TnlO Transposition and C ircle Formation In Vitro

We describe a cell-free system that promotes TnICl transposition and transposon circle formation, a related intramolecular event. TnIO circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP. The reaction requires TnIO transposase protein, and either of two E. coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA. TnIO is composed of inverted repeats of insertion sequence ISIO. Pair-wise combinations of the IS10 “outside” and “inside” ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro. In contrast to the TnIO reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.