The istA gene of insertion sequence IS21 is essential for cleavage at the inner 3′ ends of tandemly repeated IS21 elements in vitro.

Abstract

The bacterial 2.1 kb insertion sequence IS21 occurs as a tandem repeat [=(IS21)2] on the broad host range plasmid R68.45. In (IS21)2, the two IS21 elements are separated by 3 bp termed junction sequence. Plasmids carrying (IS21)2 form cointegrates with other replicons at high frequencies. The two IS21 genes, istA and istB, were found to be necessary for cointegrate formation in vivo. Since the outer ends of (IS21)2 are dispensable for cointegrate formation, we favor a transposition model according to which a plasmid carrying (IS21)2 is cleaved at the junction sequence; the opened plasmid is then inserted into a target replicon. Here we show that Escherichia coli cell extracts, which contained over‐produced IstA protein, nicked a supercoiled (IS21)2 plasmid precisely at the inner 3′ termini of IS21; the resulting staggered cut generated 5′ protrusions. The istA gene, but not the istB gene, was required for in vitro cleavage of an IS21‐IS21 junction. Because of this cleavage and our previous findings (generation of 4 bp target duplications and loss of the junction sequence after cointegrate formation in vivo) we propose that plasmids with (IS21)2 produce cointegrates by a mechanism which involves joining of the inner 3′ ends of IS21 to the 5′ ends of the target.

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

“…Both the reactive structure (the IR-IR junction) and the specific reactions mediated by the joined ends resemble those of the previously described dimers (IS2) 2 (Szeverényi et al, 1996a), (IS21) 2 (Reimmann & Haas, 1987, 1990, (IS30) 2 (Dalrymple, 1987;Farkas et al, 1996;Olasz et al, 1993) and (IS911) 2 (Turlan et al, 2000). The characteristic reaction of two directly oriented elements is the SSD leading to the formation of a dimer structure (Figs 3 and 7).…”

“…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).…”

Terminal inverted repeats of insertion sequence IS30 serve as targets for transposition

“…In the present study, we demonstrate that the terminal inverted repeats of the Escherichia coli insertion sequence IS30 are functional target sites for the transposition of the (IS30) 2 IS30, IS21, and IS2 belongs to a group of insertion sequences (IS) in which the transposition of the element is known to occur via an (IS) 2 intermediate (3,8,22,(25)(26)(27)32). The (IS30) 2 structure is formed by site-specific dimerization (Fig.…”

Formation of an inverted repeat junction in the transposition of insertion sequence ISLC3 isolated from Lactobacillus casei

“…Transpositionally active junctions have been reported experimentally for several other IS families, e.g. IS21 (Reimmann & Haas, 1990), IS30 (Kiss & Olasz, 1999;Olasz et al, 1993) and IS1 (Shiga et al, 1999;Ton-Hoang et al, 1997).…”