Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS2 transposes by an unconventional pathway

Abstract

SummaryThe Escherichia coli insertion sequence, IS2, is a member of the IS3 family of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed ¹1 translational frameshift near to the end of orfA and just after the start of orfB. We have characterized two major products of IS2 intramolecular transposition, which accumulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS2 with just a single basepair (occasionally 2 bp) separating the two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second product is a figure-eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DNA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction -the precise 3Ј end of IRR has been cleaved and joined to a target just outside the 5Ј end of IRL; the remaining vector sequences have a free 5Ј end, derived from cleavage at the 3Ј end of IRR, and a free 3Ј end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules are the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. This proposed transposition pathway provides a means for a transposase that can cleave only one strand at each IS end to produce simple insertions and avoid forming co-integrates.

Detection and analysis of transpositionally active head-to-tail dimers in three additional Escherichia coli IS elements

“…The dimer can promote the transpositional rearrangements mentioned above, but most frequently segregates into a replicon carrying only a single IS copy via a site-specific process, called dimer dissolution (DDS). This model is consistent with those based on covalently closed minicircles (Polard et al, 1992;Lewis & Grindley, 1997) and accounts for simple insertion on the basis of clear analogy between minicircles and IS-dimer-containing replicons (Kiss & Olasz, 1999). Similar to the dimer structures, the covalently joined IRs are also separated by a few base pairs in the circular form of the IS elements.…”

“…Similar to the dimer structures, the covalently joined IRs are also separated by a few base pairs in the circular form of the IS elements. These minicircles can be considered as intermediates of simple insertion, as reported for IS1 (Sekine et al, 1997a;Turlan & Chandler, 1995), IS2 (Lewis & Grindley, 1997), IS3 (Sekine et al, 1994), IS150 (Welz, 1993;Haas & Rak, 2002), IS256 (Prudhomme et al, 2002) and IS911 (Ton-Hoang et al, 1997. The synthetic IS dimer model seems to be applicable for the elements that form both minicircles and dimers (IS2, IS21, IS30, IS256 and IS911); however, its validity might also be extended to additional elements (e.g.…”

“…Such junctions have been identified in IS dimers consisting of two directly repeated elements and in minicircles formed by circularization of a single element. IS dimers have been isolated from IS2 (Szeverényi et al, 1996a), IS21 (Reimmann & Haas, 1987, 1990Reimmann et al, 1989), IS30 (Dalrymple, 1987;Olasz et al, 1993Olasz et al, , 1997, IS256 (Prudhomme et al, 2002) and IS911 (Turlan et al, 2000), and covalently closed circular IS elements have been described for IS1 (Sekine et al, 1997a, Turlan & Chandler, 1995, IS2 (Lewis & Grindley, 1997), IS3 (Sekine et al, 1994), IS30 (Kiss & Olasz, 1999), IS117 (Smokvina et al, 1994), IS150 (Welz, 1993;Haas & Rak, 2002), IS256 (Prudhomme et al, 2002) and IS911 (Polard et al, 1992(Polard et al, , 1996. For some of these elements both circular and dimeric forms have been reported, suggesting a possible connection between the formation and transposition of these structures, as described for IS30 (Kiss & Olasz, 1999).…”

Evidence that the insertion events of IS2 transposition are biased towards abrupt compositional shifts in target DNA and modulated by a diverse set of culture parameters

“…The latter are bipartite structures, containing an internal protein binding domain and an outer cleavage domain (Lewis et al 2001). orfA contains an AAAAAAG (A 6 G) slippery signal located near its stop codon, which is changed to A 7 G by a −1 translational frameshift to produce an OrfAB fusion protein (Lewis and Grindley 1997). Also shown is the outwardly directed −35 hexamer located in the IRR, which together with the terminal −10 hexamer of the catalytic domain of IRL, is involved in the formation of a junction promoter in the minicircle intermediate (Lewis et al 2004) Bacterial strains and plasmid DNA The bacterial strains Escherichia coli MG1655 ΔendA ΔrecA (F − λ − ilvG rfb-50 rph1 ΔendA ΔrecA) and DH5α (F − 80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(r k − , m k + ) phoA supE44 thi-1 gyrA96 relA1) were respectively obtained from Kristala Prather's Lab at MIT (Cambridge, MA) and from Invitrogen (Carlsbad, CA).…”

“…1). IS2 transposition occurs by a circle forming variation of the cutand-paste pathway that involves in a first step the production of a figure-8 molecule, which serves as the precursor of a minicircle intermediate, that in a second step is inserted into the target (Lewis et al 2004;Lewis et al 2001; Lewis and Grindley 1997). Despite reports in the late twentieth century of a regional preference for IS2 insertion in bacteriophage P1 genome Sengstag and Arber 1987;, the published literature in the subsequent years did not bring any conclusive evidence regarding its insertional specificity (Bernard et al 1991;Cohen et al 1993; Lewis et al 1994a; Lewis et al 1994b;Rijavec et al 2007;Szeverenyi et al 1996a;Szeverenyi et al 1996b;Whiteway et al 1998).…”