The overwhelming body of evidence indicates that IS608
transposes using ssDNA (Kersulyte et al., 2002
; Ton-Hoang et al., 2005
; Ronning et al. 2005
), and we have recently shown that ssDNA LE and RE oligonucleotides are readily cleaved, form transposon junctions and sealed donor backbones, and integrate pre-formed transposon junctions into target DNA in the presence of TnpA (Guynet et al., in press
). With our structural results demonstrating how TnpA is activated and how it recognizes the transposon ends and its cleavage sites, together with the observation that helix αD is a mobile structural unit, we propose the following model for IS608
transposition (, Movie S2
Transposition starts with TnpA locating and pairing the transposon ends by binding to the hairpins formed by the top strand LE and RE IPs (). IPLE and IPRE differ by only one base in the hairpin loop, and our structures of the TnpA dimer bound to two identical DNA molecules most likely reliably reflect features of TnpA bound to one IPLE and one IPRE. Binding the transposon IPs induces the conformational change seen in the TnpA/LE26 complex during which helix αD moves into the activated position thereby assembling the active site.
Divalent metal ion binding to the assembled active site localizes and polarizes the scissile phosphate, preparing it for nucleophilic attack by Y127 (Hickman et al., 2002
; Larkin et al., 2005
). On LE, upon cleavage, Y127 becomes covalently linked to the 5′ end of the transposon while the 3′ end of flanking donor remains bound through base-base interactions between its TTAC
and the four nt just 5′ of IPLE
(as in the TnpA/LE26/D6 complex). We have no evidence that the 15 transposon nt between this 5′ extension and the 5′ end of the transposon interact with TnpA, consistent with their variability among different IS608
isolates (Berg et al., 2002).
On RE, cleavage results in a 5′ phosphotyrosine linkage between Y127 and the flanking donor DNA while the 3′ end of the transposon stays bound through base-pairing interactions between the terminal TCAA tetranucleotide and the four nt 5′ extension of IPRE (as in the TnpA/RE35 complex). The entire RE (represented by RE35) is ordered and its DNA conformation is stabilized by internal hydrogen bonds, consistent with its complete sequence conservation.
The subsequent formation of the transposition intermediates, a transposon junction and the sealed donor backbone, would arise straightforwardly if the two αD helices now trade places, pivoting on G117 and G118 and bringing the covalently linked DNA strands with them (, Movie S2
). This model requires the 3′ OH groups to remain in their “active sites of origin” to act as the nucleophiles to resolve the swapped phosphotyrosine linkages. This immobility seems likely as our structures of both LE and the RE complexes suggest that 3′ ends would remain bound as long as the IPs with their four nt 5′ extensions are present.
By this mechanism, in one active site of the dimer, a sealed donor backbone would be generated by nucleophilic attack by the stationary LE donor flanking 3′ OH (TTAC-OH) on the swapped RE donor flank 5′ phosphotyrosine linkage. In the other active site, attack by the resident RE 3′ OH (TCAA-OH) on the 5′ phosphotyrosine linkage of the swapped transposon LE would result in a transposon junction. This proposed reaction scheme is supported by the three previously unexplainable aspects of asymmetry seen at the transposon ends: the tolerance of sequence variability at LE but not at RE; the cleavage polarities that dictate that one phosphotyrosine linkage joins TnpA to the transposon end whereas the other covalently attaches TnpA to flanking DNA; and, finally, the spacing differences between the cleavage sites and the IPs. On RE, upon cleavage TnpA becomes attached to the RE flank which is part of the donor plasmid and presumably can move freely. It is therefore easy to imagine that the traveling helix αD can bring covalently attached flanking DNA to the adjacent active site. However, on LE, it is the transposon 5′ end that is attached to helix αD. Thus, LE must have a sufficiently long and flexible spacer between the 4 nt IPLE extension and the 5′ end of the transposon that can act as a tether with some slack so helix αD can move from one active site to the other. The slack is necessary because LE cannot move as a unit as it is anchored to the TnpA dimer by the IPLE hairpin.
The notion of a switch from the trans to a cis active site arrangement is supported by the crystal structure (PDB ID 2fyx) of a closely related transposase from Deinococcus radiodurans
, which was captured in the inactive configuration but with a cis helix αD arrangement. Although we have no structural evidence that the cis configuration occurs during IS608
transposition, our biochemical data showing that mixed mutant dimers are defective in the resolution steps provides strong support that both transposon junction and sealed backbone formation occurs with helix αD in cis: in this configuration, mixed mutant dimers have no functional active sites. As shown in Movie S2
, the switch of helices from one active site to the other can be easily modeled as long as the RE flank and the nucleotides between IPLE
and the LE cleavage site are free to move.
For the next step, integration, we do not know if the excised transposon junction intermediate remains bound to TnpA or is released and rebound. If, during recombination, the junction is released upon formation, it seems likely that TnpA resets to the trans configuration before transposon junction recapture, as we have only observed uncomplexed TnpA in that state. If the transposon junction stays bound, the cleavage steps required for integration may start with TnpA in the cis configuration and switch to trans for resolution. From the point of view of the overall mechanism, the outcomes of these two possibilities are equivalent.
We propose that integration of a transposon junction intermediate into a TTAC-containing target is a mechanistic replay of excision. After transposon junction formation, the sealed donor backbone dissociates from the complex and is replaced by a TTAC-containing target sequence (). At one active site, the target DNA would bind through interactions between its TTAC tetranucleotide and the four nt 5′ extension of IPLE. Upon cleavage, the TTAC target flank would stay bound to the complex and the 5′ end would be covalently attached to Y127. At the other active site, the transposon junction would be cleaved, leaving TCAA (the RE 3′ end of IS608) bound to the active site and the LE (5′ end of IS608) linked through a 5′ phosphotyrosine linkage to Y127. A switch in the active site arrangements leads directly to the resolution steps in which the top strand of IS608 is inserted into a new target site: the stationary 3′ OH of the TTAC target flank attacks the swapped 5′ phosphotyrosine link at LE while the RE TCAA 3′ OH attacks the swapped 5′ phosphotyrosine linkage of the target flank that was created during target cleavage ().
One of the most baffling questions about IS608
transposition was how its transposase, a 155 residue, single-domain protein, could carry out all of the required steps. Part of the answer is that TnpA is sneakier than we thought: it uses bound transposon DNA to recognize its cleavage sites both on donor and target DNA obviating the need for an additional DNA binding domain. Furthermore, TnpA works only on one DNA strand and does not have to deal with the complementary strand (Kersulyte et al., 2002
; Ton-Hoang et al., 2005
; Ronning et al. 2005
; Guynet et al., in press
If TnpA acts solely on ssDNA, how do its substrates arise? ssDNA formation might be promoted by plasmid supercoiling combined with the propensity of the IPs to form stem loops; if this occurs, the cleavage sites might, with some frequency, dissociate from their complementary strands. On the other hand, IS608 might excise in vivo only when DNA becomes single stranded during a normal cellular process such as lagging strand synthesis. If transposition occurs only during DNA replication, excised TnpA-bound sealed transposon circles might readily find a suitable TTAC-containing ssDNA target. Restricting transposition to one stage in the cell cycle would also ensure that the reaction is substrate-limited, thus preventing wanton and possibly destructive movement. Since ssDNA is also generated during conjugative DNA transfer, conjugating plasmids might be preferred targets for IS608 with the added advantage of immediate horizontal transfer.
The importance of ssDNA substrates for IS200
transposition is illustrated by recent work on Deinococcus radiodurans
which can survive severe ionizing radiation damage using a mechanism called “extended synthesis-dependent strand annealing” to reconstitute its shattered chromosomes (Zahradka et al., 2006
). During recovery, as much as 15% of newly synthesized DNA is single-stranded and, remarkably, the transposition frequency of ISDra2,
family member, increases 500-fold (Mennecier et al., 2006
), suggesting that ssDNA is normally rate-limiting.
One of our most surprising results is that IS608 uses DNA sequences in the transposon itself to recognize its cleavage sites. This mode of recognition suggests that TnpA may be re-directed to new target sites, opening up an unanticipated avenue of transposon targeting with an array of genomic and biotechnological applications.
The interdependence of protein and DNA in creating suitable substrates is reminiscent of the intertwining of protein and RNA in ribosomes (Noller, 2005
). This "self-recognition" has also been reported in other RNA-dependent systems. For example, the structural basis of the matchmaking functions of mitochondrial RNA binding proteins (MRPs) has recently been reported (Schumacher et al., 2006
There is a striking parallel between the IS608
transposition pathway and the mobility of group II introns, particularly with that of the bacterial L1.LtrB system (Belfort et al., 2005; Lambowitz & Zimmerly, 2004
). For example, the mobility of RNA introns is dependent on an intron-encoded multifunctional protein which is needed to bind the RNA. Upon splicing, exons bordering the intron are joined to each other, reminiscent of the resealing of donor DNA ends by TnpA. The lariat intermediate formed during intron splicing is similar to the circular IS608
transposon junction intermediate, both of which are covalently sealed. Perhaps the most significant parallel involves targeting, as these introns select their target, or “retrohoming” site, by base-pairing interactions between segments of the lariat intermediate and the target DNA strand. This is the basis for the development of re-targeted introns (Karlberg et al., 2001) that can be used to disrupt chromosomal genes. In intron mobility, the key catalytic component is the RNA itself, which catalyzes both the splicing and reverse splicing events. Because DNA has no known self-cleavage and strand transfer activity, IS608
has to rely on the assistance of TnpA to carry out the necessary cleavage and joining reactions. Our work demonstrates that a targeting mechanism previously thought to be the property of RNA-based mobile systems also occurs in the context of a mobile DNA element.
From a structural and functional perspective, the closest characterized relatives of TnpA are IS91
-related transposases (Garcillán-Barcia et al., 2001
). These are widespread in bacteria, have eukaryotic homologs (Kapitonov & Jerka, 2001
), and have been implicated in the movement of so-called Common Regions (Stokes et al., 1997
; Toleman et al., 2006
) which may be responsible for a large fraction of the spread of antibiotic resistance genes. While IS91
-like elements transpose by a mechanism that is likely different from IS608
, they also contain subterminal IPs and insert just 3′ of short conserved sequences. It will be interesting to determine if their mode of target recognition resembles that shown here for IS608
. If so, the potential for their re-targeting could provide new possibilities toward the site-specific modification of eukaryotic genomes.