Sex

Thingy Brunette Cuntbrunette R Cuntbust 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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To detect the cleavage of the DNA fragments by integrase, reactions were set up as for the radioactive recombination assays but after incubation at 30¡ãC, reactions were heat inactivated (72¡ãC for 10 min) then incubated with 1µl of subtilisin A (Sigma 0.1 mg/ml in 1x binding buffer) for 15 min at 30¡ãC. Subtilisin was inactivated (72¡ãC for 10 min) and the reactions were loaded onto a 0.5x TBE, 5% non-denaturing polyacrylamide gel.

After electrophoresis gels were dried and exposed to a phosphorimager screen (Fuji) for 16 h and then scanned (Fuji FLA3200 phosphorimager). Quantification of radioactivity was performed using the AIDA software (Raytest, Straubenhardt, Germany).

Purification of integrase

Wild-type C31 and S12A integrase were purified as described previously (27). Integrase concentration was assayed using a method based on the dye-binding procedure of Bradford (45) employing the BioRad protein assay solution, and bovine serum albumin as a standard.

RESULTS

Identification of defective mutations in attB

The minimal attB site, according to Groth et al., (10) is 34 bp with the crossover 5'TT (abbreviated to XO) at the centre (Figure 1). Footprinting confirmed that integrase binds either side of the crossover site in all the attachment sites and, as integrase is a dimer in solution it probably binds as a dimer (29). Moreover, we have shown that integrase binds to attB and attP in a functionally symmetrical manner. Thus in order to maximize any phenotype arising from mutations in attB we generated a set of doubly mutated sites with base pair changes at symmetrical positions with respect to the crossover sequence. To aid in the description of the positions of mutations, the base pairs in the minimal attB and attP sites were annotated with either a negative number when they lie to the left of the crossover dinucleotide sequence (5'TT) i.e. B or P arm to use the terminology) or positive when it lies to the right of the crossover (B' or P' arm); the numbers count upwards as the position extends away from the crossover (Figure 1). Thus mutations in a double mutant involving the two base pairs adjacent to the crossover is at ¨C/+1 and mutations at the next position moving outwards are at ¨C/+2, etc. Mutations were chosen that would introduce sequence symmetry at the desired position. Thus each double mutant was designed to contain one of the four bases, A, T, C or G, at position ¨Cx on the B arm and at +x on the B' arm its complement, T, A, G or C, respectively, was inserted. The choice of mutation was made on the basis that the introduced bases had to be different from those present in both arms of attB and preferably also different to those seen in the pseudo-attB sites (Figure 1). For example, position 15 is a T in the B arm and a C in the B' arm and the pseudo-attB sites have a G in the B arm and a C or A in the B' arm. T-15C:C+15G and the T-15A:C+15T contain changes at ¨C/+15 on the B and the B' arm to base pairs that are different from both the wt and the pseudo-site sequences and should be functionally the same mutation in both arms. For most positions at least two mutant forms were made but for some sites (positions 4, 7, 16) only one option was available. Other positions where only a single mutant form was made are at 14, 17 and 18.

Except for mutations at positions ¨C/+3, ¨C/+8 and ¨C/+12 the activities of the double substituted attB sites was first assayed using annealed oligonucleotides. Oligonucleotides containing the double substitutions were purified by PAGE, annealed and used in an oligo-plasmid recombination assay (17). In this assay, a supercoiled plasmid containing attP was mixed with the oligonucleotide containing attB or one of the mutant forms and various concentrations of integrase. The extent of linearization of the attP plasmid indicated the extent of recombination and this was assayed after separation of the DNA in an agarose gel. A control reaction using the wild-type attB site was performed in every assay so that the activities could be compared under identical conditions. The lowest integrase concentration at which recombination could be observed was scored (Figure S1 and Table 1).

Many of the mutant attB sites showed little or only 2-fold change in activity compared to the wild-type site. These sites were changed at ¨C/+1, ¨C/+4, ¨C/+5, ¨C/+7, ¨C/+10, ¨C/+11 and ¨C/+13 (Table 1, Figure 1). The remaining mutants showed defective or partially defective activity ranging from 4-fold less active than wild type to apparently inactive. Oligos encoding sites C-2G:G+2C, C-2A:G+2T, G-6A:C+6T, (G-6T:C+6A, G-9T:C+9A, G-9A:C-9T, G-9C:C-9G, T-15C:C+15G, G-16T:G+15A and G-18C:A+18G were cloned into pGEM7 (Promega) so that the activities of the mutant attB sites could be verified by a standard recombination assay using both att sites residing on plasmids. Only one of the mutant attB sites that was partially defective (at position ¨C/+14) was not represented in the cloned mutant attB site collection; this site was instead subjected to single site substitutions (see later). T-15A:C+15T was not cloned as a plasmid containing another mutant at ¨C/+15 (T-15C:C+15G) with the same activity was quickly obtained. A plasmid encoding G-6C:C+6G was not obtained due to technical difficulties. Plasmids containing mutations in C-3T:G+3A, C-3G:G+3C, C-3A:G+3T, G-8T:C+8A, G-8C:G+8C, C-12A:G+12T and C-12T:G+12A were obtained by PCR mutagenesis as described in the Material and Methods section. The relative activities of the double substitution mutants were estimated compared to a standard reaction with wild-type attB (Table 1 and Figures 1 and 2). As for the oligo-plasmid assay the activity of each mutant site was scored as the concentration of integrase required to observe recombinants in an agarose gel stained with ethidium bromide (Table 1). The relative activities compared to the wild-type site are summarized graphically (Figure 1).

Three of the mutant attB sites were very defective for recombination and these contained substitutions at ¨C/+2, ¨C/+15 and ¨C/+16. In all cases no recombination was observed in either the oligo-plasmid or the standard assay using these double substituted attB sites (Figure 2 and Figure S1). Recombination was just detectable with attB containing substituted ¨C/+18 in the plasmid assay with 351 nM integrase (Figure 2). The low activity of the ¨C/+18 double mutant was surprising given that this position is outside the minimal attB site defined previously by Groth et al. (10). The nature of the mutations made small differences to activity in only a few mutants. The ¨C/+12 mutant containing the double transversion C-12A:G+12T was only just active with 87 nM integrase while the ¨C/+12 mutant containing the transitions C-12T:G+12A was active with 43 nM integrase (Figure 2). The G-6T:C+6A transversions had similar activity to wild-type attB but another ¨C/+6 mutant, containing transitions (G-6A:C+6T) was 2-to 4-fold less active than attB (Figure 2).

All of the mutant attB sites described in this section that were cloned into plasmids were used to test whether they would recombine with attL, attR or attB but no activity was detected in any case. Thus none of these mutant sites had any detectable gain-of-function.

The sequence on the left side of attB has a greater role in attB function than the right side

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.