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Cuntbrunette Top Wet Girls 7 El 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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The attB sites with mutations at ¨C/+15 and ¨C/+16 that were defective in the standard recombination assay did not appear to be defective in the radioactive assay to detect intermediates (Figure 4A, left panel). The amounts of cleaved covalent intermediate and shifted attL/attR products were indistinguishable from the reaction with the wild-type attB site (Figure 4A, left panel). The only observable difference was in the amount of synapse, which was reduced in the reactions with the most defective sites i.e. T-15C:C+15G, T-15G, G-16T:G+16A and G-16T and the appearance of some free product. These differences were also observed when the attB sites were labelled and incubated with integrase and cold attP (Supplementary Data¡ªFigure S2). When the S12A catalytically inactive integrase mutant was used, there was a small reduction in accumulation of the synaptic complex with T-15C:C+15G, T-15G, G-16T:G+16A and G-16T compared with wild-type attB (Figure 4A, right panel).

The inconsistency whereby mutants T-15C:C+15G and G-16T:G+16A were inactive in the recombination assay but active in the assay for intermediates was addressed. As the standard recombination assay is performed over 1 h and the synapse assay is over 2 h, time courses were performed for each assay. In the standard recombination assay, products were observed in 2 and 3 h with mutants T-15C:C+15G and G-16T:G+16A (Figure 5A). Using the more sensitive radioactive assay, cleaved substrates, T-15C:C+15G and G-16T:G+16A, and their recombinant products started to appear at 30 min of incubation and accumulated further over the next 30 min whereas with the wild-type attB, most of the substrate had been converted to intermediates or products at 15 min. Even after 60 min, less attP in the presence of T-15C:C+15G or G-16T:G+16A was converted to product compared to attP in the presence of wild-type attB (Figure 5B). Thus both the mutants T-15C:C+15G and G-16T:G+16A could undergo recombination but the reaction is considerably slower than that for the wild-type attB site. As there is a consistently reduced level of synaptic complex observed with these mutant attB sites, it is likely that changes in attB at ¨C/+15 and ¨C/+16 both result in an unstable synapse that explains the slow rate of recombination.

Figure 5. The rate of recombination with mutant attB sites T-15C:C+15G and G-16T:G+16A is greatly reduced. Panel (A) shows the appearance of products from recombination assays using T-15C:C+15G and G-16T:G+16A as substrates after prolonged incubation. Plasmids encoding the wild-type attB (wt) or the 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).