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Thingy Brunette Cuntbrunette F Bikini 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

Thingy Brunette Cuntbrunette F Bikini Szh 1 Cunt Brunette

Blonde%20youngsters%20engage%20in%20pussy%20e...i Brunette u Thingy eAV%CE%DE%C2%EB%B5%FA%C6%AC5 Thingy searchhsearch searcha Brunette e Bikini o Bikini 4e Thingy o Brunette b Cuntbrunette n Bikini t Bikini o Cuntbrunette Szh i Cuntbrunette h Szh mtsearchn Bikini Thingy tt Bikini searchi Brunette e Brunette Brunette -1 Bikini CC Cuntbrunette 1 Thingy G Bikini a Brunette dsearchGa1 Szh T Szh Gcaopom16search i retsearchy1re Cuntbrunette usearche Bikini . Bikini P Thingy nsearchl Thingy A Cuntbrunette Szh h Thingy wsearch Cuntbrunette h Bikini searchp Thingy esearchr Thingy n Szh esearchosearch r Cuntbrunette d Brunette ctsearch zooporntubevideor Szh msearchre Bikini osearchb Cuntbrunette nasearchi Szh na1ssearchy Brunette s Cuntbrunette n Bikini search-1C: Thingy +search5 Thingy ndsearchG Szh 1 Thingy T Cuntbrunette G Szh 1searchAsearchasearch searchu Szh ssearchr Bikini te Brunette Thingy ft Bikini r Cuntbrunette prlo Bikini g Thingy d Szh i Szh cusearchasearchi Thingy nDirty+mature+bitch+busted+by+spy...+ Szh lsearchs Szh i Bikini s Brunette ec Cuntbrunette dng the Cuntbrunette wiAsian+bitch+gets+pleasure+from+vibr...+dAsian%20bitch%20gets%20pleasure%20from%20vibr...tsearchp Szh Szh tt Asian%2Bbitch%2Bgets%2Bpleasure%2Bfrom%2Bvibr...wt Thingy or1t Cuntbrunette e indicated attB mutants were incubated with pRT702 (attP) for 1, 2 or 3 h at 30¡ãC and then the products analysed by restriction and agarose gel electrophoresis. After 2 and 3 h some product (attL) is visible in the lanes containing the ¨C/+15 and ¨C/+16 mutations. Panel (B) shows the time-dependent appearance of recombination intermediates when wild-type attB (wt) was used compared to C-2A:G+2T, T-15C:C+15G or G-16T:G+16A. The ¨C/+2 mutant site rapidly forms a synapse (Int:synapse attP/B) and thereafter the reaction is blocked. The ¨C/+15 and ¨C/+16 attB sites slowly accumulated the cleaved intermediate (Int:cleaved attP/B) and some shifted product complexes (Int:attL/R). The remaining complexes on the gel are as described in Figure 4.

We reasoned that altered recombination conditions might partially suppress the defect in T-15C:C+15G and G-16T:G+16A by stabilizing the putative protein¨Cprotein interface. Recombination was observed when the NaCl concentration was increased to 500 mM or 1 M in recombination buffer (Figure 6A). However, increasing the concentration of NaCl did not increase the amount of synaptic complex observed with these mutant attB sites (Figure 6B). Indeed at 1 M NaCl there was a reduction in the level of synapse observed with the mutants at ¨C/+15 and ¨C/+16 and a slight reduction in the affinity for the attB site by integrase (Figure 6B and C).

Figure 6. High NaCl concentrations can partially suppress the recombination defective phenoytpe of the mutations. Panel (A) shows the appearance of products from recombination assays using T-15C:C+15G, G-16T:G+16A, G-16T and T-15C as substrates after incubation in either 500 mM or 1 M NaCl. Plasmids encoding the wild-type attB (wt), or the above mutants were incubated with pRT702 (attP) in recombination buffer adjusted to 100 mM, 500 mM or 1 M NaCl. After digesting with HindIII the DNA was separated in an agarose gel. The appearance of the 5435 bp fragment encoding attL is indicative of recombination. Panel (B) shows the synapse assays using the wild-type attB (wt), C-2A:G+2T, T-15C:C+15G and G-16T:G+16A under different NaCl conditions with wild type (left panel) or S12A integrase (right panel) with labelled attP. The complexes are annotated as described in Figure 4. ¨C/+15 and ¨C/+16 mutant attB sites accumulated both the cleaved complex (Int:cleaved attP/B), the shifted products (Int:attL/R) and released some free product (attL/R) with 500 mM and 1 M NaCl with the wild-type integrase. The synapse however as indicated using the S12A integrase did not become more abundant in high NaCl buffer, if anything it reduced. Panel (C) shows that the binding affinity of attB sites for integrase in the presence of different NaCl concentrations. Radiolabelled attB sites were incubated in binding buffer containing 50 mM, 500 mM or 1 M NaCl. Integrase was added at 66 nM. The shifted attB complexes are indicated by arrows. Only at 1 M NaCl, there was a slight increase in the free DNA for all four attB sites.

DISCUSSION

The interactions between C31 integrase and its attachment sites are critical in determining the directionality of recombination. In vitro integrase only recombines attB and attP to form the hybrid products, attL and attR. We have shown previously that, in vitro, integrase selectively brings attP and 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).

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.