FORMATION OF THE TANDEM REPEAT (IS30)2 AND ITS ROLE IN IS30-MEDIATED TRANSPOSITIONAL DNA REARRANGEMENTS

Plasmids carrying two IS30 elements in the same orientation, as in the composite transposon Tn2706, are structurally unstable in Escherichia coli. A primary segregation product is formed by site-specific deleti on of the sequences carried between the two IS30 elements. The resulti ng covalently closed replicon carries the two IS30 elements as tandem repeats separated by only 2 bp. This (IS30)2 Structure is extremely un stable, but it can nevertheless be isolated on its vector plasmid and, after purification, can be reintroduced into host cells by transforma tion. Among the descendants of transformants of recA- bacteria, replic ated copies of the introduced (IS30)2 structure are still present, tog ether with various kinds of segregation products which provide evidenc e for the efficient generation of DNA rearrangements. Most abundant is the product of another site-specific recombination between two identi cal ends of the IS30 elements involved, which results in the presence of just one intact IS30 on the plasmid. Apart from this, and depending on the presence of appropriate targets for IS30 transposition, variou s transposition products of (IS30)2 are also seen. Intramolecular reac tions lead to DNA inversions and deletions with breakpoints other than IS30 ends. In intermolecular reactions inverse transposition occurs a t high frequency and one also obtains simple transposition and cointeg ration. A mutational study revealed the requirement in cis of one inta ct IS30 transposase gene and of both proximal ends of the two IS30 ele ments concerned not only for the formation of (IS30)2, but also for it s further rearrangement reactions, including the efficient formation o f site-specific deletions. A model is proposed, which postulates that (IS30)2 intermediates play a key role in IS30 transposition pathways i n which the formation of (IS30)2 may be rate-limiting. Once this struc ture is formed, it gives rise to a burst of transpositional rearrangem ents in the subclone carrying (IS30)2. Evolutionary implications of th ese findings are discussed.