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Two abundant intramolecular transposition products, resulting from reactions initiated at a single end, suggest that IS 2 transposes by an unconventional pathway
Author(s) -
Lewis Leslie A.,
Grindley Nigel D. F.
Publication year - 1997
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1997.4871848.x
Subject(s) - minicircle , transposase , biology , transposable element , insertion sequence , inverted repeat , cleavage (geology) , transposition (logic) , tn10 , plasmid , genetics , dna , microbiology and biotechnology , gene , mutant , paleontology , linguistics , philosophy , genome , fracture (geology)
The Escherichia coli insertion sequence, IS 2 , is a member of the IS 3 family 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 characterized two major products of IS 2 intramolecular transposition, which accumulate in cells that express the IS 2 OrfAB fusion protein at elevated levels. The more abundant product is a minicircle composed of the complete IS 2 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 IS 2 end (IRL). The second product is a figure‐eight molecule that contains all the IS 2 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 3′ end of IRR has been 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 at 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 are the precursor to IS 2 minicircles and that the formation of these two products is the initial step in IS 2 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.