During Tn10 transposition, both transposon ends have identical IHF binding sites adjacent to the inverted repeats. However, we have previously shown that the transpososome is asymmetric and that the IHF sites do not have identical structural or functional roles within the complex (Fig. 1B; Sewitz et al., 2003; Crellin et al., 2004). We therefore refer to these as the α and β IHF binding sites. IHF binding is required only at the α site to stimulate assembly of the paired ends complex (PEC). After assembly of the complex in vitro, IHF is immobilized and locked into the α side of the complex until it is released by the addition of the catalytic metal ion (Sewitz et al., 2003). In contrast, an IHF binding site is not required on the β side of the complex during assembly. However, if one is present, it remains unlocked and IHF is free to associate or dissociate, independently of the presence or absence of divalent metal ion. As the IS10 elements within Tn10 have IHF binding sites at one end only, the α and β sides of the complex therefore appear to represent the outside and inside-ends of IS10 respectively. This structural asymmetry has been shown to affect the cleavage step of the reaction, which is slightly biased towards initiating on the α side of the complex (Fig. 1C; Liu et al., 2005).
We have also described a substantial conformational change in the transpososome, which renders the nucleotides at bp +1 and −1 on the transferred strand of the transposon end hypersensitive to hydroxyl radical footprinting (Crellin and Chalmers, 2001). Although the precise significance of the hypersensitivity is unknown, it almost certainly reflects a conformational change in preparation for the excision step of the reaction. Excision starts with a nick located between the two hypersensitive nucleotides, and continues with formation and resolution of the hairpin intermediate. This conformational change was originally observed in response to the addition of Ca++, an analogue of the catalytic metal ion (Fig. 2, panels 1 and 2). However, further experiments showed that hypersensitivity could not be attributed directly to the effects of Ca++ (Crellin et al., 2004). Instead, Ca++ was required to license the release of IHF from the α side of the complex, converting the bottom-PEC (bPEC) to the top-PEC (tPEC) as the transposon arm(s) unfolded. It was therefore the unfolding of the folded α transposon arm that produced the conformational change, rather than the divalent metal ion per se. This mechanical view of the reaction is supported by the phenotype of point mutations in transposase that alter transpososome unfolding and inhibit the hairpin formation step (Humayun et al., 2005). The amino acid residues in question are distant from the active site and, based on the Tn5 cocrystal structure, are predicted to interact directly with the folded transposon arm distal to the IHF binding site.
Figure 2. Coupled conformational changes on either side of the PEC. The indicated complexes were assembled and footprinted with hydroxyl radicals as described in Experimental procedures. The experiment reveals that the conformational change responsible for the hyperreactivity is present on both sides of the complex and is produced by unfolding of the α transposon arm. The radioactive label was either present on both sides of the complex or was placed specifically on the α or the β side of the complex, as indicated by the asterisk. The even-end cannot form a PEC on its own because it cannot interact with IHF. In reactions containing a labelled even-end and a cold outside-end, only the mixed complex is therefore detected. In reactions containing a labelled outside-end and a cold even-end, mixed complexes are formed along with complexes containing two outside-ends. In this case, the different complexes are resolved at the gel shift stage of the experiment because the DNA fragments are of different sizes. The asterisk indicates the location of radioactive label.
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Structural coupling between opposite sides of the transpososome
As described above, the α and β transposon arms have different structural and functional roles during assembly and subsequent unfolding of the Tn10 PEC. These observations raise further interesting questions regarding whether the asymmetry of the transposon arms is reflected in the structure of the active site. For example, whether or not the structural change responsible for the appearance of hydroxyl radical hypersensitivity is symmetric on each side of the complex. We are able to address this question using a mixed complex in which only one of the two transposon arms contains an IHF binding site (Fig. 2).
We have previously developed the technique for assembly of mixed complexes using an artificial transposon end in which the DNA between bp +19 and +47 was replaced by tandem repeats of the bases 5′-CTGA (Crellin and Chalmers, 2001). We refer to this as an ‘even-end’ because the hydroxyl radical cleavage profile in the presence of a saturating concentration of IHF is completely even, indicating that IHF binding is negligible. The even-end fails to form PEC on its own because it lacks an IHF binding site. However, it is efficiently recruited into a mixed PEC when the assembly reaction is supplemented with an outside-end fragment. In these experiments the even-end is the equivalent of the inside-end of IS10 which also lacks an IHF binding site. In such mixed complexes, the outside-end DNA fragment, with its IHF binding site, is always located on the α side of the PEC.
As alluded to above, the conformational change responsible for the hydroxyl radical hypersensitivity takes place after unfolding of the α transposon arm, as the bPEC is converted to the tPEC. When the α transposon arm is ≤ 73 bp, this can be achieved in the absence of divalent metal ion by using heparin instead of competitor DNA to remove the IHF (Fig. 1A). As the aim of the experiment was to determine whether the hypersensitivity is symmetric or asymmetric, aliquots of the mixed complexes were treated with hydroxyl radicals either before or after treatment with heparin to promote IHF dissociation and unfolding of the α transposon arm. The complexes were then purified using the standard electrophoretic mobility shift assay (EMSA) and the hypersensitivity of the transposon ends was determined by analysis on a DNA sequencing gel (Fig. 2). The experiment was performed twice with the radioactive label present either on the outside-end or on the even-end (equivalent to the α and β sides of the complex respectively). When the radioactive label was on the α side of the complex, the hypersensitivity was identical to the standard bPEC with two outside-end fragments (compare panels 2 and 4). Likewise, identical hypersensitivity was observed when the radioactive label was present on the β side of the complex (compare panels 2 and 6). Taken together, these results demonstrate that unfolding of the α transposon arm promotes a conformational change on both sides of the complex. This suggests that there is communication or structural coupling between the active sites at each transposon end. This idea is supported by further evidence presented below.
Functional coupling between opposite sides of the transpososome
Hydroxyl radical footprints have shown that transposase contacts the DNA up to 80 bp inside the transposon arm (Fig. 1A; Crellin and Chalmers, 2001). These extensive contacts are facilitated by the 180° IHF-induced bend that folds the transposon arm during PEC assembly. Previous structural modelling suggested that the subterminal contacts between bp +48 and bp +80 were located on either side of the transposase dimer interface, providing a possible mechanism to couple opposite sides of the complex (Fig. 1A; Crellin and Chalmers, 2001; Sewitz et al., 2003).
When a highly truncated 52 bp outside-end is used in the transposition reaction, PEC assembly is very inefficient (Crellin and Chalmers, 2001). Once formed, the PEC is also functionally defective and the reaction can progress only as far as the single end break (SEB) stage. This is a particularly interesting phenotype as the hairpin intermediate on the cleaved transposon end remains unresolved (Crellin et al., 2004). These defects appear to be the result of abolishing the contacts between the transposon arm and the transposase monomer most distal to the dimer interface (illustrated in Fig. 1A). The location of the missing contacts on one side of the dimer interface, taken together with the generation of a SEB product, suggests that this phenotype may result from a failure of communication or coupling between the active sites within the complex. We were again able to address this problem using the mixed complex in which only one transposon end has an IHF binding site. In this way we are able to ask whether a short transposon end imparts the SEB phenotype only when present on one specific side of the complex, or whether it imparts an intrinsic defect irrespective of its location.
When the PEC is assembled using two standard outside-ends, addition of Mg++ induces cleavage of the flanking DNA and the PEC is converted to a double end break (DEB) (Fig. 3, panel 1) (Sakai et al., 1995). If a mixed complex is assembled by replacing one of the outside-end partners with an even-end that is unable to interact with IHF, the PEC behaves in the same way and addition of Mg++ produces DEB (Fig. 3 panel 4). In the method used to assemble these mixed complexes, a radioactively labelled even-end is supplemented with a cold outside-end. Note that complexes containing two outside-ends are also formed in such a mixture, but that these are invisible because they contain no radioactive label.
Figure 3. A hairpin resolution defect reveals functional coupling on either side of the PEC. Transposition reactions were analysed using the standard EMSA and visualized by autoradiography. To form the mixed complexes, the unlabelled outside-end DNA fragment was added in excess, which accounts for the apparently low level of PEC in some lanes. Transposition reactions were incubated for 3 h which is sufficient time for the standard reaction, with full-length transposon arms (panel 1), to reach completion. Reactions with truncated outside-end fragments on the α side of the complex fail to reach completion (panels 2 and 3). Long OE, outside-end > 85 bp; long EE, even-end > 85 bp; short OE, short outside-end < 56 bp; short EE, short even-end < 56 bp. The asterisk indicates the location of the radioactive label.
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As explained above, a PEC with two short (52 bp) outside-ends fails to complete the cleavage step of the reaction when Mg++ is added (Fig. 3, panel 2). In this case no DEB complex is produced. However, some flanking DNA is released owing to the cleavage of one transposon end to produce the SEB complex (Crellin et al., 2004).
If the two active sites are in communication, or are mechanistically coupled, then the cleavage defect of the short outside-end PEC in panel 2 may be rescued by a full-length partner. Furthermore, the rescue may also depend on whether the full-length partner is on the α or β side of the complex. Of course, the structural considerations outlined above would suggest that rescue is more likely for a full-length partner that can fold and unfold normally on the α side of the complex.
To address this issue, a mixed PEC was assembled in which a short outside-end is paired with a long even-end partner (Fig. 3, panel 3). As the even-end is unable to interact with IHF, it will always be present on the β side of the complex. When Mg++ is added, no DEB is produced and the reaction is almost identical to that in panel 2 where the complex contains two short (52 bp) outside-ends. This result demonstrates that a full-length transposon arm on the β side of the complex is unable to rescue the cleavage defect produced by the truncated transposon arm on the α side. Flanking DNA is again detected owing to the cleavage at one transposon end that produces the SEB.
The symmetry of the SEB complex was examined by reversing the labelling strategy (not shown). This experiment revealed that the product of this reaction is a mixture of α and βSEBs. Thus, even though the defect is produced by truncation of the α transposon arm, the SEB phenotype can be manifest on either side of the complex.
Finally, a mixed PEC was assembled in which a short (52 bp) even-end on the β side of the complex was paired with a full-length α outside-end (Fig. 3, panel 5). When Mg++ is added, this complex behaves normally and completes the cleavage steps of the reaction, releasing the flanking DNA to produce the DEB complex. This is an important result because the presence of the 52 bp even-end in the complex shows that a truncated transposon arm is not sufficient in itself to confer a cleavage defect. This experiment therefore demonstrates functional coupling between the two active sites in the PEC that depends on a full-length IHF-folded transposon arm on the α side of the complex.
The base pair 1G mutation
Based on the genetic phenotypes of mutations, the inverted repeat at the end of Tn10 can be divided into two domains (Huisman et al., 1989; Haniford and Kleckner, 1994; Sakai et al., 1995; Kleckner et al., 1996). Transposase binding is mediated by sequence-specific interactions with bp +6–13. In contrast, mutations at bp +1–3 cause defects in the chemical steps of the reaction, subsequent to synapsis of the transposon ends. Earlier genetic evidence had demonstrated that some of these mutations could be partially rescued by a wild-type partner (Huisman et al., 1989). We were therefore interested to discover whether the physical basis for these intriguing genetic observations is provided by the asymmetry and communication between the α and β sides of the complex. As a starting point we chose the 1C to G mutation (subsequently referred to as 1G). We chose this mutation because it has a strong phenotype in vivo when present at both ends, reducing transposition to 0.07 of the wild-type level. However, when present at only one transposon end, the wild-type partner rescues the activity back to 0.7 of the wild-type level.
As a reference point, a standard bPEC was assembled with two wild-type outside-end fragments (Fig. 4A, left panel). This is prefixed by the term fully folded (ff) because both transposon arms remain bound by IHF throughout the reaction (see illustration in Fig. 1B). When the reaction is initiated by addition of Mg++, the SEB intermediate appears early and is gradually converted into DEB product during the time-course. The DEB complex is the end-point of the reaction as no suitable DNA was included in these reactions to act as a target or IHF competitor. When both of the transposon ends contain the 1G mutation (Fig. 4A, right panel), the kinetics of SEB formation are also slow. After 15 min, SEB is clearly visible in the wild-type reaction, but very little has appeared in the 1G reaction. At later time points no DEB is produced, and the reaction appears to stall at the SEB intermediate stage (Fig. 4A, indicated by the letter X). This is consistent with previous work with this mutation showing that it is proficient for the first nick in the reaction, but partially defective for hairpin formation (Allingham and Haniford, 2002).
Figure 4. Cleavage and hairpin resolution defect of the 1G transposon end mutation. A and B. The indicated complexes were assembled using different combinations of wild-type and mutant transposon ends as indicated above and below the panels. The reactions were initiated by the addition of Mg++, stopped at the indicated time point by addition of EDTA, and visualized using the standard EMSA. All of the DNA substrates have full-length transposon arms (> 85 bp), which either have the wild-type sequence or have the 1G mutation at bp +1 of the transposon end, as indicated. In (A), the complexes contain a pair of identical outside-ends. In (B), mixed complexes were assembled with outside-end and even-end fragments on the α and β sides of the complex respectively. The asterisk indicates the location of the radioactive label. C. The SEB complexes labelled as X and Y in (A) and (B), respectively, were recovered from the gel by the crush and soak method and deproteinated by SDS treatment. The DNA component of the complexes was analysed on a denaturing DNA sequencing gel. Complex X contains the unreacted OE.1G substrate plus the cleavage product and the hairpin intermediate. Complex Y contains the unreacted EE.1G substrate. The cleaved transposon end on the opposite side of complex Y is not detected because it is unlabelled. D. A diagram representing the structures of the SEB complexes X and Y deduced from the DNA components visualized in (C).
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Although the SEB product in this experiment indicates that the 1G defect may involve communication between the two active sites, it provides no information regarding the symmetry because it is not possible to distinguish the α and β sides when the complex contains two identical outside-ends. The experiment was therefore repeated using a mixed complex in which an outside-end is paired with an even-end partner (Fig. 4B). As the radioactive label is present only on the even-end, only mixed complexes are detected in this experiment. In this case, the mixed bPEC is prefixed by the term ‘semi-folded’ (sf) because only the α transposon arm is bound by IHF.
When the sfbPEC contains two wild-type transposon ends, the SEB intermediates appear early and are converted to the DEB product at later time points (Fig. 4B, left panel). In this experiment, with the semi-folded complex, the EMSA is able to resolve the α and β isomers of the SEB intermediate which co-migrate in the gel when using the fully folded form of the complex (the αSEB and βSEB are illustrated in Fig. 1C; Sewitz et al., 2003). The slight bias in the reaction towards the αSEB intermediate (Fig. 1C) can be seen quite clearly at the 15 min time point. When the outside-end and even-end partners both contain the 1G mutation, no DEB is produced and the reaction again stalls at the SEB intermediate stage (indicated by the letter Y in Fig. 4B, right panel). The location in the gel suggests that this is the αSEB intermediate, as will be shown in more detail below.
The SEB complexes X and Y from Fig. 4A and B were recovered from the gel, deproteinated by SDS treatment and analysed on a denaturing DNA sequencing gel (Fig. 4C). As expected, the SEB represented by band X contains both the unreacted and the cleaved outside-end of the transposon. In addition, it contains a substantial amount (83%) of the hairpin intermediate, which will accumulate if the hairpin resolution step is blocked following the double strand cleavage to release the flanking DNA. This indicates a catalytic defect because the hairpin intermediate has a short half-life in wild-type reactions, and very little would normally persist after 120 min (Kennedy et al., 1998).
The analysis of band X provides no information about the symmetry of the complex because the transposon ends on both sides are identical and cannot be distinguished. However, band Y is derived from a mixed PEC in which the radioactively labelled even-end is by definition always present on the β side of the complex. Figure 4C shows that this SEB complex contains only the unreacted even-end, with no hairpin intermediate or cleavage product detected. The cleaved transposon end in this SEB complex must therefore be on the α side of the complex. Taken together, these results show that bands X and Y represent the αSEB complex (summarized in Fig. 4D). Furthermore, the cleaved transposon end on the α side of the complex is 83% hairpin. As the hairpin intermediate is barely detectable in equivalent wild-type reactions, this indicates that the hairpin resolution step of the reaction has been severely compromised.
It is noteworthy that the SEB-hairpin product obtained with the 1G mutation is very similar to that obtained using the truncated 52 bp outside-end substrate (Fig. 3; Crellin et al., 2004). The key difference is that the 1G mutation produces only αSEB, whereas the short α transposon arm produces a mixture of α and βSEBs. Thus, although the SEB-hairpin represents a mechanistically significant intermediate, the reaction is sufficiently flexible that this point can be reached by more than one pathway. (For another example of the flexibility of the reaction pathway see Fig. 7 of Liu et al., 2005.)
Rescue of the 1G mutation
Genetic experiments had previously demonstrated that a wild-type partner could rescue the phenotype of the 1G mutation (above). We were therefore interested to determine whether rescue was dependent on the location of the wild-type partner, on one side of the complex or the other. To address this point we assembled a mixed PEC in which a wild-type transposon end was paired with the 1G mutation either on the α or β side of the complex (Fig. 5A). These mixed complexes are referred to as α1G and β1G respectively.
Figure 5. Rescue of the 1G mutation cleavage defect. A. Mixed PECs were assembled in which wild-type and 1G mutant transposon ends were placed on either the α or the β side of the complex. Reactions were initiated by the addition of Mg++, stopped at the indicated time point with EDTA and visualized in the standard EMSA. In the left panel, the reaction fails to reach completion and produces an αSEB. In the right panel, the reaction reached completion via the βSEB intermediate. In these gels the αSEB migrates only slightly faster than the βSEB. However, the identities of these complexes are confirmed in (B). The asterisk indicates the location of the radioactive label. B. The αSEB (A, left panel) and the βSEB (A, right panel) were recovered from the gel by the crush and soak method and deproteinated by SDS treatment. The DNA component of the complexes was analysed on a denaturing DNA sequencing gel. The identities of the complexes assigned in (A) were confirmed because an unreacted even-end was recovered from the αSEB, and a cleaved even-end fragment was recovered from the βSEB complex. No hairpin intermediate is detected in either complex. C. Identical complexes to those in (A) were assembled, except that the labelling scheme was reversed, placing the radioactive label on the outside-end fragment. The complexes were purified using the standard EMSA, located by autoradiography and excised from the gel. The gel slices containing the respective complexes were soaked in reaction buffer. After incubation under reaction conditions, complexes were recovered from the gel by the crush and soak method and deproteinated by SDS treatment. The DNA component of the complexes was analysed on a denaturing DNA sequencing gel. The extent of the reaction is low because of the inherent inefficiencies of the in-gel cleavage strategy. However, both samples contained cleaved transposon ends but no signal from the hairpin intermediate, indicating that hairpin resolution had been achieved. The question mark indicates an unidentified cleavage product. D. A diagram summarizing the structures of the αSEB and the DEB complexes in (A), as deduced from the DNA components identified in (B) and (C). No hairpin intermediates are present.
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In the β1G situation, the reaction stalls at the SEB stage (Fig. 5A, left panel). A wild-type partner on the α side of the complex is therefore unable to rescue the reaction. The location in the gel suggests that the product is an αSEB and this will be demonstrated in more detail below.
In the α1G situation, when the wild-type partner is placed on the β side of the complex, the mutant phenotype is rescued and the complexes complete the cleavage steps of the reaction, producing DEB (Fig. 5A, right panel). A wild-type partner on the β side of the complex is therefore able to rescue the reaction. This was surprising as it is the opposite placement of the wild-type partner needed to rescue reactions with the truncated 52 bp transposon end (Fig. 3). There are also two notable differences compared with the wild-type reactions presented in Fig. 4B. First, the kinetics of the reaction are much slower, which is the reason the time-course of the reaction has been extended in this experiment. Second, the vast majority of the SEB intermediate appears to be the β isomer. It therefore appears that the normal slight bias in the reaction towards the αSEB intermediate has been completely reversed in favour of the βSEB. Presumably, both of these effects arise from an inhibition of cleavage on the α side by the 1G mutation, and the promotion of cleavage on the β side of the complex by the wild-type sequences.
The slightly unusual nature of these results prompted us to confirm the identities of the SEB isomers assigned in Fig. 5A. The respective bands were therefore recovered from the gel, deproteinated by SDS treatment and analysed on a DNA sequencing gel (Fig. 5B). Note that in both cases the radioactive label was present only on the even-end which is always on the β side of the complex. As expected, only unreacted even-end is present in the αSEB lane. Also, > 95% of the signal in the βSEB lane corresponds in size to the cleaved even-end. These results confirm the identities of the respective complexes. Furthermore, as no hairpin intermediate is detected in the βSEB lane, the resolution step has been achieved.
In these experiments with mixed complexes, the radioactive label is usually present only on the β side of the complex. It is therefore unknown whether the αSEB and the DEB products, shown in Fig. 5A, are trapped at the hairpin intermediate stage. This question can be addressed by reversing the labelling strategy for the mixed complexes, placing the radioactivity on the outside-end present on the α side of the complex. However, this approach is problematic because, in addition to the desired mixed complexes, homogeneous complexes containing two labelled outside-ends will also be detected. Although the two types of complexes can be resolved in the standard EMSA, owing to the different length of the DNA fragments employed, the products of the reaction after addition of Mg++ cannot be resolved completely or identified unambiguously. To circumvent this problem, the unreacted mixed complexes were resolved in the standard EMSA and excised in a gel slice. The gel slice was then soaked in reaction buffer to initiate catalysis. After extended incubation, the reactions were stopped by addition of EDTA, deproteinated by SDS treatment, and the DNA was recovered and analysed on a DNA sequencing gel (Fig. 5C). The in-gel reactions produced the expected αSEB and the DEB products, as judged by the presence of the unreacted and cleaved outside-end fragments. Furthermore, there was no signal from the hairpin intermediate, indicating that resolution had been achieved. Taken together these results show that the β1G and α1G complexes yield fully resolved DEB and αSEB products respectively (summarized in Fig. 5D).
The defect and rescue of the 8C mutation
Transposase binding to the transposon end is mediated primarily via sequence-specific interactions with bp +6–13 (Huisman et al., 1989; Haniford and Kleckner, 1994; Sakai et al., 1995; Kleckner et al., 1996). Unfavourable mutations at these positions are thought to affect assembly of the transpososome, prior to the first catalytic step. We have examined the behaviour of the bp +8A to C mutation (subsequently referred to as 8C). When present on both transposon ends in vivo, this mutation reduces transposition to 0.003 of the wild-type frequency. However, a wild-type partner end rescues the activity back to 0.1 of the wild-type level (Huisman et al., 1989).
A mixed bPEC was assembled with the 8C mutation present on both transposon ends (Fig. 6, panel 1). When the reaction is initiated by Mg++, most of the complexes dissociate, although a low level of SEB and DEB products are detected (compare with the wild-type control in Fig. 4B, left panel). Dissociation probably results from a combination of the weakened end binding caused by the 8C mutation and the mechanical forces that appear to operate during the cleavage step of the reaction (Crellin et al., 2004).
Figure 6. The cleavage defect and rescue of the 8C mutation. The indicated complexes were assembled using different combinations of wild-type and mutant transposon ends, as indicated above and below the panels. The reactions were initiated by the addition of Mg++, stopped at the indicated time point by addition of EDTA, and visualized using the standard EMSA. All of the DNA substrates have full-length transposon arms (> 85 bp), which either have the wild-type sequence or have the 8C mutation at bp +8 of the transposon end as indicated. The asterisk indicates the location of the radioactive label.
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We next assembled a mixed PEC in which a wild-type transposon end was paired with the 8C mutation either on the α or β side of the complex. These are referred to as α8C and β8C when the mutant end is present on the α and β side of the complex respectively (Fig. 6, panels 2 and 3). DEB production in both of these mixed complexes is rescued compared with the fully mutant situation (compare the relative amounts of PEC and DEB at the last time point in each of the three sets of reactions). This is immediately different from the 1G mutation in which rescue is specific to a wild-type partner on the β side of the complex (Fig. 5A).
Visual assessment of the time-courses suggests that α8C is more rescued than β8C (Fig. 6, panels 2 and 3). Quantification of the gel further reveals that this is due to the fact that a larger proportion of the β8C complex present at the zero time point dissociates at the start of the reaction. This effect is particularly noticeable in the double mutant situation (panel 1). Indeed, even in the wild-type situation, a significant fraction of the PEC dissociates before cleavage in response to the addition of Mg++. Although never specifically addressed, this is a well-documented effect, observed since the earliest work in this experimental system (Sakai et al., 1995). However, four different classes of metal ion-sensitive PECs, including the 8C mutation used here, have been addressed specifically, and can all be construed as a result of weakened end-binding (Sakai et al., 1995; Kennedy and Haniford, 1996; Crellin et al., 2004). The present results suggest that although this effect operates on both sides of the complex, the β side is more sensitive.
A further unusual aspect of this experiment is that the normal bias of the reaction towards the αSEB is exaggerated in β8C (Fig. 6, panel 3), and almost completely reversed α8C (panel 2). The simplest explanation is that the SEB complex is even more sensitive than the PEC to metal ion-dependent dissociation when the 8C mutation is present on the side of the complex that cleaves first. Thus, α and βSEBs are under represented in α8C and β8C respectively. Finally, the products of the reactions with the 8C-mutant complexes were examined on a DNA sequencing gel (not shown). No signal corresponding to the hairpin intermediate was detected, showing that hairpin resolution had been achieved. This demonstrates that the defect caused by the bp +8 mutation does not affect the catalytic steps and is likely caused by a simple reduction in transposase affinity for the transposon end.