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A major focus in our lab has been to understand why C31 integrase can only recombine attB and attP in vitro. We have shown previously that integrase cannot synapse pairs of recombination sites other than attP with attB indicating that the formation of the synapse is the major block to excision in vitro (27). We and others have proposed that integrase adopts specific conformations when bound to attP and attB sites that enable the formation of a synapse, but when bound to attL and attR disable or destabilize the synapse (26,27,29). In this model, the interactions between integrase and attP and attB are central to the formation of the synaptic interface. Some clues as to the preferred sequences of attP and attB have been obtained previously through studies that have characterized the substrates used by integrase when one of the cognate sites is not present (9,41,42). Pseudo-attB sites in the bacterial host, Streptomyces coelicolor and other actinomycetes show a strong preference for certain bases . Similarly, pseudo-attP sites have been characterized in mammalian genomes and these also show base specific preferences (Figure 1). Many of the bases that are conserved in the pseudo-attP and pseudo-attB sites are also conserved between attP and attB (Figure 1).
To examine the integrase¨CattB interaction in more detail, the minimal attB site was subjected to mutagenesis and the activities of the mutants assayed in recombination and binding assays. Recombination defective attB mutants that could still bind to integrase with affinities not dissimilar to the wild-type attB site were found to be blocked either at synapsis or at DNA cleavage. The most likely explanation is that there are two separate recognition events that occur between integrase and the attB site. The first event results in a protein¨Cprotein interface that enables synapsis and the second post-synapsis event results in activation of DNA cleavage.
MATERIALS AND METHODS
Bacterial strains and plasmids
Escherichia coli strains DH5 and DS941 were used as general cloning hosts and were grown in LB or 2xYT (44). E. coli transformation, plasmid preparations and DNA manipulation were performed as described previously (44).
Plasmids pRT600 and pRT700 were constructed previously by insertion of annealed oligonucleotides RM1/RM2 containing attB (51 bp) and RM3/RM4 attP (50 bp) sites inserted into pGEM7 cut with EcoRI and Csp45I (29). For this work, the attP site from pRT700 was excised with BamHI and EcoRI and inserted into BamHI and EcoRI cut pSP72 to form pRT702. Plasmids containing mutant attB sites at all positions except for ¨C/+3, ¨C/+8 and ¨C/+12 were constructed as for pRT600; annealed oligonucleotides (see Supplementary Data, Table S1) were inserted into pGEM7 cut with EcoRI and Csp45I. Plasmids containing mutations at ¨C/+3, ¨C/+8 and ¨C/+12 were constructed differently; PCR amplification using primers containing a randomized base at positions 3, 8 or 12 (Supplementary Data, Table S2) resulted in fragments that could be inserted into pGEM7 and these were then sequenced to determine the nature of the mutations. To create the double mutants with mutations at symmetrical positions, fragments containing the two single mutations were spliced together using the unique StyI site in the centre of the attB site. All the plasmids containing the mutant attB sites were subjected to confirmation by sequencing.
Recombination assays
Standard recombination assays between two attachment sites located on two separate plasmids were performed as described previously. Plasmids (100 ng each) containing attB (or the mutant attBs) and attP were mixed with 18 µl of recombination buffer (10 mM Tris pH 7.5, 1 mM EDTA pH 8, 100 mM NaCl, 5 mM DTT, 5 mM spermidine, 4.5% glycerol and 0.5 mg/ml bovine serum albumin) and C31 integrase was added to the recombination reaction to final concentrations 0, 441, 110, 55, 27 or 14 nM unless otherwise stated. Reactions were incubated at 30¡ãC for 1 h unless otherwise stated and terminated by incubation at 65¡ãC for 10 min. After addition of an equal volume of 2x restriction buffer (20 mM Tris-HCl pH 7.9, 100 mM NaCl, 20 mM MgCl2, 2 mM DTT) the plasmids were treated with HindIII restriction endonuclease (37¡ãC for 2 h) and the fragments were separated by electrophoresis through 0.8% agarose gels in 1x TBE buffer (100 V). HindIII linearizes the substrates containing attB (or mutant attBs) and attP to give DNA molecules of 3035 and 2491 bp, respectively. The recombination product is a cointegrate of the two substrate plasmids and is cut by HindIII into two fragments; 5435 bp containing attL and 91 bp containing attR. Only the attL fragment is detected routinely after electrophoresis.
Recombination reactions were also performed using a plasmid encoding the attP site, pRT702, and annealed oligonucleotides containing the attB sequence or its mutant derivatives (the ¡®oligo-plasmid¡¯ assay; see Table S2 in the Supplementary Data for the sequences of the oligos) (17). pRT702 (100 ng) was mixed with 4.5 ng of annealed oligonucleotides encoding a mutant attB site and 18 µl of recombination buffer. C31 integrase (1 µl) was added to give final concentrations as described above (i.e. 0, 441, 110, 55, 27 and 14 nM) and the reactions were incubated at 30¡ãC for 1 h. The recombination reactions were terminated by heat inactivating the samples at 65¡ãC for 10 min and the products of recombination were analysed on 0.8% agarose gels in 1x TBE buffer (100 V). The products of recombination were identified as linear DNAs (2546 bp).
DNA binding and radioactive recombination assays
DNA affinity and synapse assays were performed as described previously (29). DNA fragments for radioactive labelling were prepared by digestion of pRT600 (encoding wt attB), pRT700 (encoding attP), or plasmids containing cloned annealed oligonucleotide pairs encoding mutant attB sites with HindIII and XhoI restriction enzymes. The 72 bp fragments containing the att sites were separated on 4% agarose gel (Nusieve agarose) and then purified using gel extraction columns (QIAGEN) as per the manufacturer's protocol. The concentration of the purified fragment was determined on 4% agarose gels following which the fragments were end-labelled using DNA polymerase I large (Klenow) fragment in the presence of dCTP (as described previously in Sambrook et al. (44)). Unless otherwise stated, binding affinity assays were performed with 1.0 ng labelled probe in binding buffer (20 mM Tris-HCl pH 8.0, 0.1 mM EDTA, 50 mM KCl, 5% glycerol), 1 µg sonicated salmon sperm DNA and integrase added to final concentrations of 0, 351, 87, 43, 21 and 10 nM. Reactions with no integrase contained 1 µg BSA. Reactions were incubated at 30¡ãC for 30 min prior to electrophoresis following which the reaction mix were separated on 5% non denaturing 0.5x TBE polyacrylamide gels in 0.5x TBE running buffer (200 V, 5 W for 2 h).
For radioactive recombination assays, unlabelled ¡®partner¡¯ fragments prepared by PCR amplification of pRT600 and pRT700 with SP6 and T7 primers containing either attP (193 bp) or wt or mutant attBs (194 bp) were added to the radiolabelled attachment site in the presence of integrase. Complexes containing either the uncleaved synapse, the cleaved intermediates with integrase bound covalently to the att sites, and integrase bound to the labelled substrate and products were observed by non-denaturing PAGE as described previously (27). These assays were performed using 1.5 ng of labelled probe (72 bp), 20 ng of the unlabelled fragment containing a ¡®partner¡¯ attachment site and 66 nM integrase in binding buffer. Unless otherwise stated, all reactions were incubated for a period of 2 h at 30¡ãC prior to electrophoresis on 5% non-denaturing polyacrylamide gels (200V, 5W for 2 h).
To detect the cleavage of the DNA fragments by integrase, reactions were set up as for the radioactive recombination assays but after incubation at 30¡ãC, reactions were heat inactivated (72¡ãC for 10 min) then incubated with 1µl of subtilisin A (Sigma 0.1 mg/ml in 1x binding buffer) for 15 min at 30¡ãC. Subtilisin was inactivated (72¡ãC for 10 min) and the reactions were loaded onto a 0.5x TBE, 5% non-denaturing polyacrylamide gel.
After electrophoresis gels were dried and exposed to a phosphorimager screen (Fuji) for 16 h and then scanned (Fuji FLA3200 phosphorimager). Quantification of radioactivity was performed using the AIDA software (Raytest, Straubenhardt, Germany).
Purification of integrase
Wild-type C31 and S12A integrase were purified as described previously (27). Integrase concentration was assayed using a method based on the dye-binding procedure of Bradford (45) employing the BioRad protein assay solution, and bovine serum albumin as a standard.
RESULTS
Identification of defective mutations in attB
The minimal attB site, according to Groth et al., (10) is 34 bp with the crossover 5'TT (abbreviated to XO) at the centre (Figure 1). Footprinting confirmed that integrase binds either side of the crossover site in all the attachment sites and, as integrase is a dimer in solution it probably binds as a dimer (29). Moreover, we have shown that integrase binds to attB and attP in a functionally symmetrical manner. Thus in order to maximize any phenotype arising from mutations in attB we generated a set of doubly mutated sites with base pair changes at symmetrical positions with respect to the crossover sequence. To aid in the description of the positions of mutations, the base pairs in the minimal attB and attP sites were annotated with either a negative number when they lie to the left of the crossover dinucleotide sequence (5'TT) i.e. B or P arm to use the terminology) or positive when it lies to the right of the crossover (B' or P' arm); the numbers count upwards as the position extends away from the crossover (Figure 1). Thus mutations in a double mutant involving the two base pairs adjacent to the crossover is at ¨C/+1 and mutations at the next position moving outwards are at ¨C/+2, etc. Mutations were chosen that would introduce sequence symmetry at the desired position. Thus each double mutant was designed to contain one of the four bases, A, T, C or G, at position ¨Cx on the B arm and at +x on the B' arm its complement, T, A, G or C, respectively, was inserted. The choice of mutation was made on the basis that the introduced bases had to be different from those present in both arms of attB and preferably also different to those seen in the pseudo-attB sites (Figure 1). For example, position 15 is a T in the B arm and a C in the B' arm and the pseudo-attB sites have a G in the B arm and a C or A in the B' arm. T-15C:C+15G and the T-15A:C+15T contain changes at ¨C/+15 on the B and the B' arm to base pairs that are different from both the wt and the pseudo-site sequences and should be functionally the same mutation in both arms. For most positions at least two mutant forms were made but for some sites (positions 4, 7, 16) only one option was available. Other positions where only a single mutant form was made are at 14, 17 and 18.