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Cunt Brunette Cuntbrunette M Fresh Ru 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 effects of mutations at positions ¨C/+2, ¨C/+14, ¨C/+15, ¨C/+16 and ¨C/+18 were studied further. Oligonucleotides were synthesized that had single mutations at either the ¨Cx position in the B arm or in the +x position in the B' arm. Recombination was performed with the oligo-plasmid assay and with the standard recombination assay using the sites cloned into pGEM7. The attB sites containing the single mutations C-2G and G+2C regained much of the activity of the wild-type attB site suggesting that a correct interaction on one or other side of the crossover at this position is sufficient for recombination (Figure 2). Similarly the single mutation at ¨C18 or +18 also regained some activity compared to wild-type attB (Figure S1). Single mutations at the 15 and 16 positions behaved differently. Mutants at ¨C15 or ¨C16 had much greater effects on recombination than the mutants at +15 or +16. The single mutations C+15G and G+16A regained some activity compared to the double mutants T-15C:C+15G and G-16T:G+16A whereas the single mutants at T-15C and G-16T did not (Figure 2). A similar difference, but less so, was also observed at position 14 where the left B arm was more sensitive to mutation than the right B' arm (Table 1). To test this further we experimented with partially symmetrical sites. The B arm of attB that included the region from ¨C12 to ¨C18 was replaced with the +12 to +18 sequence from the B' side . The 2L (+12 to +18) attB site was as active as the wild-type attB site whereas the 2R (¨C12 to ¨C18) site was inactive (Figure 2). These data indicate that the sequence in the left arm of attB plays a major role in attB function and its loss removes all activity. A mutant attB site RL, with the straight swap of the B arm sequence between ¨C12 and ¨C18 with the B' arm sequence at +12 to +18 was inactive (Figure S1) indicating that whatever positive role the ¨C12 to ¨C18 sequence plays in attB function, it is not acting independently of other sequences in the attB site.

Mutant attB sites have little or no reduction in affinity for integrase

This mutational analysis of attB showed that double mutations at three positions ¨C/+2, ¨C/+15 ¨C/+16 and the single mutants at ¨C15 and ¨C16 were particularly defective for recombination.

We have shown previously that it is possible to assay several intermediate steps in recombination i.e. DNA binding, formation of the synapse and cleavage of the DNA to form the covalent intermediate in which integrase is covalently bound to its cleaved substrate (27). The mutant attB sites were used first in affinity assays with integrase. As seen previously integrase bound to the wild-type attB site with an affinity of 60 nM (27,29). Most of the mutant attB sites bound with a similar affinity to the wild-type attB site including the severely recombination defective sites C-2A:G+2T and G-16T:G+16A (Figure 3, Table 2). The mutant T-15C:C+15G had a slightly lower affinity for integrase (128 nM) but this loss of affinity was abolished in the single mutant at ¨C15 (T-15C) which was still defective in recombination (Table 2, Figures 1 and 2). Differences in binding affinities by integrase for mutant attB sites C-2A:G+2T, G-16T:G+16A, T-15C and G-16T cannot therefore account for the defectiveness of these sites in recombination. Mutations involving position 18 from the crossover dinucleotide showed an 3-fold lower affinity for integrase than wild-type attB which could contribute to the observed decrease in recombination activity (Figure 2). It seems likely that attB sites with mutations at ¨C/+2, ¨C/+15, ¨C/+16 were blocked elsewhere in the recombination pathway.

Figure 3. Binding affinities by integrase for the wild-type and mutant attB sites. Integrase was incubated with radiolabelled wild type (panel A) and mutant attB sites (panels B¨CF). In each panel, the phosphorimage shows the complexes obtained with increasing integrase concentrations and, below, 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.