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ti Szh el Forum Forum rsearchn Forum s Terms a Terms tsearch a Terms d attB together to form the synapse and no other combination of sites forms a stable synapse under these conditions (27). These observations have led to the proposal that integrase adopts specific conformations when bound to attP or attB that permit formation of the protein:protein interface required for stable synapsis (27,29). Here we showed that mutations in attB can significantly affect the ability of integrase to form a stable synapse or to cleave the substrates. These perturbations in the reaction are likely to be due to the absence of important interactions between integrase and attB and could be indicative of ¡®non-permissive¡¯ conformations of integrase that block recombination at these different stages.
The mutations at ¨C/+2 in attB showed a failure to cleave the DNA but these substrates could still form a stable synapse (Figures 4¨C6). These data show clearly that there is a post-synaptic activation step required for recombination by C31 integrase. This activation step depends on an interaction that has been disrupted in the attB ¨C/+2 mutants, C-2G:G+2C or C-2A:G+2T. The nature of the interaction is not known but could be a specific base-pair contact or a DNA conformation that is recognized. The block in DNA cleavage occurred in both the mutant attB sites themselves and in the wild-type attP sites (Figures 4¨C6 and Figure S2). This behaviour is consistent with concerted DNA cleavage in the reaction with wild-type recombination sites. Possibly the block in cleavage in reactions containing the ¨C/+2 mutant sites could be due to failure to undergo a conformational change in the whole synaptic complex which would normally lead to cleavage. Alternatively the DNA conformation of the mutant sites prevents the catalytic sites gaining access to the scissile phosphate. As reversion of just one of the bases from the double mutant back to the wild type was sufficient to regain most of the attB activity it would seem that activation only requires a ¡®correct¡¯ interaction at one half-site of attB.
The mutants T-15C:C+15G and G-16T:G+16A were able to recombine but at a slow rate compared to wild-type attB (Figure 5). There was a consistent reduction in the amount of synapse observed during recombination with these mutants suggesting that the synaptic complex was unstable (Figures 4 and 5). Raising the concentration of NaCl partially suppressed the defect in recombination with the ¨C/+15 and ¨C/+16 mutants but it is not clear which step was affected by NaCl (Figure 6). The stability of the synapse did not increase in the presence of a higher concentration of NaCl, if anything the binding affinity and the level of synapse was reduced at 1 M NaCl (Figure 6). Despite this, suppression was still observed suggesting that high NaCl activates or stabilizes an event later in the recombination pathway. The single point mutants at ¨C15 and ¨C16 were sufficient to severely affect recombination while mutations at +15 or +16 had a lesser effect (Table 1, Figures 2, 4¨C6). Thus the single mutations at positions ¨C15 and ¨C16 accounted for most of the defect in the ¨C/+15 and ¨C/+16 double mutants. These data argue that there could be a specific interaction between the B arm and integrase that contributes significantly to the activity of the attB site. The partial symmetrization of the attB sites (with either the sequence from the B arm ; Figure 2, panel H) showed that the B arm was indeed more active than the B' arm. However, it is known from previous work that the attB and attP sites act with integrase in a functionally symmetrical manner as integrase does not control the relative orientation of the sites when they come together at synapsis (28). Thus the interactions by each subunit of integrase bound to each arm of attB are not independent of each other and we propose that a specific integrase conformation that results from the ¨C15, ¨C16 interactions in the B arm is communicated through both subunits.
These conclusions can be combined with information from other large serine recombinases and the resolvases to generate a model that focuses on substrate recognition and formation of the synapse by integrase (adapted from that published previously for Bxb1 integrase, 26; Figure 7). In the resolvases, the DNA is contacted in the minor groove in the centre of each binding site and through specific contacts in the major groove towards the outer flank of the site via the C-terminal DNA binding domain (24,25). The geometry of DNA-binding is such that the C-terminal domain of resolvase extends around the DNA and contacts on the opposite side of the DNA to the catalytic serine (25). As in Bxb1 and TnpX, C31 integrase has a proteolytically sensitive site between the N and C terminal domains (K152, unpublished data)(26,30,46). Moreover, the C-terminal domains of Bxb1 and TnpX have been shown previously to be capable of binding specifically to DNA (26,30,46). Thus we propose that the C-terminal domains interact with the outer flanks of the att sites, that these interactions determine the conformations of integrase bound to each site and therefore whether they are compatible for synapsis. In attB this information is ¡®read¡¯ at least in part from ¨C15 and ¨C16 where disruption of this interaction disables the ability of integrase to form a stable synapse (Figures 4¨C6). The model predicts that there is communication between the putative DNA-binding motifs in the C-terminal domain and the regions of integrase that generate the protein-protein interface for synapsis. We currently envisage this communication as an allosteric switch mediated by conformational changes. In resolvase, the synaptic interface is located at the DNA distal surface of the catalytic domain and it is likely that the serine integrases use the equivalent of this interface for synapsis, although it is possible that the C-terminal domain may also have a role in synapsis. After synapsis an activation step is required for DNA cleavage and in attB this depends on the base pairs at position ¨C/+2. In attB only one of the ¨C/+2 bases needs to be wild type for activity and this can be either on the B or B' arm. Given the proximity of ¨C/+2 to the scissile phosphate, position 2 is more likely to interact with the catalytic domain than with the C-terminal domain. As in resolvase there may be significant conformation changes that occur with activation of recombination (34,37).