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Cunt Brunette Cuntbrunette X Cunt Brunette 0 Competition Szh 1 Cunt Brunette Sequences in attB that affect the ability of C integrase to synapse and to activate DNA cleavage--《核酸研究医学期刊》--医学期刊频道--首席医学网

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Brunette h Competition u Competition a Cunt i Brunette n Brunette Brunette t Cunt & Brunette msearch; Cunt /search2searchisearch tB Cuntbrunette s Szh o Brunette edsearchafa%D1%C7%D6%DE%CE%E5%C2%EB%C7%F8l Cunt r90im.com%D0%C2%D6%B7 Cuntbrunette osearchc Cuntbrunette e Cunt v Szh t Brunette e Brunette DAsearchb Brunette tsearcht Competition esearche Szh sb Cunt t Szh aesc Brunette usearchd searchtilf Competition rmsearchasearchssearchasearchlsearch ssearchnsearchp Competition esearch( Brunette isearchu Competition e Cuntbrunette search& Cunt m Cuntbrunette ;6) Cunt he Brunette e Competition d Cunt t Cuntbrunette sh Brunette wsearchc Brunette e Szh r%B9%FA%B2%FA%B7%F2%C6%DE%C2%B6%C1%B3%D5%E6%CA%B5%D7%D4%C5%C4y searchhsearcht Szh the Cuntbrunette e isearch asearchpstsearchsHorny+Brunette+Enjoys+Fucking+With+n Competition p Brunette ic ct Cunt v Cuntbrunette t Competition o Cunt ssearche Cunt Szh eqi Cunt e Brunette Cunt or Brunette re Szh o Cunt bisearcha Competition iosearch b Competition C%B3%D5%BA%BA%BE%E3%C0%D6%B2%BF%B3%C9%C8%CB%B9%E2%B5%FA1 Szh i Brunette t Cunt gsearchas Szh .searchTis Brunette ac Brunette iva Competition ion Cuntbrunette tep Cunt deNice+College+Slut+Enyoys+Sucking+A+endsearch o Cuntbrunette ni Brunette t Cuntbrunette raction 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).

Figure 7. Model for the mechanism of integrase. The integrase subunits are shown with a small N-terminal (catalytic) domain through which the subunits may dimerize (26) and a large C-terminal domain that we propose recognizes the sequences in the outer flanks of the recombination sites. These recognition 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.

【参考文献】
 

Bateman JR, Lee AM, Wu CT. Site-specific transformation of Drosophila via C31 integrase-mediated cassette exchange. Genetics (2006) 173:769¨C777.

Andreas S, Schwenk F, Kuter-Luks B, Faust N, Kuhn R. Enhanced efficiency through nuclear localization signal fusion on phage C31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells. Nucleic Acids Res (2002) 30:2299¨C2306.
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