IS911 transposon circles give rise to linear forms that can undergo integration in vitro

High levels of expression of the transposase OrfAB of bacterial insertion s equence IS911 leads to the formation of excised transposon circles, in whic h the two abutted ends are separated by 3bp, Initially, OrfAB catalyses onl y single-strand cleavage at one 3' transposon end and strand transfer of th at end to the other. It is believed that this molecule, in which both trans poson ends are held together in a single-strand bridge, is then converted t o the circular form by the action of host factors. The transposon circles c an be integrated efficiently into an appropriate target in vivo and in vitr o in the presence of OrfAB and a second IS911 protein OrfA. In the results reported here, we have identified linear transposon forms in vivo from a tr ansposon present in a plasmid, raising the possibility that IS911 can also transpose using a cut-and-paste mechanism, However, the linear species appe ared not to be derived directly from the plasmid-based copy by direct doubl e-strand cleavages at both ends, but from preformed excised transposon circ les. This was confirmed further by the observation that OrfAB can cleave a cloned circle junction both in vivo and in vitro by two single-strand cleav ages at the 3' transposon ends to generate a linear transposon form with a 3'-OH and a three-nucleotide 5' overhang at the ends. Moreover, while signi ficantly less efficient than the transposon circle, a precleaved linear tra nsposon underwent detectable levels of integration in vitro. The possible r ole of such molecules in the IS911 transposition pathway is discussed.