IDENTIFICATION AND CHARACTERIZATION OF THE LINEAR IS3 MOLECULES GENERATED BY STAGGERED BREAKS

Insertion sequence IS3 encodes two, out-of-phase, overlapping open rea ding frames, orfA and orfB. The OrfAB transframe protein that is IS3 t ransposase is produced by -1 translational frameshifting between orfA and orfB. Efficient production of the IS3 transposase in the cells har boring the IS3-carrying plasmid has been shown to generate miniplasmid s as well as characteristic minicircles, called IS3 circles, consistin g of the entire IS3 sequence and one of the 3-base pair sequences flan king IS3 in the parental plasmid. Here, we show that the IS3 transposa se also generates the linear molecules of IS3 with 3-nucleotide overha ngs at the 5'-ends. The nucleotide sequences of the overhangs are the same as those flanking IS3 in the parental plasmid, suggesting that th e linear IS3 molecules are generated from the parental plasmid DNA by staggered double strand breaks at the end regions of IS3. The linear I S3 molecules are likely to be the early intermediates in the transposi tion reaction, which proceeds in a non-replicative manner.