Formation and transposition of the covalently closed IS30 circle: the relation between tandem dimers and monomeric circles

Abstract

In the present study, we demonstrate that a circular IS30 element acts as an intermediate for simple insertion. Covalently closed IS and Tn circles constructed in vitro are suitable for integration into the host genome. Minicircle integration displays all the characteristics of transpositional fusion mediated by the (IS30 )2 dimer regarding target selection and target duplication. Evidence is provided for in vivo circularization of the element located either on plasmids or on the genome. It is shown that circle formation can occur through alternative pathways. One of them is excision of IS30 from a hot spot via joining the IRs. This reaction resembles the site‐specific dimerization that leads to (IS30 )2 establishment. The other process is the dissolution of (IS30 )2 dimer, when the element is excised from an IR–IR joint. These pathways differ basically in the fate of the donor replicon: only dimer dissolution gives rise to resealed donor backbone. Analysis of minicircles and the rearranged donor replicons led us to propose a molecular model that can account for differences between the circle‐generating processes. Our focus was to the dissolution of IR–IR joints located on the host genome, because these events promoted extensive genomic rearrangements and accompanied minicircle formation. The results present the possibility of host genome reorganization by IS30‐like transposition.

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.…”

“…This phenomenon may hint at a replicative manner of dimer formation for IS3, while a conservative manner seems more likely for IS186. It is worth noting that both pathways are possible, as suggested for IS911 (Polard et al, 1996) and IS30 (Kiss & Olasz, 1999).…”

“…This product presumably arose by SSD or by transposition of the IRR-IRL junction to the IR end of the full IS copy, as shown for IS911 (Turlan et al, 2000) and IS30 (Kiss & Olasz, 1999;Olasz et al, 1997). The C5 dimers were similar to those that arose from C3 and the SR was identical with the sequence originally separating the truncated IRs in C4 (Fig.…”

“…Plasmid constructs were cloned into pBluescript KS+ (pKS) (Stratagene), pEMBL19 (Dente et al, 1983) or pJKI88 (Kiss & Olasz, 1999), respectively. Full IS copies were derived from pAW1326 : : IS plasmids (Szeverényi et al, 1996b).…”

“…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).…”