Protein-DNA interactions define the mechanistic aspects of circle formation and insertion reactions in IS2 transposition
© Lewis et al; licensee BioMed Central Ltd. 2012
Received: 22 September 2011
Accepted: 26 January 2012
Published: 26 January 2012
Transposition in IS3, IS30, IS21 and IS256 insertion sequence (IS) families utilizes an unconventional two-step pathway. A figure-of-eight intermediate in Step I, from asymmetric single-strand cleavage and joining reactions, is converted into a double-stranded minicircle whose junction (the abutted left and right ends) is the substrate for symmetrical transesterification attacks on target DNA in Step II, suggesting intrinsically different synaptic complexes (SC) for each step. Transposases of these ISs bind poorly to cognate DNA and comparative biophysical analyses of SC I and SC II have proven elusive. We have prepared a native, soluble, active, GFP-tagged fusion derivative of the IS2 transposase that creates fully formed complexes with single-end and minicircle junction (MCJ) substrates and used these successfully in hydroxyl radical footprinting experiments.
In IS2, Step I reactions are physically and chemically asymmetric; the left imperfect, inverted repeat (IRL), the exclusive recipient end, lacks donor function. In SC I, different protection patterns of the cleavage domains (CDs) of the right imperfect inverted repeat (IRR; extensive in cis) and IRL (selective in trans) at the single active cognate IRR catalytic center (CC) are related to their donor and recipient functions. In SC II, extensive binding of the IRL CD in trans and of the abutted IRR CD in cis at this CC represents the first phase of the complex. An MCJ substrate precleaved at the 3' end of IRR revealed a temporary transition state with the IRL CD disengaged from the protein. We propose that in SC II, sequential 3' cleavages at the bound abutted CDs trigger a conformational change, allowing the IRL CD to complex to its cognate CC, producing the second phase. Corroborating data from enhanced residues and curvature propensity plots suggest that CD to CD interactions in SC I and SC II require IRL to assume a bent structure, to facilitate binding in trans.
Different transpososomes are assembled in each step of the IS2 transposition pathway. Recipient versus donor end functions of the IRL CD in SC I and SC II and the conformational change in SC II that produces the phase needed for symmetrical IRL and IRR donor attacks on target DNA highlight the differences.
KeywordsCurvature propensity plot data extensive sequence-specific binding figure-of-eight transposition intermediate hydroxyl radical footprinting minicircle junction selective binding synaptic complex transpososome
The ends of IS2 are 41 bp and 42 bp right and left imperfect inverted repeats (IRR and IRL; Figure 1B) respectively; between these ends the IS encodes two overlapping reading frames, OrfA and OrfB (Figure 1B, i). OrfA is a 14 kDa protein which has been reported in IS2 to bind to a sequence just upstream of the weak indigenous extended-10 promoter (PIRL-) located just inside the left end (IRL) of the element (Figure 1C, ii). This weak promoter regulates the expression of IS2 proteins in Step I. The function of OrfB is unknown but a fusion protein OrfAB, the functional transposase (TPase), is generated by programmed -1 translational frameshifting [13, 21, 22] at a sequence of slippery codons (the A6G frameshift window in IS2), located near the 3' end of orfA (Figure 1B, i). Mutation of this window to A7G in IS2 (Figure 1B, ii) produces OrfAB as the predominant species [11, 23]. When the IS2 ends are aligned (Figure 1C, i, ii), they show four non-conserved elements (I, A, B and C) and two conserved elements (II and III) which play critical roles in the transposition mechanism. Elements A and I comprise a cleavage domain (CD) and B, II, C and III, a protein binding domain (PBD). The differences in the sequences of the two ends are related to their donor and recipient end functions (see below) in Step I .
Several features distinguish circle formation and its consequences in IS2 from those in other IS3 family members. The reaction is physically as well as chemically asymmetric in that the right end functions uniquely as the donor or transferred end and the left end serves exclusively as the functional recipient end. This asymmetry is not unique to IS2, having also been demonstrated in copies of IS256 in Tn4001. Recipient end function in IS2 is partially defined by the accuracy with which the joining reaction occurs. Abutted ends at the MCJ (Figure 1C, iii) are separated by a one or two base pair spacer with a ratio of 90% to 10% [11, 24] but functional minicircles are limited to those with a single base pair spacer. This is so because creation of the MCJ in IS2 assembles a promoter, Pjunc,  which has an absolute requirement for a 17-nucleotide promoter spacer (Figure 1C, i, ii) that is conferred by the one base pair MCJ spacer. This more powerful Pjunc is essential for and drives transposase reactions in Step II . MCJ promoters with spacers of two or more base pairs are completely non-functional.
We concluded from earlier studies that differences in length and sequence of the two ends of IS2 in Step I are responsible for the restriction of donor and recipient end functions to IRR and IRL respectively . Differences in length are related to the correct positioning of the shorter donor end (IRR) in the catalytic pocket. However, random mutation in the A element of the AIRR sequence in an IRR CD eliminated minicircle production, while similar changes in AIRL in an IRL CD had no effect on the efficiency of minicircle formation; this result implied that extensive sequence-specific protein affinity for the A element was important in defining donor function but not recipient end function. For the B element, mutations in the BIRR sequence also eliminated minicircle formation, implicating sequence-specific protein affinity. Additional domain swapping experiments involved the substitution of a 6 bp BIRL sequence in an IRR derivative, which did not change the length of IRR (Figure 1C, i and 1C, ii). This reduced but did not eliminate IRR donor activity, implying that the protein had a weaker affinity for BIRL. Further evidence for some protein interaction with the BIRL sequence is that in IRL, its mutation (a triplet of point mutations) all but eliminated minicircle formation. These results suggested that the degree of sequence-specific interaction of the protein for sequences in or near the CDs may also be related to donor and recipient end functions; in an IRR end, extensive interaction of the protein with AIRR and BIRR would be required for the donor function; however, in IRL the lack of extensive interaction of the protein with AIRL and a weak affinity for BIRL may contribute to recipient end identity.
Additional data from experiments with AIRL threw light on this supposition. First, in an IS2 mutant with two IRR ends, the increase in length of one IRR by a single base pair alone was necessary and sufficient to convert it to a recipient end with no donor function. However, the addition of AIRL was absolutely essential for the accuracy of recipient end function. Furthermore, alteration of any one of three non-conserved nucleotides in positions 2, 5 and 7 in AIRL (Figure 1C, ii) that made the sequence more like that of the IRR CD reduced the accuracy of the joining reaction in Step I by increasing MCJ spacer size. We posited then, that the non-conserved base pairs in AIRL, through some interaction with the protein, were responsible for the accuracy of recipient end function by correctly positioning the IRL CD in trans in the vicinity of the IRR CD to generate a single interstitial base pair between the abutted ends. (See the Results and discussion section for a complete analysis of all factors which define recipient end function.) It is interesting that mutation of position 2 of IRL, which converted the TA3' terminal dinucleotide to the CA3' consensus in the IS3 family, did not confer functional donor activity on IRL , due, among other factors, to its incorrect positioning in the cognate catalytic center (CC). Finally, although the features described above for IRL define its accuracy as a recipient end, the sequence of the flanking host DNA can also play a role in determining spacer size , implying that the host DNA sequence adjacent to IRL is also involved in some kind of interaction with the TPase.
For IS2, however, we proposed that in SC I, given the donor-inactive IRL, the 5 bp distance between the two CCs and the one base pair MCJ spacer size, the IRL CD would have to be positioned near the single active CC at which the IRR CD was bound to facilitate the joining reaction. For SC II, we proposed that the abutted CDs separated by a single base pair would also be complexed at a single active cognate IRR CC (the first transition state) and that a series of cleavage-triggered conformational changes would result in each CD cis-bound at its cognate CC (as shown in Figure 2B). It is important to note, however, that other factors may play an important part in this process in SC II, such as the role of the IS911 OrfA, which has been shown in in vitro assays to stimulate insertion principally into DNA targets devoid of IS911 end sequences . Nevertheless, in these ISs, the assembly of intrinsically different SC I and SC II transpososomes appears to be necessary [24, 26]. This conclusion is applicable to circle forming elements in the IS3 family which use the two-step pathway, for example, IS3[12, 13] IS150 and IS911[7, 8], where MCJ spacer size is similar (2 bp to 4 bp) to that of the direct repeat (see Figure 2). It is particularly true for the SC II in IS2 and in IS256 in Tn4001, where physically asymmetric Step I reactions have been described and where the acquisition of donor function by the recipient end, lacking in Step I, is essential. Similar thinking would apply to IS21 and IS1665 where, as is the case in IS256, the interstitial MCJ distance is less than the size of the direct repeat. In this study we have tested these hypotheses with hydroxyl radical footprinting analyses of Step I complexes of IS2 and by comparative footprinting analyses of covalently joined and pre-cleaved (or nicked) MCJ substrates in SC II.
We show here that the footprinting patterns of both IRR and IRL single ends of IS2 reveal bipartite structures. They differ in that the IRR CD is strongly and extensively protected while the IRL CD is only selectively or intermittently bound by the protein. We propose a model in which non-specific and/or selective binding to the adjacent host sequence and selective binding to the IRL CD act additively in SC I to promote binding of the IRL CD in trans at the active cognate IRR CC. In SC II, extensive protection of both the IRL and the abutted IRR CDs, separated by a single base pair, suggests binding at a single active cognate IRR CC with the IRL CD bound in trans, creating the first phase of the SC. Our data suggest that sequential cleavages (associated with small conformational changes) at the 3' termini of IRR and IRL at this active CC trigger a conformational change that leads to transition to a second phase; that is, each CD complexed in cis to its own active cognate CC. In addition, the location of enhanced residues indicative of distortion or bending of DNA, corroborated by curvature propensity plot data, have helped gain insight into the paths of the IRL DNA which facilitate binding in trans within the architecture of SC I and SC II transpososomes.
Results and discussion
Footprinting the single ends of IS2
Hydroxyl radical footprinting was carried out using 87 bp (R87) and 79 bp (L79) radio-labeled dsDNA substrates containing the 41 bp sequence of IRR and the 42 bp sequence of IRL, respectively. The substrates were prepared as annealed oligonucleotides with the labeled strand as the footprinting target (see the Methods section). The transposase was overexpressed from pLL2522, the plasmid with the orfAB::GFP fusion construct, and partially purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography . Mutational studies with this partially purified protein (specifically null mutants with a complete loss of binding proficiency), indicated strongly that the observed binding reactions did not result from trace amounts of the IS2 Tpase from chromosomal copies of the element. In addition, two sets of results suggest that the presence of the GFP tag affected neither the binding properties nor the activity of OrfAB. First, in vivo transposition frequencies of the tagged protein are statistically identical to those of the native protein ; secondly, in a cleavage assay , complexes formed in-gel with a mixture of 87-mer IRR and 50-mer IRR substrates, generated the 95 nucleotide and 114 nucleotide high molecular weight recombination products predicted for paired-ends complexes formed by a chemically active protein activated with Mg2+ (Additional file 1). This latter result and footprinting data from complexes formed with the MCJ substrates in which both ends are protected along their lengths, indicate that fully formed complexes are generated by the OrfAB-GFP protein and that paired-ends complexes composed of at least dimers are being formed. For footprinting reactions, the protein-DNA complexes, initially visualized in the gel retardation assays shown in Figure 3A, were formed in solution and subjected to cleavage reactions at room temperature (20°C) prior to fractionation on 8% polyacrylamide sequencing gels.
Data summarized in Figure 6 indicate that an 11 bp sequence at the outside end of IRR that makes up the cleavage domain (the A and I elements) is strongly protected by the transposase. Strong protection is also observed for the B element at the outside end of the PBD of IRR, although a gap at base pairs 12 and 13 separates the IRR cleavage domain from the BIRR (Figure 6II, i). Extensive but weaker interactions are associated with elements at the inside terminus of the end. On the other hand, the first 11 bp of the IRL CD (elements A and I) are only intermittently contacted by the protein (Figure 5), at positions 2, 5 and 7, the same residues shown from earlier mutation studies to affect the accuracy of the joining reaction. In addition, in BIRL, the residues are more weakly bound than those of BIRR (summarized schematically in Figure 6II, ii). Thus, the cleavage domain of IRL is not extensively protected by the transposase in Step I. We refer to the intermittent binding of the IRL CD as selective binding, which describes the interaction of the protein with a few residues of the sequence of the recipient end in order to ensure the accuracy of the joining reaction. These results support conclusions reached from earlier mutational studies  that AIRR and BIRR are important binding targets in IRR for the TPase, that BIRL would be bound with lower affinity, that AIRL would not be the subject of sequence-specific binding and that its residues 2, 5 and 7 might have a unique type of interaction with the TPase.
The different bipartite footprinting patterns of the single IRR and IRL ends are related to their functions in the Step I transpososome
Our results provide physical confirmation of earlier genetic data that the functionally bipartite ends are composed of an outer 11 bp cleavage domain and an inner protein binding domain . We conclude that the strong protection of the CD of IRR, likely protected at two major grooves (Figure 6II, i), results from sequence-specific binding by the catalytic center of the protein and propose that it creates a stable complex which enables accurate cleavage of the donor end to take place. We arrive at this conclusion by taking into account the recent results of mutations in the catalytic center of the transposase, in which alteration of three residues in the beta strands and alpha helices of the CC generated partially dissociated complexes in electrophoretic mobility shift assays (EMSAs). This suggested that a loss of affinity of this part of the protein for the DNA substrate had occurred. Similar mutant phenotypes were also observed for mutations in the binding domain of the protein, indicative of two distinct but interdependent binding capabilities of the protein .
We propose that in both IRR and IRL, the B elements, which are also bound extensively at major grooves, together with the II elements comprise the major targets of the BD of OrfAB (Figure 6II). This is not unlike the situation in IS911 and IS30. In the former, the β domain of the ends was specifically bound and protected by a truncated N-terminal fragment of the transposase, whereas in IS30 the central region of the ends was protected by a similarly truncated derivative. In IRL of IS2, binding of BIRL is weaker than that of BIRR, a result that is supported by data from earlier mutation studies which showed the inability of BIRL to maintain normal levels of donor activity in an IRR end . This weaker protection pattern may be related to the need to allow the tip of IRL (that is, the CD) to be bent (see below).
The differences in the protection patterns of IRR and IRL in SC I correspond to their functions. While the extensive protection of the B element and of the CD of IRR creates and stabilizes an enzymatically competent complex, we propose that the selective binding to the IRL CD and non-specific and/or selective binding to the adjacent host DNA (Figure 6, positions -1 to -8) act additively to direct the CD away from a cis interaction with its cognate CC by bending the DNA to facilitate binding in trans at the active CC, while simultaneously determining the accuracy of the joining reaction. An additional aspect of the data in Figure 6 appears to support this idea. The cleavage domain of IRL shows a relatively high frequency of enhanced residues (six of the eleven positions), compared to its PBD. This suggests that the IRL CD in SC I is distorted because it may need to be bent by the protein. It is interesting that in both substrates L79 (residues 1,3,6,7,8 and 10) and R87 (residues 9 and 10), the enhanced residues are associated with a series of base pairs comprising a guanine/cytosine-rich tract within the CDs (positions 7 to 13 in IRL and 8 to 12 in IRR; Figure 6), a sequence which facilitates bending of the DNA [38, 39]. In support of this idea are results from an IS2 derivative with multiple transversion mutations at positions 8 to 12 of IRL (IIRL), in which minicircle formation was completely abolished , although current results do not show protection of these residues by the protein.
We interpret these data as suggesting that the IRL CD is positioned in trans and juxtaposed to the active CC occupied by the cis-bound IRR CD, in a tract which is probably that used for non-specific binding to the host DNA. The importance of non-specific and/or selective binding of the adjacent host DNA by the protein receives support from our earlier studies, which indicated that the nature of the host DNA flanking the recipient end can play a role in determining MCJ spacer size , as well as from a more recent report of the binding efficiency of a truncated version of the IS911 OrfAB (residues 1 to 149). This derivative bound much less efficiently to a 36 bp substrate containing only the IRR sequence than to a longer 100 bp substrate, due, they proposed, to the non-specific binding capability of the transposase . This interpretation of the architecture of the IS2 SC I is further supported by data from studies in which mutated IS2 derivatives with two left ends produced no minicircles . When complexes are formed in vitro with only DNA of the left end, several factors would then work against either IRL functioning as a donor (that is, bound in cis at its cognate CC): selective rather than extensive binding of their CDs; the non-specific and/or selective binding of adjacent host DNA; their longer length (one bp) than donor IRRs; the reduced affinity of the TPase for the adjacent BIRL element; and the tendency of the CDs to be bent by the protein. These factors would prevent minicircle formation and therefore define the identity of the recipient end in the wild type element.
In elements with two right ends, however, both function as donors with equal probability and produce minicircles in which approximately 90% of the MCJs have interstitial sequences of 2 bp to 3 bp. This would not be the case if both donor CDs were complexed in cis at their cognate CCs (Figure 2A) when the majority of minicircles would have 5 bp interstitial sequences. In complexes formed with two right ends, the CD of one end is bound in cis and that of the other bound in trans, both at a single active CC. Binding in trans would be facilitated by the non-specific and/or selective binding of the adjacent host DNA coupled with the bending of the CD by the protein as indicated by enhancements at residues 9 and 10.
Different conformational states define the protein-DNA interactions of IRR and IRL not only at their outside ends but also at their inside ends, primarily due to the different functions of the ends. At the inside ends of IRR and IRL, different protection patterns involve the two most distal elements (C and III) of the PBD (Figure 6). The stronger protection pattern in elements CIRL and IIIIRL is a manifestation of the location of the docking site, 5'TAAATAA3', for the repressor function of OrfA, (Figure 1C, ii; [20, 40]). The transposase bound to IRL (Figure 5) shows strong protection of the last 4 bp at the inside end of IRL, T/A, T/A, A/T, A/T (residues 39 to 42 of element III), and the 6 bp sequence A/T, T/A, A/T, A/T, G/C, T/A (residues 43 to 48) located immediately adjacent to the inside end and just upstream of PIRL, the extended-10 promoter . These two sequences together appear to form a 10 bp sequence which includes the site to which the 14 kDa OrfA binds competitively in carrying out its repressor function. It is interesting that the truncated 17 kDa derivative of the IS30 TPase (the structural equivalent of OrfA) has also been shown to overlap the promoter region, likely repressing transcription , but that OrfA in IS911 does not have this function. Instead, it has been shown to modify the stoichiometry of complexes formed with the 1-149 truncated forms of OrfAB . In addition, in IS911 OrfA is involved with both heteromultimerization with OrfAB , as well as with its own homomultimerization and with the ability to stimulate minicircle insertion in vitro into target DNA not associated with the IS911 ends . It is likely that these heteromultimers may also exist in our preparations, which consist of a mixture of OrfA and OrfAB . Speculatively, in IS2, the three-dimensional configuration of OrfAB could allow the BD of the protein to target the B and II elements in the PBD, whereas (as a regulatory mechanism) the BD in OrfA, with a slightly different configuration, would target the promoter.
Three previous studies have reported footprinting analyses of the IS3 family and related elements that hint at the bipartite nature of the ends. Earlier, Hu et al. , using cell-free extracts of the IS2 Tpase, reported in situ 1, 10 phenanthroline-copper ion footprinting data for the bottom strand of the right end (5'-TGG... TTAA-3') and the top strand of the left end (5'-TAG.... TTAA-3') of IS2. They showed essentially identical patterns of protection of residues 16 to 41 in the case of IRR and 16 to 42 in the case of IRL with additional protection of residue 43 in the former and protection of residues 43 to 46 in the latter. They reported no binding to the outer base pairs, 1 to 15, for either end, due perhaps to the prevalence of truncated N-terminal species in the preparation of the protein  or to the imprecise folding of the C-terminus, a process which appears to have been avoided in our GFP-tagged version .
Normand et al.  reported DNase I and Cu(OP)2 (copper-1,10phenanthroline) footprinting data for IRR and IRL single-ends of IS911 using a truncated version of OrfAB (residues 1 to 149) from which the carboxy-terminus was deleted; the protein thus consisted primarily of its binding and dimerization domains. Their deletion-gel retardation analyses of the ends of IS911 showed that they are composed of three conserved blocks of residues α, β and γ; β and γ comprise the PBD of IRR and IRL whereas the α motif comprises the CD. Footprinting experiments with both IRR and IRL showed that the truncated OrfAB bound efficiently in an extensive manner to the PBDs of the ends. Finally, DNase I footprinting experiments with the 17 kDa N-terminal derivative of the IS30 Tpase containing only the BD of the protein, showed binding to the central region of an inner, presumed PBD, leaving the outer termini of both right and left ends unprotected .
The bipartite nature of the ends of transposable elements has been well documented by mutational analysis and DNA footprinting studies. The two domains, originally identified through mutational studies in IS903, IS50 (Tn5) [43, 44] and IS10, were subsequently shown in early DNA footprinting studies to be a unique inner binding sequence for the transposase and an outer unbound sequence assigned to post binding cleavage functions. This was shown to be true for simple insertion sequences IS30, IS1, IS903, IS50 and IS911 as well as for the more complex transposons, Tn3[49–51] and Mu [52, 53]. Binding to both domains, however, was shown to occur in fully formed SCs in Mu [54–56] and in IS50. We conclude from these analyses that the bipartite binding pattern exhibited in IS2 protein/DNA complexes is the result of a fully formed Step I SC.
Footprinting results in SC I correlate well with those of previous mutational analyses of the PBD of the single IRL end
An earlier mutational analysis of the IRL sequence indicated that, while residues 12 to 19 (primarily the B element) played an important role in protein recognition, an anchoring sequence for the transposase was also located at residues 20 to 42 (elements II, C and III; ). In general, the footprinting data (Figure 6) support these conclusions. We assessed the effect of seven single base deletion mutations on the efficiency of minicircle formation and found that there is a good correlation with current binding efficiency data. Deletion of base pairs at positions 13, 19, 21 and 36 had no effect on minicircle efficiency. In these footprinting studies, only position 21 was protected by the protein. Deletions of base pairs at positions 14, 26 and 29 eliminated minicircle formation and only residue 26 was not protected by the protein.
Footprinting the IS2 MCJ
In Step II of the IS2 transposition pathway, donor function of each of the abutted ends at the MCJ is a prerequisite for insertion of the element into the target sequence. In an earlier model [10, 24], we proposed for the sake of simplicity that the complex involved a dimer of transposase molecules with the PBD of each end bound at its own monomer. Initial cleavage of the abutted CDs of the MCJ would occur at the 3' end of the IRR CD, bound in cis at its cognate CC (a first transition state). As a result of a conformational change the partially cleaved junction would be relocated to permit cis-binding of the IRL CD at its cognate CC (a second transition state). There, cleavage at its 3' terminus would occur, permitting the reacquisition of cis binding by the IRR CD.
To test these ideas, we asked here whether a covalently joined MCJ (substrate MJcj) and a precleaved MCJ (substrate MJpc) would produce different SC II footprinting patterns for the IRR and IRL CDs. The covalently joined MCJ was prepared from two annealed 114 nucleotide oligomers (substrate MJcj in the Methods section) containing an 84 bp sequence of the abutted right and left ends separated by a single guanine/cytosine base pair. For footprinting experiments, the bottom strand (3' to 5') was labeled at its 3' end with alpha 32P-labelled di-deoxy adenosine triphosphate ([α32P] ddATP). Substrate MJpc (see the Methods section) containing the precleaved MCJ was prepared using a bottom strand identical to that in the MJcj substrate and labeled as described above. The top strand consisted of two oligomers; at the 5'end was a 56 nucleotide oligonucleotide, containing the 41 nucleotide donor strand of IRR ending in its CA-3' terminal dinucleotide. The second component was a 58 nucleotide oligonucleotide containing the 42 nucleotide strand of IRL, with a single nucleotide (C) at its 5' end representing the spacer base between the two abutted ends. The result of the annealing reaction was a double-stranded MCJ with a one base pair spacer, nicked at the CA-3' terminus of the IRR CD. Very efficient binding of the protein to both substrates was observed in EMSA gels (Figure 3B). The slight difference in the running patterns of the two complexes may be attributed to the differences in the structure of the two substrates.
CDs in the MJcj substrate are complexed to the same catalytic center followed by a cleavage-triggered conformational change
Secondly, in the MJcj substrate, the CDs of both IRL and IRR are protected in a similar manner by the protein (compare L2, L5, L6 and L9 to 11 with R2, R3, R7 and R11 in Figure 9 and 7B, lane 3). This result suggests that the CD of IRL in the MJcj substrate is extensively bound at the same CC as the IRR CD. Since the two CDs in the IS2 MJcj substrate are separated by a single base pair, their observed extensive protection (summarized in Figure 9) should result from initial binding of both CDs (the IRL in trans and the IRR in cis) at a single active CC (Figure 10B). This represents the first phase of the SC II complex in IS2. A similar scenario may apply to IS21, IS1665 and IS256 in Tn4001.
Thirdly, the MJpc substrate shows evidence of disengagement of the IRL CD from the TPase (Figure 9). In the covalently joined substrate, three sets of residues within the IRL CD are bound moderately tightly (L2 (T), L5 and L6 (GA) and L9 to L12 (GGGG)); of these, only two residues (L9 and L10) are protected in the precleaved substrate (see also the protection patterns labeled (a) in Figures 7B and 8A). This is not the case for the IRR CD where binding is even more extensive than in the MJcj. Based on two lines of evidence, we conclude that the partially cleaved junction is not positioned at the IRL CC after right end cleavage, as suggested in our original hypothesis. First, the apparent disengagement of the IRL CD suggests that it is not bound extensively at its cognate CC. Secondly, the IRR CD in the precleaved substrate remains bound at its cognate CC as judged by the similarity of its protection pattern to that of the single-end substrate. We propose then, that the protection patterns observed for the MJpc substrate represent those of a temporary (and artifactual) transition state and that complete disengagement of the IRL CD would follow the two sequential single-strand cleavages at the IRR CC (Figure 10B). After a conformational change, re-engagement of the IRL CD at a new site, its cognate CC, would then occur to produce a second complex in SC II (Figure 10C).
There is additional evidence for this temporary transition state. Two differences within the CDs of the two MCJ substrates at residues R1 (T) and R5 to R8 (TTTC) make the profile of the IRR CD in the MJpc substrate almost identical to that of the single-end substrate (Figure 9; see also the gel in Figure 7B, lane 4, protection patterns labeled (b) and (c)). Also, there are subtle differences in the protection patterns of the IRR PBD in the two MCJ substrates; residues R11 to R15, which are protected in the MJcj substrate, are disengaged in the MJpc substrate (Figure 7B, compare lanes 3 and 4, protection patterns labeled (d)), again making its protection pattern almost entirely like that of the single-end substrate (Figure 9). We note that major changes in protection patterns do not affect the PBDs. There is a basic similarity but not identity in the protection patterns within the PBDs IRR (R17 to R19 and R26 to R31) and IRL (L15 to L18 and L21 to L28) in each of the three substrates (Figure 9).
It is now well understood that the process of transpositional recombination is controlled by a series of conformational changes within the transpososome that drive the process forward unidirectionally. These may be triggered by cleavages , host proteins , divalent cations , the role of terminal cognate nucleotides  and associated transposition proteins . It appears here that sequential cleavages at the abutted IRR and IRL CDs of the first phase in the SC II transpososome of IS2 would provoke the conformational change that is required for the establishment of the second phase that is needed for final strand transfer reactions into the target DNA.
The sequence of the IRL CD has evolved to permit selective binding in SC I without compromising extensive sequence-specific binding in SC II
In earlier studies we proposed that the non-conserved base pairs in IRL were necessary for efficient recipient end function and were sufficient to prevent binding of the CD in cis to its cognate active site in SC I, without compromising binding proficiency in SC II . Footprinting data for the MJcj substrate support these suppositions. Six of the eleven residues within IRL CD in the bottom strand of the MJcj substrate are bound by the protein. The non-conserved residues at positions L2 and L5 are protected, as is the run of guanines at positions L9 (non-conserved) to L12 (Figure 9). Protection of this guanine/cytosine run is characteristic of strong extensive binding in the single-end IRR substrate and is not observed in the single-end IRL substrate (Figure 6). Thus, two of the three residues in the IRL CD that are involved in selective binding in SC I, are also utilized in extensive sequence-specific binding in SC II. It seems likely that this extensive sequence-specific binding of the IRL CD in the MJcj substrate results partially from its proximity to the extensively bound IRR CD. In addition, given the proximity of selective binding of the IRL CD and the non-specific and/or selective binding of the adjacent host DNA in the L79 single-end substrate (Figure 6), we propose that the nature of (or the absence of) the DNA adjacent to the cleavage domain of IRL plays a decisive role in determining whether it is involved in selective binding or extensive sequence-specific binding.
Evidence for bending of the DNA in MCJ and single-end substrates from footprinting data is corroborated by curvature propensity plot data from the MJcj sequence
The differences in the protection patterns of the MJcj and MJpc may be due to perturbation, generated as suggested above by the binding of both CDs at a single active site. Perturbation in the MJcj substrate is indicated by the presence of five enhanced residues in the PBD of IRR and four enhanced residues in the CD and the PBD of IRL. In contrast, in the MJpc substrate, only a single residue (R24) is enhanced in the PBD of IRR (Figure 9; see also Figure 7B, lanes 3 and 4 and Figure 8), suggesting not only that perturbation of the IRR DNA is relieved following cleavage but that the remaining enhanced R24 residue in both substrates is indicative of intrinsic bending at that site. In addition, the location of the enhanced region within the PBD of the IRR single end substrate (see residues R25 and R29 to R30; Figure 6) is similar to that in the two MCJ substrates (see residue R24; Figure 9), suggesting a single bend in IRR DNA in all three substrates. In a similar vein, enhanced residues in IRL are observed at two identical locations in the MJcj and MJpc substrates, that is, residue L3 in the CD and L14 at the outer end of the PBD (Figures 8 and 9). An enhanced residue is also observed at L15 in MJpc. In addition the enhanced region in the CD of the IRL single-end substrate (see residues L1, L3, L6 to L8 and L10; Figure 6) is in the same location in the MCJ substrates (see residues L3 and L8; Figure 9), suggesting that the tip of IRL is bent in all three substrates to accommodate binding in trans to its CD at the active IRR CC as illustrated in Figure 10A, B. Thus the presence of enhanced residues that are at common or near common locations in the single end, MJcj and MJpc substrates may be indicative of bending at intrinsically bent sites (see below).
We have asked whether the curvature maxima identified within the MJcj sequence are a reflection of the intrinsic curvature resulting from the interaction of the two CDs or curvature associated with the powerful promoter within the MCJ sequence. Since the curvature maxima at L6 and R25 correspond to enhancements in both the single-end substrates and the MCJ substrates as described above, we interpret the enhancements in the L79 (IRL) substrate as the result of bending to accommodate binding of both IRL CDs in trans in SC I (see the bend in IRL in Figure 10A) and in the MJcj substrate as bending to accommodate binding of the abutted CDs of the MCJ to a single CC in SC II (Figure 10B); indeed, observed perturbations of the DNA in the footprinted gels of the MJcj and MJpc substrates or of the single ends may result from the DNA being bent by the protein in the same direction as the intrinsic sequence-dependent curvature . We note that there is no enhancement of the residues in the PBD of the L79 (IRL) single-end substrate corresponding to the L25 maximum of the MCJ sequence (see below). In addition, the weak curvature maximum at R9 in the MJcj sequence is probably a relic of the type of IRR CD to CD interaction described earlier, for elements with two right ends. Thus the intrinsic curvature data not only corroborate the footprinting data but also support the idea that interaction of the CDs at a single active CC requires the adoption of a bent structure.
Intrinsic curvature of IS2 target sites
Because binding of the IRL CD in trans at the active CC within SC I and SC II seems to require the DNA to adopt a bent structure, we wondered whether IS2 target sites could also be structurally constrained. Therefore we decided to look at the predicted curvature of 200 bp-sized sequences from several IS2 target sites reported in the literature. A representative sample of these is shown in Figure 11B (the remaining curvature profiles are presented in Additional file 3). An interesting feature of the data is a consistent periodic behavior of the predicted curvature of sequences flanking the target sites. Subsequent analysis led to the division of the target sites into four regions (R1 to R4), a profile which was roughly similar in all of the DNA sequences examined. R1 holds a local minimum in predicted curvature, R2 a local maximum harboring a shoulder peak that sometimes appears as two well resolved peaks and regions, and R3 and R4 hold a local minimum and maximum respectively. IS2 insertion sites (black dots) mapped preferentially within the sub-sequences of R2 with a mean curvature of 4.4 ± 1.9 degrees per 10.5 bp helical turn. It is thus tempting to assume that the choice of insertion site might depend on DNA curvature at the target with the decision for integration based on subsequences of R2 having a certain range of curvature values. This similarity between curvature profiles is reflected in the three-dimensional structure of each region (Figure 11C) where an S-like (and sometimes L-like) structure is preferentially adopted. IS2 insertion sites were found to be located between two bent regions ([65, 66]; Figure 11C, i, ii) or alternatively exactly at a bent region ([67, 68]; Figure 11C, iii, iv). Additional three-dimensional representations of the curvature profiles are also presented in Additional file 4.
A model for the two-step transposition pathway of IS2; CD to CD interactions require that IRL adopt a bent structure in SC I and SC II
We describe here a refined version of our model for SC I and SC II . For SC I, single bends of the IRR PBD and of the IRL CD are required to synapse the CDs in two different orientations, I and II respectively, at the single active CC as illustrated in Figure 10A. For the first phase of the SC II complex, binding of the two CDs separated by a single base pair suggest that the CDs are complexed in orientation I at the active IRR CC. Two bends of IRL, at the CD and the outer end of the PBD and a single bend of the IRR PBD are needed to achieve this binding arrangement (Figure 10B), where sequential cleavage reactions would occur to generate the second phase of the complex (Figure 10C).
Intrinsic curvature data have indicated that both MCJ DNA and target sites adopt bent structures that apparently share identical profiles (compare Figure 11A (i) and 11B). Given the large number of target sites analyzed (Additional file 4), it is tempting to assume that curving propensity might play some role in target site selection although it is not clear how and to what extent this would affect the mechanics of transposition. A similar dependence between transposition and target curvature has been shown to exist for IS231, where target sites contain alternate guanine/cytosine- and adenine/thymine-rich tracts that promote bending in opposite directions of the regions flanking the consensus target sequence. In a more recent example, Kobori et al.  reported a target site for the spontaneous insertion of IS10 located within an intrinsically bent DNA region of the commonly used vector pUC19. Likewise, we observe from Figure 11C that IS2 preferentially inserts in the close vicinity of curved regions or specifically at a bent region. This concept has been incorporated into the model of the second phase of the SC II complex, where curved target DNA is now bound non-specifically across each CC permitting strand transfer to the target by each donor end (Figure 10C).
Bacterial strains and media
Escherichia coli strain JM105 was used for cloning and for most procedures involving plasmid DNA preparation. DNA transformation was carried out into supercompetent XL1 Blue cells (Stratagene Inc., Santa Clara, CA, USA) for reactions requiring cloning and expression of pLL2522, the plasmid with the fused orfAB and GFPuv genes.
Cultures were routinely grown in lysogeny broth media at 37°C, supplemented where necessary with carbenicillin (Cb, 50 μg/mL) or chloramphenicol (Cm, 20 μg/mL). For the overexpression of the fused orfAB::GFP genes in plasmid pLL2522, cultures were grown at 28°C in a 2× yeast extract and tryptone (2 × YT) medium supplemented with Cm, Cb and arabinose (6 mg/mL).
pLL2522, which contained the fused orfAB and GFPuv genes, has been described in detail previously .
Preparation of the OrfAB-GFP fusion protein under native conditions
Plasmid pLL2522 was transformed into BL21(DE3)pLysS cells (Stratagene Inc.). Single colonies were inoculated into 40.0 mL of 2 × YT medium supplemented with Cm, Cb and arabinose and inoculated in baffled flasks overnight at 28°C. Harvested pellets were checked for bright fluorescence, washed with 3.0 mL Native Wash Buffer (Qiagen, Valencia, CA, USA) and frozen at -70°C for 15 min. Three milliliters of B-PER Protein Extraction Reagent (Thermo Scientific, Pierce Protein Research Products, Rockford, IL, USA), supplemented with 4.0 μL of Benzonase (Novagen-EMD4Biosciences, La Jolla, CA, USA), per 40 mL of overexpressed culture and 3.0 mL Protease Arrest (Calbiochem/EMD La Jolla, CA, USA) per milliliter of lysate was added to the frozen pellet, which was allowed to thaw on ice on a horizontal rotary shaker for 60 min. The lysate was nutated at 4°C for 1 h and subjected to a hard spin at 10,000 ×g for 45 min at 4°C. It was then purified through Ni-NTA His-tag technology. 6 × His-tag purification of the protein was achieved by gravity flow affinity chromatography using Ni-NTA agarose (Qiagen) under native conditions essentially following the manufacturer's instructions. The crude lysate was loaded on to a 1.0 mL bed of the nickel-charged resin in a 5.0 mL column for chromatographic separation followed with UV light. The protein bound as a tight brightly fluorescing band at the top of the column and remained bound through washings with 10 mM to 60 mM imidazole, when a slight dissociation of the band was observed. To circumvent continued dissociation, the band was eluted with 250 mM imidazole and its progress through the column followed. Peak fractions (fluorometrically determined) were subjected to diagnostic 12% PAGE using acrylamide and bis-acrylamide (Ac:Bis; 30%:8%, respectively) polyacrylamide gels . Fractions showing both the 74-kDa OrfAB::GFP and the 17-kDa OrfA proteins were pooled (approximately 700 μL), concentrated to about 75 μL in a YM-10 Microcon Centrifugal Filter Device (Millipore, Billerica, MA, USA), dialyzed overnight in Slide-A-Lyzer cassettes (Thermo Scientific, Pierce Protein Research Products) and stored in 50% glycerol at -20°C. The concentration of the fused OrfAB-GFP protein was measured with spectrophotometry at 397 nm and that of the control GFP at 280 nm and 397 nm. Comparative levels of fluorescence of GFP and the fusion proteins were measured with fluorometry and used to confirm the concentration data.
Oligonucleotides used in gel retardation and DNA footprinting experiments
The right single end (IRR) was represented by an 87-bp substrate R87. The IRR sequence is shown between the brackets. Top strands were labeled at the 5' end and bottom strands at the 3' end. The top strand (primer A) sequence was as follows: 5'GCTGACTTGACGGGACGGGGATCC[TTAAGTGATAACAGATGTCTGGAAATATAGGGGCAAATCCA]ATCGACCTGCAGGCATATAAGC3'; the bottom strand (primer B) sequence was as follows: 5'GCTTATATGCCTGCAGGTCGAT[TGGATTTGCCCCTATATTTCCAGACATCTGTTATCACTTAA]GGATCCCCGTCCCGTCAAGTCAGC3'.
The left single end (IRL) was represented by the 78-bp substrate L79. The IRL sequence is shown between the brackets. The top strand sequence (primer A) was as follows: 5'ACGCGGAGTGAATTCGAGCTC[TAGACTGGCCCCCTGAATCTCCAGACAACCAATATCACTTAA]ATAAGTGATAGTCTTA3'; bottom strand (primer B) sequence was as follows: 5'TAAGACTATCACTTAT[TTAAGTGATATTGGTTGTCTGAAGATTCAGGGGGCCAGTCTA]GAGCTCGAATTCCACTCCGCGT3'.
The covalently closed MCJ was represented by the 114-bp substrate, MJcj. The abutted IRR (bold) and IRL sequences, shown in the brackets, are separated by a single base pair guanine/cytosine spacer. The top strand sequence (primer A) was as follows: 5'GGTACCCGGCCATGG[ttaagtgataacagatgtctgggaaatataggggcaaatcca]C[TAGACTGGCCCCCTGAATCTCCAGACAACCAATATCACTTAA]ATAAGTTATAGTCTT3'; bottom strand (primer B) sequence was as follows: 5'AAGACTATAACTTAT[TTAAGTGATATTGGTTGTCTGGAGATTCAGGGGGCCAGTCTA]G[TGGATTTGCCCCTATATTTCCAGACATCTGTTATCACTTAA]GGATCCCCGGGTACC3'.
The precleaved (nicked) MCJ was represented by the 114-bp MJpc substrate. Two oligonucleotides were needed to create the top strand. The first, a 56-mer oligonucleotide contained the IRR sequence (bold font) terminated with an A-3'OH at the junction and was labeled at its 5' end. The sequence for the top strand (primer A1) was as follows: 5'GGTACCCGGGGATCC[TTAAGTGATAACAGATGTCTGGAAATATAGGGGCAAATCCA]3'.
The second primer, a 58-mer oligonucleotide, terminated at its 5' end with a cytosine representing the single spacer nucleotide. It was labeled at its 5' end. Its sequence (Primer A2) was: 5'C[TAGACTGGCCCCCTGAATCTCCAGACAACCAATATCACTTAA]ATAAGTTATAGTCTT3'. The bottom strand was identical to that described for the MJcj substrate.
5'- and 3'- end labeling and annealing of the oligonucleotides
5'-end labeling of the primers: A 20-μL labeling reaction contained 30 units of T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA), 2.0 μL of 10X T4 polynucleotide kinase reaction buffer, 20 μM of the primer, 50 μCi of the gamma 32P-labeled adenosine triphosphate (γ32PATP) (6000 Ci/mmole). The reaction was incubated at 37°C for 30 min and heat killed at 90°C for 5 min.
3'-end labeling of the primers: The 50-μL reaction contained 20 units of terminal transferase in 1X reaction buffer (USB Corp, Cleveland, OH, USA), 20 μM of the oligonucleotide and 50 μCi of α32PddATP. The reaction was incubated at 37°C for 1 h, terminated with 10 μL 2 M ethylenediaminetetraacetic acid (EDTA) and heat killed at 70°C for 10 min.
A 100-μL annealing reaction contained 10 ρmol and 13 ρmol of the labeled and unlabeled strands respectively, 20 mM tris(hydroxymethyl)aminomethane-chloride (Tris-Cl) pH 8.0, and 100 mM sodium chloride. The reaction was placed in a boiling water bath, cooled to 65°C, held there for 15 min and allowed to cool to room temperature. Annealed oligonucleotides were stored at -20°C.
Protein-DNA complex formation and EMSA
Protein-DNA binding reactions were carried out in 20-μL reaction mixtures with 20 mM Tris-Cl, pH 8.0.Cl, 1 mM EDTA, 1.0 μg/mL calf thymus DNA, 2 nM of the radioactively labeled annealed primers and 80 nM of the partially purified preparation of the OrfAB-GFP fusion protein. Reactions were incubated for 30 min at room temperature and electrophoresed through 5% 19:1 Ac:Bis native polyacrylamide gels at 4°C for 1,000 Vhr.
In-gel cleavage assays of OrfAB complexed with IRR substrates
DNA substrates used in complex formation: An 87-bp IRR substrate (see description of oligonucleotides) and a 50-bp IRR substrate  were used in the preparation of protein-DNA complexes. Three types of complexes were formed: (a) with the 50-bp substrate alone, (b) with the 87-bp substrate alone and (c) with a mixture of the 50-bp and 87-bp substrates. Complexes were electrophoresed as described above.
In-gel excision of the complexes and activation of the TPnase: Complexes were excised and activation effected based partly on the protocol of Bhasin et al. . Essentially the gel was wrapped and exposed to X-ray film for 30 min. It was then superimposed over the developed film and complexes excised based on the location of the images. Each excised gel slice was cut in half and placed into separate 2.0-mL eppendorf tubes. To one tube, 1 mL of an activation buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 100 mM K glutamate and 10 mM magnesium chloride or magnesium acetate) was added. To the second control tube, 1 mL of the same buffer lacking Mg++ was added. Gels were incubated at 37°C for 5 min and rinsed twice with 1.0 mL nuclease free water (Ambion/Life Technologies, Grand Island, NY, USA).
Elution of DNA from gel slices: The gels were crushed with a micro pestle in 1.0 mL of a 'crush and soak' buffer (10 mM Tris.Cl, 1% SDS and 10 mM EDTA) and nutated at 4°C overnight. The gel pieces were pelleted at 14 K rpm in a microcentrifuge at room temperature for 10 min then rinsed in 500 μL of the same buffer. The resulting 1.5 mL supernatant was then reduced to about 400 μl with three consecutive 14 K rpm spins in a YM-10 Microcon Centrifugal Filter Device (Millipore). Each sample was then subjected to seven buffer exchange (topped up with 400 mL Tris-EDTA pH 8.0) spins at 14 K rpm for 16 min at room temperature. Samples were dried down to a pellet in a Savant SpeedVac DNA concentrator (Savant Instruments, Inc., Holbrook, New York, USA) and resuspended in 2.5 μL nuclease-free water, 2.5 μL of gel loading buffer (GE Healthcare Biosciences, Piscataway, NJ, USA), placed in a boiling water bath for 5 min and stored at -20°C.
Hydroxyl radical footprinting protocols
Two reactions, one for the footprinting experiment and the other for the free DNA control, were prepared for each substrate as described for the EMSA protocol but with two modifications. Hydroxyl radicals were generated by the Fenton reaction . Reactions were carried out in 70-μL volumes and protein was added to the footprinting tube only, at a final concentration of 225.7 nM. The tubes were incubated at room temperature for 30 min and then subjected to OH radical cleavage. Final concentrations of 5 mM ferrous ammonium sulfate ((NH4)2 Fe(SO4)2.6H2O), 10 mM EDTA and 0.05% hydrogen peroxide were added to each tube to bring the final volume to 100 μL. These reactants were added as three drops to the side of the tube, then mixed and immediately combined with the sample. The reaction was incubated at room temperature for 2 min and stopped by adding an equal volume of stop buffer consisting of 4% glycerol, 0.6 mM sodium acetate (NaOAc)and 50 μg/mL tRNA. Thiourea was also added as a stop reagent to a final concentration of 11.4 mM.
Purification of the DNA was initiated by removing the protein by the addition of an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1; Sigma-Aldrich, St. Louis, MO, USA), vortexing for 10 s and centrifuging at 15,000 ×g for 2 min. Aqueous layers were removed from each of two repetitions and the DNA was precipitated by adding first NaOAc and glycogen to final concentrations of 100 mM and 0.3 μg/mL, respectively, and then twice the reaction volume of 100% ethanol kept at -20°C. The reaction was stored at -70°C overnight and pellet recovery followed standard procedures . The pellet was dissolved in 10 μL formamide-based loading buffer and stored at -20°C. G+A Maxam-Gilbert sequencing reactions followed the standard procedure . The three reactions, footprinting, free DNA and Maxam-Gilbert, were run side by side in 8.0% polyacrylamide sequencing gels at 1400 v 40 W. The results were quantified on a Typhoon phosphorimager 9400 (GE Healthcare).
In silico prediction of intrinsic DNA curvature
Curvature propensity plots were obtained using the BEND algorithm  by submission of DNA sequences to the bend.it server (http://hydra.icgeb.trieste.it/dna/bend_it.html; ) using the DNAse I-based parameters of Brukner et al. . This server calculates DNA curvature as a vector sum of dinucleotide geometries (roll, tilt and twist angles) and expresses it as degrees per helical turn (10.5° per helical turn = 1° per base pair). DNA sequences were submitted in raw format and the predicted curvature was collected through email in ASCII format. Three-dimensional representation of the curvature profiles was performed with the model.it server (http://hydra.icgeb.trieste.it/dna/model_it.html; ) and the output was displayed and visualized with MOLEGRO Molecular Viewer http://www.molegro.com/mmv-product.php. A literature search was performed to analyze the intrinsic curvature of IS2 target sites and a detailed list of several DNA sequences from genomic, phage and plasmid DNA encompassing different IS2 target sites was gathered. Each of these sequences was analyzed in 200 bp-sized windows by bend.it and model.it. The mean curvature of all IS2 target sites was also computed.
electrophoretic mobility shift assay
green fluorescent protein
right and left imperfect, inverted repeats
covalently joined minicircle junction substrate
precleaved minicircle junction substrate
open reading frame
protein binding domain
- 2 × YT:
2× yeast extract and tryptone.
We thank NDF Grindley for useful discussions, T Seymour for help with Figure 1 and J Lopino for help with Figure 10. We especially thank T. Paglione and N. Khandaker for use of facilities in the York College Department of Earth and Physical sciences. This research was supported by US Public Health Service grant NIGMS/MBRS GMO8153, a York College FDSP award 990110 to LAL and by Fundação para a Ciência e a Tecnologia (PPTDC/EBB-BIO/113650/2009, BD/22320/2005) to PHO.
- Chandler M, Mahillon J: Insertion sequences revisited. Mobile DNA II. Edited by: Craig NL, Craigie R, Gellert M, Lambowitz AM. 2002, Washington, DC: ASM Press, 305-366.View ArticleGoogle Scholar
- Rousseau P, Normand C, Loot C, Turlan C, Alazard R, Duval-Valentin G, Chandler M: Transposition of IS911. Mobile DNA I. Edited by: Craig NL, Craigie R, Gellert M, Lambowitz AM. 2002, Washington, DC: ASM Press, 367-383.View ArticleGoogle Scholar
- Duval-Valentin G, Marty-Cointin B, Chandler M: Requirement of IS911 replication before integration defines a new bacterial transposition pathway. Embo J. 2004, 23: 3897-3906. 10.1038/sj.emboj.7600395.PubMed CentralView ArticlePubMedGoogle Scholar
- Craig NL: Unity in transposition reactions. Science. 1995, 270: 253-254. 10.1126/science.270.5234.253.View ArticlePubMedGoogle Scholar
- Haren L, Ton-Hoang B, Chandler M: Integrating DNA: transposases and retroviral integrases. Annu Rev Microbiol. 1999, 53: 245-281. 10.1146/annurev.micro.53.1.245.View ArticlePubMedGoogle Scholar
- Turlan C, Chandler M: Playing second fiddle: second-strand processing and liberation of transposable elements from donor DNA. Trends Microbiol. 2000, 8: 268-274. 10.1016/S0966-842X(00)01757-1.View ArticlePubMedGoogle Scholar
- Polard P, Chandler M: An in vivo transposase-catalyzed single-stranded DNA circularization reaction. Genes Dev. 1995, 9: 2846-2858. 10.1101/gad.9.22.2846.View ArticlePubMedGoogle Scholar
- Polard P, Ton-Hoang B, Haren L, Betermier M, Walczak R, Chandler M: IS911-mediated transpositional recombination in vitro. J Mol Biol. 1996, 264: 68-81. 10.1006/jmbi.1996.0624.View ArticlePubMedGoogle Scholar
- Ton-Hoang B, Betermier M, Polard P, Chandler M: Assembly of a strong promoter following IS911 circularization and the role of circles in transposition. Embo J. 1997, 16: 3357-3371. 10.1093/emboj/16.11.3357.PubMed CentralView ArticlePubMedGoogle Scholar
- Lewis LA, Cylin E, Lee HK, Saby R, Wong W, Grindley ND: The left end of IS2: a compromise between transpositional activity and an essential promoter function that regulates the transposition pathway. J Bacteriol. 2004, 186: 858-865. 10.1128/JB.186.3.858-865.2004.PubMed CentralView ArticlePubMedGoogle Scholar
- Lewis LA, Grindley ND: Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway. Mol Microbiol. 1997, 25: 517-529. 10.1046/j.1365-2958.1997.4871848.x.View ArticlePubMedGoogle Scholar
- Sekine Y, Aihara K, Ohtsubo E: Linearization and transposition of circular molecules of insertion sequence IS3. J Mol Biol. 1999, 294: 21-34. 10.1006/jmbi.1999.3181.View ArticlePubMedGoogle Scholar
- Sekine Y, Eisaki N, Ohtsubo E: Translational control in production of transposase and in transposition of insertion sequence IS3. J Mol Biol. 1994, 235: 1406-1420. 10.1006/jmbi.1994.1097.View ArticlePubMedGoogle Scholar
- Haas M, Rak B: Escherichia coli insertion sequence IS150: transposition via circular and linear intermediates. J Bacteriol. 2002, 184: 5833-5841. 10.1128/JB.184.21.5833-5841.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Kiss J, Olasz F: Formation and transposition of the covalently closed IS30 circle: the relation between tandem dimers and monomeric circles. Mol Microbiol. 1999, 34: 37-52. 10.1046/j.1365-2958.1999.01567.x.View ArticlePubMedGoogle Scholar
- Szabo M, Kiss J, Nagy Z, Chandler M, Olasz F: Sub-terminal sequences modulating IS30 transposition in vivo and in vitro. J Mol Biol. 2008, 375: 337-352. 10.1016/j.jmb.2007.10.043.View ArticlePubMedGoogle Scholar
- Berger B, Haas D: Transposase and cointegrase: specialized transposition proteins of the bacterial insertion sequence IS21 and related elements. Cell Mol Life Sci. 2001, 58: 403-419. 10.1007/PL00000866.View ArticlePubMedGoogle Scholar
- Prudhomme M, Turlan C, Claverys JP, Chandler M: Diversity of Tn4001 transposition products: the flanking IS256 elements can form tandem dimers and IS circles. J Bacteriol. 2002, 184: 433-443. 10.1128/JB.184.2.433-443.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Ton-Hoang B, Polard P, Haren L, Turlan C, Chandler M: IS911 transposon circles give rise to linear forms that can undergo integration in vitro. Mol Microbiol. 1999, 32: 617-627. 10.1046/j.1365-2958.1999.01379.x.View ArticlePubMedGoogle Scholar
- Hu ST, Hwang JH, Lee LC, Lee CH, Li PL, Hsieh YC: Functional analysis of the 14 kDa protein of insertion sequence 2. J Mol Biol. 1994, 236: 503-513. 10.1006/jmbi.1994.1161.View ArticlePubMedGoogle Scholar
- Polard P, Prere MF, Chandler M, Fayet O: Programmed translational frameshifting and initiation at an AUU codon in gene expression of bacterial insertion sequence IS911. J Mol Biol. 1991, 222: 465-477. 10.1016/0022-2836(91)90490-W.View ArticlePubMedGoogle Scholar
- Vogele K, Schwartz E, Welz C, Schiltz E, Rak B: High-level ribosomal frameshifting directs the synthesis of IS150 gene products. Nucleic Acids Res. 1991, 19: 4377-4385. 10.1093/nar/19.16.4377.PubMed CentralView ArticlePubMedGoogle Scholar
- Hu ST, Lee LC, Lei GS: Detection of an IS2-encoded 46-kilodalton protein capable of binding terminal repeats of IS2. J Bacteriol. 1996, 178: 5652-5659.PubMed CentralPubMedGoogle Scholar
- Lewis LA, Gadura N, Greene M, Saby R, Grindley ND: The basis of asymmetry in IS2 transposition. Mol Microbiol. 2001, 42: 887-901. 10.1046/j.1365-2958.2001.02662.x.View ArticlePubMedGoogle Scholar
- Szeverenyi I, Bodoky T, Olasz F: Isolation, characterization and transposition of an (IS2)2 intermediate. Mol Gen Genet. 1996, 251: 281-289.PubMedGoogle Scholar
- Normand C, Duval-Valentin G, Haren L, Chandler M: The terminal inverted repeats of IS911: requirements for synaptic complex assembly and activity. J Mol Biol. 2001, 308: 853-871. 10.1006/jmbi.2001.4641.View ArticlePubMedGoogle Scholar
- Rousseau P, Tardin C, Tolou N, Salome L, Chandler M: A model for the molecular organisation of the IS911 transpososome. Mob DNA. 2010, 1: 16-10.1186/1759-8753-1-16.PubMed CentralView ArticlePubMedGoogle Scholar
- Rousseau P, Loot C, Guynet C, Ah-Seng Y, Ton-Hoang B, Chandler M: Control of IS911 target selection: how OrfA may ensure IS dispersion. Mol Microbiol. 2007, 63: 1701-1709. 10.1111/j.1365-2958.2007.05615.x.View ArticlePubMedGoogle Scholar
- Reimmann C, Moore R, Little S, Savioz A, Willetts NS, Haas D: Genetic structure, function and regulation of the transposable element IS21. Mol Gen Genet. 1989, 215: 416-424. 10.1007/BF00427038.View ArticlePubMedGoogle Scholar
- Kiss J, Nagy Z, Toth G, Kiss GB, Jakab J, Chandler M, Olasz F: Transposition and target specificity of the typical IS30 family element IS1655 from Neisseria meningitidis. Mol Microbiol. 2007, 63: 1731-1747. 10.1111/j.1365-2958.2007.05621.x.View ArticlePubMedGoogle Scholar
- Lewis LA, Astatke M, Umekubo PT, Alvi S, Saby R, Afrose J: Soluble expression, purification and characterization of the full length IS2 transposase. Mob DNA. 2011, 2: 14-10.1186/1759-8753-2-14.PubMed CentralView ArticlePubMedGoogle Scholar
- Haren L, Normand C, Polard P, Alazard R, Chandler M: IS911 transposition is regulated by protein-protein interactions via a leucine zipper motif. J Mol Biol. 2000, 296: 757-768. 10.1006/jmbi.1999.3485.View ArticlePubMedGoogle Scholar
- Nagy Z, Szabo M, Chandler M, Olasz F: Analysis of the N-terminal DNA binding domain of the IS30 transposase. Mol Microbiol. 2004, 54: 478-488. 10.1111/j.1365-2958.2004.04279.x.View ArticlePubMedGoogle Scholar
- Szabo M, Kiss J, Olasz F: Functional organization of the inverted repeats of IS30. J Bacteriol. 2010, 192: 3414-3423. 10.1128/JB.01382-09.PubMed CentralView ArticlePubMedGoogle Scholar
- Hennig S, Ziebuhr W: Characterization of the transposase encoded by IS256, the prototype of a major family of bacterial insertion sequence elements. J Bacteriol. 2010, 192: 4153-4163. 10.1128/JB.00226-10.PubMed CentralView ArticlePubMedGoogle Scholar
- Bhasin A, Goryshin IY, Steiniger-White M, York D, Reznikoff WS: Characterization of a Tn5 pre-cleavage synaptic complex. J Mol Biol. 2000, 302: 49-63. 10.1006/jmbi.2000.4048.View ArticlePubMedGoogle Scholar
- Stalder R, Caspers P, Olasz F, Arber W: The N-terminal domain of the insertion sequence 30 transposase interacts specifically with the terminal inverted repeats of the element. J Biol Chem. 1990, 265: 3757-3762.PubMedGoogle Scholar
- Hallet B, Rezsohazy R, Mahillon J, Delcour J: IS231A insertion specificity: consensus sequence and DNA bending at the target site. Mol Microbiol. 1994, 14: 131-139. 10.1111/j.1365-2958.1994.tb01273.x.View ArticlePubMedGoogle Scholar
- Vinogradov AE: DNA helix: the importance of being GC-rich. Nucleic Acids Res. 2003, 31: 1838-1844. 10.1093/nar/gkg296.PubMed CentralView ArticlePubMedGoogle Scholar
- Lei GS, Hu ST: Functional domains of the InsA protein of IS2. J Bacteriol. 1997, 179: 6238-6243.PubMed CentralPubMedGoogle Scholar
- Haren L, Polard P, Ton-Hoang B, Chandler M: Multiple oligomerisation domains in the IS911 transposase: a leucine zipper motif is essential for activity. J Mol Biol. 1998, 283: 29-41. 10.1006/jmbi.1998.2053.View ArticlePubMedGoogle Scholar
- Derbyshire KM, Hwang L, Grindley ND: Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats. Proc Natl Acad Sci USA. 1987, 84: 8049-8053. 10.1073/pnas.84.22.8049.PubMed CentralView ArticlePubMedGoogle Scholar
- Johnson RC, Reznikoff WS: DNA sequences at the ends of transposon Tn5 required for transposition. Nature. 1983, 304: 280-282. 10.1038/304280a0.View ArticlePubMedGoogle Scholar
- Makris JC, Nordmann PL, Reznikoff WS: Mutational analysis of insertion sequence 50 (IS50) and transposon 5 (Tn5) ends. Proc Natl Acad Sci USA. 1988, 85: 2224-2228. 10.1073/pnas.85.7.2224.PubMed CentralView ArticlePubMedGoogle Scholar
- Huisman O, Errada PR, Signon L, Kleckner N: Mutational analysis of IS10's outside end. Embo J. 1989, 8: 2101-2109.PubMed CentralPubMedGoogle Scholar
- Zerbib D, Prentki P, Gamas P, Freund E, Galas DJ, Chandler M: Functional organization of the ends of IS1: specific binding site for an IS 1-encoded protein. Mol Microbiol. 1990, 4: 1477-1486. 10.1111/j.1365-2958.1990.tb02058.x.View ArticlePubMedGoogle Scholar
- Derbyshire KM, Grindley ND: Binding of the IS903 transposase to its inverted repeat in vitro. Embo J. 1992, 11: 3449-3455.PubMed CentralPubMedGoogle Scholar
- Jilk RA, York D, Reznikoff WS: The organization of the outside end of transposon Tn5. J Bacteriol. 1996, 178: 1671-1679.PubMed CentralPubMedGoogle Scholar
- Ichikawa H, Ikeda K, Amemura J, Ohtsubo E: Two domains in the terminal inverted-repeat sequence of transposon Tn3. Gene. 1990, 86: 11-17. 10.1016/0378-1119(90)90108-4.View ArticlePubMedGoogle Scholar
- Ichikawa H, Ikeda K, Wishart WL, Ohtsubo E: Specific binding of transposase to terminal inverted repeats of transposable element Tn3. Proc Natl Acad Sci USA. 1987, 84: 8220-8224. 10.1073/pnas.84.23.8220.PubMed CentralView ArticlePubMedGoogle Scholar
- New JH, Eggleston AK, Fennewald M: Binding of the Tn3 transposase to the inverted repeats of Tn3. J Mol Biol. 1988, 201: 589-599. 10.1016/0022-2836(88)90640-7.View ArticlePubMedGoogle Scholar
- Craigie R, Mizuuchi M, Mizuuchi K: Site-specific recognition of the bacteriophage Mu ends by the Mu A protein. Cell. 1984, 39: 387-394. 10.1016/0092-8674(84)90017-5.View ArticlePubMedGoogle Scholar
- Zou AH, Leung PC, Harshey RM: Transposase contacts with mu DNA ends. J Biol Chem. 1991, 266: 20476-20482.PubMedGoogle Scholar
- Lavoie BD, Chan BS, Allison RG, Chaconas G: Structural aspects of a higher order nucleoprotein complex: induction of an altered DNA structure at the Mu-host junction of the Mu type 1 transpososome. Embo J. 1991, 10: 3051-3059.PubMed CentralPubMedGoogle Scholar
- Mizuuchi M, Baker TA, Mizuuchi K: DNase protection analysis of the stable synaptic complexes involved in Mu transposition. Proc Natl Acad Sci USA. 1991, 88: 9031-9035. 10.1073/pnas.88.20.9031.PubMed CentralView ArticlePubMedGoogle Scholar
- Surette MG, Harkness T, Chaconas G: Stimulation of the Mu A protein-mediated strand cleavage reaction by the Mu B protein, and the requirement of DNA nicking for stable type 1 transpososome formation. In vitro transposition characteristics of mini-Mu plasmids carrying terminal base pair mutations. J Biol Chem. 1991, 266: 3118-3124.PubMedGoogle Scholar
- Gueguen E, Rousseau P, Duval-Valentin G, Chandler M: The transpososome: control of transposition at the level of catalysis. Trends Microbiol. 2005, 13: 543-549. 10.1016/j.tim.2005.09.002.View ArticlePubMedGoogle Scholar
- Chalmers R, Guhathakurta A, Benjamin H, Kleckner N: IHF modulation of Tn10 transposition: sensory transduction of supercoiling status via a proposed protein/DNA molecular spring. Cell. 1998, 93: 897-908. 10.1016/S0092-8674(00)81449-X.View ArticlePubMedGoogle Scholar
- Crellin P, Chalmers R: Protein-DNA contacts and conformational changes in the Tn10 transpososome during assembly and activation for cleavage. Embo J. 2001, 20: 3882-3891. 10.1093/emboj/20.14.3882.PubMed CentralView ArticlePubMedGoogle Scholar
- Yanagihara K, Mizuuchi K: Progressive structural transitions within Mu transpositional complexes. Mol Cell. 2003, 11: 215-224. 10.1016/S1097-2765(02)00796-7.View ArticlePubMedGoogle Scholar
- Lemberg KM, Schweidenback CT, Baker TA: The dynamic Mu transpososome: MuB activation prevents disintegration. J Mol Biol. 2007, 374: 1158-1171. 10.1016/j.jmb.2007.09.079.PubMed CentralView ArticlePubMedGoogle Scholar
- Hagerman PJ: Sequence-directed curvature of DNA. Annu Rev Biochem. 1990, 59: 755-781. 10.1146/annurev.bi.59.070190.003543.View ArticlePubMedGoogle Scholar
- Plaskon RR, Wartell RM: Sequence distributions associated with DNA curvature are found upstream of strong E. coli promoters. Nucleic Acids Res. 1987, 15: 785-796. 10.1093/nar/15.2.785.PubMed CentralView ArticlePubMedGoogle Scholar
- Nickerson CA, Achberger EC: Role of curved DNA in binding of Escherichia coli RNA polymerase to promoters. J Bacteriol. 1995, 177: 5756-5761.PubMed CentralPubMedGoogle Scholar
- Lewis LA, Gopaul S, Marsh C: The non-random pattern of insertion of IS2 into the hemB gene of Escherichia coli. Microbiol Immunol. 1994, 38: 461-465.View ArticlePubMedGoogle Scholar
- Sengstag C, Shepherd JC, Arber W: The sequence of the bacteriophage P1 genome region serving as hot target for IS2 insertion. EMBO J. 1983, 2: 1777-1781.PubMed CentralPubMedGoogle Scholar
- Oliveira PH, Prazeres DM, Monteiro GA: Deletion formation mutations in plasmid expression vectors are unfavored by runaway amplification conditions and differentially selected under kanamycin stress. J Biotechnol. 2009, 143: 231-238. 10.1016/j.jbiotec.2009.08.002.View ArticlePubMedGoogle Scholar
- Whiteway J, Koziarz P, Veall J, Sandhu N, Kumar P, Hoecher B, Lambert IB: Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to 5-nitrofuran derivatives in Escherichia coli. J Bacteriol. 1998, 180: 5529-5539.PubMed CentralPubMedGoogle Scholar
- Kobori S, Ko Y, Kato M: A target site for spontaneous insertion of IS10 element in pUC19 DNA located within intrinsically bent DNA. Open Microbiol J. 2009, 3: 146-150. 10.2174/1874285800903010146.PubMed CentralView ArticlePubMedGoogle Scholar
- Dixon WJ, Hayes JJ, Levin JR, Weidner MF, Dombroski BA, Tullius TD: Hydroxyl radical footprinting. Methods Enzymol. 1991, 208: 380-413.View ArticlePubMedGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning, A Laboratory Manual. 1989, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2Google Scholar
- Goodsell DS, Dickerson RE: Bending and curvature calculations in B-DNA. Nucleic Acids Res. 1994, 22: 5497-5503. 10.1093/nar/22.24.5497.PubMed CentralView ArticlePubMedGoogle Scholar
- Vlahovicek K, Kajan L, Pongor S: DNA analysis servers: plot.it, bend.it, model.it and IS. Nucleic Acids Res. 2003, 31: 3686-3687. 10.1093/nar/gkg559.PubMed CentralView ArticlePubMedGoogle Scholar
- Brukner I, Sanchez R, Suck D, Pongor S: Sequence-dependent bending propensity of DNA as revealed by DNase I: parameters for trinucleotides. EMBO J. 1995, 14: 1812-1818.PubMed CentralPubMedGoogle Scholar
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