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t Szh e Cuntbrunette s Mobile qu Brunette nesearch nthe ousearche fsearcha Szh ks o Cuntbrunette Mobile h Brunette recaoliushenquomb%B6%ED%C2%DE%CB%B9%C9%D9%C5%AE%28HD%C4%DA%C9%E4%C6%C6%B4%A6%29+495MBnsearcht Szh on s Mobile t Brunette s Mobile Tsearches Cuntbrunette searchec Mobile gnition events, specifically ¨C15 and ¨C16 (annotated as blue bars) in attB, lead to an ¡®induced fit¡¯ or stabilization of a specific conformation of integrase that enables synapsis with integrase bound to attP. Different conformations of integrase bound to either attB or attP are shown as different colours. The synaptic interface via the N-terminal catalytic domains is indicated based on the resolvase precedent; there is no evidence to indicate that the C-terminal domains could also participate in a synaptic interface. Mutations at positions ¨C15 or ¨C16 (such as in T-15C:C+15G, G-16T:G+16A), T-15C or G-16T do not induce the conformation of integrase that can form a stable synapse with attP so the rate of reaction decreases (thin arrow). Mutations at ¨C/+2 in attB (red bars) are severely inhibited in cleavage but are capable of forming a stable synapse. These mutants indicate that the formation of the synaptic complex is followed by a well-defined activation step that results in concerted DNA cleavage. After strand exchange integrase is bound to the hybrid sites and adopts a conformation that cannot synapse attL and attR. The putative tetrameric complex rapidly dissociates to binary complexes containing integrase and either attL or attR. See text for more details.
The data presented here provides a source of information that could be used for the design of alternative attB sites for genome engineering using C31 integrase. Indeed we have already used this information to create a non-methylatable attB site for use in vertebrate cell lines (4). In this site, attBm, the CpG steps have been replaced with bases that we have shown here were neutral with respect to recombination activity. It is noteworthy that the positions in attB that are critical for recombination other than position 2 i.e. positions 15, 16 were not highlighted as being particularly preferred in the pseudo-attB sites (Figure 1). However all of the pseudo-attB sequences have either the wild-type C at ¨C2 or a wild-type G at +2 (Figure 1) (41,43). It is also noticeable that the regions where there are most identities between attB and attP are not particularly sensitive to mutation (Figure 1). A plausible explanation for both these observations is that the 24 bp of att site DNA from about position 11 to the crossover site is a core sequence that integrase binds to specifically. We propose that the role of positions 15 and 16 in attB revealed by this study is to greatly enhance the efficiency of the reaction and effectively discriminate between the pseudo-sites and the cognate attB site. It is envisaged that this data and a similar analysis with attP will enable an understanding of the optimal sequences to target for continued application of C31 integrase.
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We are grateful for comments on this manuscript from Paul Rowley and Paul Hoskisson. M.G was funded by a scholarship from the University of Aberdeen. This work was funded by the Biotechnology and Biological Sciences Research Council, UK. Funding to pay the open access publication charges for this article was provided by the Biotechnology and Biological Sciences Research Council, UK.
Conflict of interest statement. None declared.
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