2 ABUNDANT INTRAMOLECULAR TRANSPOSITION PRODUCTS, RESULTING FROM REACTIONS INITIATED AT A SINGLE END, SUGGEST THAT IS2 TRANSPOSES BY AN UNCONVENTIONAL PATHWAY

The Escherichia coil insertion sequence, IS2, is a member of the IS3 f amily of bacterial transposable elements. Its transposase is a fusion protein, OrfAB, made by a programmed -1 translational frameshift near to the end of orfA and just after the start of orfB. We have character ized two major products of IS2 intramolecular transposition, which acc umulate in cells that express the IS2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the com plete IS2 with just a single basepair (occasionally 2bp) separating th e two IS ends. In all cases, this basepair is derived from the vector sequence immediately adjacent to the left IS2 end (IRL). The second pr oduct is a figure-eight molecule that contains all the IS2 and vector sequences present in the parental plasmid. One DMA strand contains the parental sequences unrearranged. The other contains a single-stranded version of the minicircle junction - the precise 3' end of IRR has be en cleaved and joined to a target just outside the 5' end of IRL; the remaining Vector sequences have a free 5' end, derived from cleavage a t the 3' end of IRR, and a free 3' end, released upon cleavage of the target site adjacent to IRL. We propose that figure-eight molecules ar e the precursor to IS2 minicircles and that the formation of these two products is the initial step in IS2 intermolecular transposition. Thi s proposed transposition pathway provides a means for a transposase th at can cleave only one strand at each IS end to produce simple inserti ons and avoid forming co-integrates.