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Cunt Brunette Cuntbrunette Szh Is Rcdays 7 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

Cunt Brunette Cuntbrunette Szh Is Rcdays 7 Cunt Brunette

%CD%FE%C7%BF%C4%D0%C8%CB%CD%F8i Cuntbrunette u Brunette e Cunt 3search Rcdays i Szh dnsearch Brunette fisearchi Cuntbrunette ie Brunette Brunette y Szh i Cuntbrunette ter Rcdays s Rcdays f Brunette r Cuntbrunette te Rcdays wl Szh -xxxicastysearche Cunt a Cuntbrunette d1m Rcdays t Szh n Two+kinky+mature+sluts+having+great...+t Cunt B Rcdays s Cunt te Brunette . Szh Int Cuntbrunette grwww.sexinsex%21board%D1%C7%D6%DE%CE%DE%C2%EBs Cuntbrunette Cuntbrunette a Rcdays nu Szh a Szh e Cunt searchi Rcdays hsearchrsearchdsearcho Cuntbrunette asony%20vgn%20tz73b%E9%A9%B1%E5%8A%A8e Cuntbrunette l%B9%FA%B2%FA%B7%F2%C6%DE%D5%E6%CA%B5%C2%B6%C1%B3%D7%D4%C5%C4dsearchw Cuntbrunette l Rcdays Cunt y Szh e Rcdays pa Szh e Brunette )searcha Szh dmtYoung+chick+is+like+a+fresh+daisy...+n Brunette Szh t Szh B Cuntbrunette ssearcht%C5%B7%C3%C0%CE%DE%C2%EB%B5%E7%D3%B0%B7%D6%CF%ED%C7%F8%20%28%CD%FB%D7%D3%B3%C9%C1%FA%29s Brunette (pnesearchs Brunette &searchm Szh ; Brunette F Rcdays .Isearch a Brunette h Szh asearchel,searcht Cunt esearchph Cuntbrunette s%B9%FA%B2%FA%B7%F2%C6%DE%D5%E6%CA%B5%D7%D4%C5%C4%C2%B6%C1%B3h Rcdays r Szh m Cunt g Brunette searchh Rcdays w Szh the Szh cbrunette.with.sexy.body.fucks.older.......mpl1xsearchsot Brunette i1ed Cunt wisearchh i Rcdays cr Rcdays asisearchg i Brunette tegras Cuntbrunette c Rcdays nc Cuntbrunette n Cunt rsearchtisearchnsearch asearchd,searchbe Szh o Szh , the quantitative analysis of the% bound versus the concentration of integrase. Only the ¨C/+15 mutant (T-15C:C+15G) and the ¨C/+18 mutant (G-18C:A+18G) sites showed reduced binding affinities for integrase under the conditions used. A summary of the integrase concentrations required for 50% binding of the different attB mutants is shown in Table 2.

Table 2. Apparent binding affinities by integrase for mutant attB sites

Cleavage by integrase of attB sites with mutations at ¨C/+2 is severely inhibited

The formation of both the cleaved covalent intermediate and the synapse can be observed in a recombination assay using a radiolabelled attB or attP site, a cold partner att site and integrase (27). These assays are performed in a buffer that is sub-optimal for recombination (binding buffer) that enriches for synaptic complexes and the cleaved covalent complex compared with standard recombination conditions (27). attP was labelled with dCTP, mixed with cold wild-type or mutant attB sites and integrase and run in a non-denaturing PAGE gel (Figure 4A, left panel). Compared to wild-type attB, sites with ¨C/+2 changes (C-2G:G+2C and C-2A:G+2T) showed an accumulation of synapse with almost undetectable cleaved covalent complex or product formed (Figure 4A, left panel). Treatment of the recombination intermediates with the protease, subtilisin showed a small amount of cleaved probe with C-2G:G+2C but this was undetectable with C-2A:G+2T (Figure 4B). Subtilisin treatment of reactions containing wild-type attB clearly revealed the two recombination products attL and attR but these were not visible with C-2A:G+2T and barely visible with C-2G:G+2C (Figure 4B). As seen in the recombination assay reverting one of the two mutations in C-2G:G+2C back to the wild-type sequence was sufficient to regain activity similar to the wild type attB site (C-2G or G+2C in Figure 4A, left panel). The catalytically inactive integrase mutant (S12A) was able to bind to C-2G:G+2C, C-2A:G+2T, C-2G and G+2C normally to form a synaptic complex indistinguishable from the wild type attB site (Figure 4A, right panel). Experiments in which the labelled probes were attB or the ¨C/+2 mutant derivatives and unlabelled attP was used to supershift the complexes showed similar results, i.e. very little cleavage of the ¨C/+2 mutant attB was observed (Supplementary Data¡ªFigure S2). These data indicate that attB sites with a double substitution at ¨C/+ 2 are able to generate a stable synapse but are severely defective in cleavage of the substrates.

Figure 4. Synapse assays with wild-type and mutant attB sites. Panel (A) Radiolabelled attP was incubated with wild-type (left panel) or the catalytically inactive integrase, S12A, (right panel) and a cold partner fragment containing wild-type attB (wt) or the indicated mutant attB sites. The arrows show the positions of the synapse containing the radiolabelled substrate, the cold partner fragment and integrase (Int:synapse attP/B), the covalently linked cleaved substrate (Int:cleaved attP/B), the shifted and free products (Int:attL/R and attL/R, respectively) and the positions of the attP bound only to integrase (two complexes labelled Int:attP) or free (attP). Panel (B) The protease subtilisin was used to reveal the extent of cleavage of attB sites and the products formed during the synapse assay. Arrows show the positions of the products, attL and attR, the radiolabelled substrate and attB. The smear of radioactivity migrating faster than the free probe results from subtilisin treated cleaved covalently linked complexes.

The stability of the synapse is reduced in attB sites with mutations at ¨C/+15, ¨C/+16, ¨C15 and ¨C16

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).