-His-tagged wild-type Int. Lanes 5 to 7 contain His-tagged Int in the absence or presence of the specified bending protein. Reaction mixtures contained, when specified, IHF or HU at 37 nM and either of the Int proteins at 100 nM.

Determination of Int's multimeric state during recombination. We directly investigated the multimeric state of Int using several homobifunctional cross-linking reagents which react either with primary amines or with cysteines. The primary amine-reactive compound which gave the greatest extent of cross-linking was DSG, a nonreversible cross-linking agent with a 7.7-Å tether between reactive groups. The most effective sulfhydryl-reactive compound was BMH, also a nonreversible agent but with a 16.1-Å tether between reactive groups. We have obtained very similar results by oxidizing Int with glutathione (data not shown).

View larger version (60K): [in this window] [in a new window]   FIG. 4.   BMH and DSG cross-linking of wild-type Int and IntC25S. (A) Effect of specific attL DNA on multimerization as assayed by BMH cross-linking. Reaction mixtures were treated with 0.3 mg of BMH per ml for the number of minutes indicated and were then quenched with excess -mercaptoethanol. Lanes 1 to 4 show reaction mixtures containing wild-type (wt) Int (see Materials and Methods). Lanes 5 to 8 show reaction mixtures containing IntC25S. At least one of the "trimer" species is in fact a dimer with slower mobility (refer to the text). ns DNA, nonspecific DNA (i.e., salmon sperm DNA). (B) Multimers formed in different recombination pathways as assayed by DSG cross-linking. Each reaction mixture was treated with 0.5 mg of DSG per ml for 10 min. Cross-linking was stopped by addition of excess Tris, pH 8. Lanes 1 to 6 show reaction mixtures containing wild-type Int, while lanes 7 to 12 show reaction mixtures containing IntC25S.

The N-terminal domain is involved in multimerization. In order to test directly the involvement of the N-terminal domain in multimerization, we compared the cross-linking patterns of wild-type Int with those of a mutant protein, IntC25S. The IntC25S mutation, constructed in the Landy lab, was shown not to affect recombination via the integrative or excisive pathways (41). When the IntC25S protein was cross-linked with DSG, it yielded the same multimerization pattern as the wild-type protein (Fig. 4B). However, when the protein was cross-linked with BMH, the predominant cross-linked species migrated as a trimer (Fig. 4A). Although it migrates more slowly than expected, this trimer is in fact a dimer, since sulfhydryl-reactive cross-linkers tether this species in the cases of two Int proteins which contain a single cysteine each (IntC25S-C197A-C217A and IntC25S-C217A-C262A) (L. Jessop, J. Boldt, and A. Segall, unpublished data). Because IntC25S forms the same multimers as wild-type Int but BMH fails to tether some of them, the cysteine at position 25 must be at or near the interface involved in the formation of tetramers. These results provide further evidence that the N-terminal domain of Int is involved in protein-protein interactions.

Effect of cross-linking on Int activity. How does tethering Int monomers to each other affect the assembly of recombination intermediates and recombination? Bent-L recombination reaction mixtures containing Int or IntC25S were assembled, and cross-linking reagents were added. The extent of recombination was determined by PAGE. Similar amounts of recombination products were obtained after 90 min from reaction mixtures with either wild-type Int or IntC25S (Fig. 5B, lanes 2 and 7; quantitation data are shown in Fig. 5C). When BMH was added to reaction mixtures containing either wild-type Int or IntC25S, recombination was inhibited significantly whether cross-linkers were quenched after 15 min (Fig. 5B, compare lane 2 with lane 3 and lane 7 with lane 8) or were active throughout the 90-min incubation (Fig. 5B, lanes 4 and 9). Experiments carried out with DSG gave similar results (data not shown).

View larger version (56K): [in this window] [in a new window]   FIG. 5.   Effect of cross-linking on bent-L recombination and formation of pathway intermediates. Reaction mixtures in lanes 2 to 6 contain wild-type Int, while reaction mixtures in lanes 7 to 11 contain IntC25S. Cross-linkers in DMSO were added at time zero to the reaction mixtures in lanes 3 to 6 and 8 to 11. After the specified time, DTT was added to a final concentration of 20 mM to quench the cross-linkers. All reaction mixtures were incubated a total of 90 min at room temperature. (A) Separation of recombination intermediates on a native polyacrylamide gel in the presence or absence of cross-linking reagents. (B) Conversion of substrate DNA to recombinant products on a Tris-Tricine-SDS gel (see Materials and Methods). (C) Quantitation of the bands of the gel shown in panel B; percent recombination values were normalized such that the amount of recombination products (Rec Pdts) seen in the absence of cross-linker was 100%. Solid black bars represent reaction mixtures in lanes 2 and 7 (DMSO only); vertically striped bars represent reaction mixtures in lanes 3 and 8 (15-min BMH treatment); horizontally striped bars represent reaction mixtures in lanes 4 and 9 (90-min BMH treatment); open bars represent reaction mixtures in lanes 5 and 10 (15-min DTME treatment followed by breakage of the tether); and stippled bars represent reaction mixtures in lanes 6 and 11 (90-min DTME treatment followed by breakage of the tether). The actual values of recombination were as follows: lane 1, 0.4%; lane 2, 21.2%; lane 3, 7.3%; lane 4, 7.4%; lane 5, 9.2%; lane 6, 3.0%; lane 7, 15.7%; lane 8, 7.4%; lane 9, 4.8%; lane 10, 7.0%; and lane 11, 3.9%. This experiment is representative of 10 repetitions.

	DISCUSSION

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We have described a series of experiments that begin to localize the regions of Int involved in protein-protein contacts and directly address the nature of the multimers assembled by Int during recombination. Taken together, our results from a genetic protein fusion assay, from biochemical assays of an Int protein modified at the N terminus, and from physical cross-linking assays show that the amino terminus of Int is involved in protein-protein interactions between Int monomers in addition to mediating protein-DNA interactions.

The genetic protein fusion assay identified amino acids 80 through 169 as the minimal region of Int (among the Int segments tested) that confers detectable protein-protein contacts. However, addition of the N-terminal 80 amino acids (Int fragment 1 to 169) improves the activity of the fusion protein, and comparison of the activities of the Int_1-262::AraC_D and Int_80-262::AraC_D fusion proteins confirms that the N terminus is involved in protein-protein contacts. Although we have not yet identified mutations in the N-terminal region of Int that affect multimerization, we have found that addition of a six-His tag (total of 20 amino acids) or even three new amino acids (Gly, Ser, and His) at the N terminus markedly impairs cooperative interactions between Int monomers without significantly affecting interactions between Int and DNA. Amino acid residues 169 to 262 also contribute to multimerization properties (compare the activities of fusions Int_1-169::AraC_D and Int_1-262::AraC_D). Within this segment of Int, we have identified one mutation, T236I, that decreases the activity of the Int_1-262::AraC_D protein to background level and decreases cooperative interactions between Int monomers in vitro. This mutation was isolated in a screen for recombination-defective Int proteins, although it remains catalytically active (11, 38). In contrast, a mutation that affects the active site of Int, R212Q, has no effect on the activity of the Int_1-262::AraC_D protein. These data suggest that protein-protein contacts are spread over at least two-thirds of the Int polypeptide.

Although the amino-terminal domain appears to mediate multimerization properties in vivo and in vitro, it is not essential for multimerization of Int in vitro. The His-tagged protein, although it has lost significant DNA binding cooperativity, can still multimerize as assayed by either DSG or BMH cross-linking (data not shown). It is not surprising that the N-terminal intermonomer contacts can be dispensable, since the fusion assay shows that amino acid residues throughout a large fraction of Int contribute to protein-protein contacts. Differences between the in vivo and in vitro effects of the N terminus on multimerization may be due to less stable folding on the part of the fusion proteins, lower concentration of the protein in vivo, or differences in the ionic environment in vivo versus in vitro. We are currently looking for mutations in the N terminus that affect multimerization. Crystallographic analysis of Int-related proteins shows that large protein-protein interfaces also exist in the catalytic domain (9, 13). Because the catalytic domains of the tyrosine recombinases are the most highly conserved (6, 29), these strong protein-protein interactions probably exist in Int as well (Fig. 2B and L. Jessop, J. Boldt, and A. Segall, unpublished data). (Fig. 2B).

Strong cooperativity at the arm sites is correlated with Int's ability to mediate strong contacts with the core sites and to assemble high levels of synaptic complexes (Fig. 3); both of these properties are necessary to carry out efficient recombination. Although the His-tagged protein can bind to arm sites, this binding is much less cooperative than that of the non-His-tagged protein and results in assembly of fewer Int-DNA complexes. In turn, cooperative interactions between the N termini of Int monomers bound at the arm sites of attL stabilize interactions of the catalytic domain with the core sites, near the loci of strand exchange. When these cooperative interactions are weakened, Int's recombination activity is compromised.

Cross-linking assays with the wild-type Int and IntC25S mutant proteins showed that Int assembles predominantly dimers, "trimers," and tetramers during the recombination process (Fig. 4). At least one (and perhaps all) of the apparent trimers is in fact a dimer with slower mobility, based on the fact that proteins lacking all but Cys 197 (or all but Cys 262) can form this species (L. Jessop, J. Boldt, and A. Segall, unpublished data). Although multimerization is not triggered by DNA, att sites in particular and nonspecific DNA to a lesser extent do increase the extent of multimerization. The fact that the C25S mutation changes the observed cross-linking patternspecifically, only one type of dimer and few if any tetramers are trapped with BMHshows that this residue is found at an interface required to form a subset of Int dimers and that the amino terminus is involved in protein-protein interactions.

The phenotype of cross-linking the wild-type Int at C25 is in some ways similar to that caused by modification of the protein by NEM, which has been reported to occur exclusively at this N-terminal cysteine (41). Indeed, BMH and DTME treatments of wild-type Int strongly inhibit interactions between the arm binding domain of Int and the DNA, and the IntC25S protein becomes immune to the adverse effects of cross-linkers on arm binding (data not shown). Nevertheless, IntC25S is inactivated for recombination by either BMH or DTME; this loss of recombination activity must result from modification of one or more of the remaining three cysteines (C197, C217, and C262). We are currently investigating where this modification occurs and whether it depresses protein-protein and/or protein-DNA interactions.

Our results indicate that cross-linking IntC25S blocks recombination by preventing the assembly of UMC2 (Fig. 5). The UMC2 complex is formed by addition of an unstably bound Int monomer to the UMC1 intermediate (35), and we hypothesize that this Int is held in place by low-affinity protein-DNA interactions at the C' core site and by protein-protein interactions. It is possible that modification of one or more of the three cysteines in the catalytic domain interferes with protein-protein interactions important for loading this incoming Int. While these interactions may be dispensable for cooperative binding to the arm sites, they might be essential for formation of UMC2 and for recombination. Alternatively, cross-linking may immobilize multimers, preventing rearrangements important for assembly of UMC2. A similar situation, in which tethering protein multimers resulted in destabilized DNA binding, was found in the case of the Hin recombinase (22). We disfavor this interpretation, however, because cleavage of the tether in DTME does not restore UMC2 formation.

Multimerization is an integral part of the mechanism of site-specific recombination (8, 12, 14, 21 [and references therein], 22, 25, 42). The coordination of catalytic events may involve communication between different protomers in the synaptic complex. This appears to be the case for the Cre protein, where the two monomers that have carried out cleavage occupy a similar position with respect to the complex but differ from the two monomers which have not carried out catalysis (9). Catalysis by individual Int protomers is less concerted in bent-L recombination than in excisive or integrative recombination (G. Cassell and A. Segall, unpublished data; 17). This may be due to subtle differences among the pathways in protein-DNA contacts, protein-protein contacts, or both. We are investigating these differences by fine-mapping the interfaces between Int protomers in different pathways of  recombination. We hope that understanding differences in the conformations of intermediate complexes among the four pathways and the relationship between the geometry of intermediates and catalytic events will lead to an understanding of the molecular events that trigger catalysis. In turn, this will lead us to a more complete understanding of how the efficiency of site-specific recombination is achieved.