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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.
Except for mutations at positions ¨C/+3, ¨C/+8 and ¨C/+12 the activities of the double substituted attB sites was first assayed using annealed oligonucleotides. Oligonucleotides containing the double substitutions were purified by PAGE, annealed and used in an oligo-plasmid recombination assay (17). In this assay, a supercoiled plasmid containing attP was mixed with the oligonucleotide containing attB or one of the mutant forms and various concentrations of integrase. The extent of linearization of the attP plasmid indicated the extent of recombination and this was assayed after separation of the DNA in an agarose gel. A control reaction using the wild-type attB site was performed in every assay so that the activities could be compared under identical conditions. The lowest integrase concentration at which recombination could be observed was scored (Figure S1 and Table 1).
Many of the mutant attB sites showed little or only 2-fold change in activity compared to the wild-type site. These sites were changed at ¨C/+1, ¨C/+4, ¨C/+5, ¨C/+7, ¨C/+10, ¨C/+11 and ¨C/+13 (Table 1, Figure 1). The remaining mutants showed defective or partially defective activity ranging from 4-fold less active than wild type to apparently inactive. Oligos encoding sites C-2G:G+2C, C-2A:G+2T, G-6A:C+6T, (G-6T:C+6A, G-9T:C+9A, G-9A:C-9T, G-9C:C-9G, T-15C:C+15G, G-16T:G+15A and G-18C:A+18G were cloned into pGEM7 (Promega) so that the activities of the mutant attB sites could be verified by a standard recombination assay using both att sites residing on plasmids. Only one of the mutant attB sites that was partially defective (at position ¨C/+14) was not represented in the cloned mutant attB site collection; this site was instead subjected to single site substitutions (see later). T-15A:C+15T was not cloned as a plasmid containing another mutant at ¨C/+15 (T-15C:C+15G) with the same activity was quickly obtained. A plasmid encoding G-6C:C+6G was not obtained due to technical difficulties. Plasmids containing mutations in C-3T:G+3A, C-3G:G+3C, C-3A:G+3T, G-8T:C+8A, G-8C:G+8C, C-12A:G+12T and C-12T:G+12A were obtained by PCR mutagenesis as described in the Material and Methods section. The relative activities of the double substitution mutants were estimated compared to a standard reaction with wild-type attB (Table 1 and Figures 1 and 2). As for the oligo-plasmid assay the activity of each mutant site was scored as the concentration of integrase required to observe recombinants in an agarose gel stained with ethidium bromide (Table 1). The relative activities compared to the wild-type site are summarized graphically (Figure 1).