Differential roles of the transposon termini in IS91 transposition

Abstract

Insertion sequence 91 (IS91) inserts specifically at GTTC or CTTG target sequences without duplication of the target. After insertion, the right inverted repeat (IRR) lies adjacent to the 3' end of the target sequences (or 5' to the complementary sequence CAAG or GAAC). We have analyzed the effects of alteration of each terminus of IS91 on transposition activity in Escherichia coli. IRR is absolutely required for transposition. Deletion analysis indicates that a 14-bp segment is not sufficient, but an 81-bp sequence within the IRR region is sufficient. Furthermore, the GTTC/CTTG target site is also required. The left inverted repeat (IRL) of IS91 is dispensable. Plasmid fusions originated by one-ended transposition of IS91 derivatives lacking IRL occur at about the same frequency as cointegrate formation observed for the wild-type element. In the one-ended-type fusions, the inserted fragment of donor DNA is flanked at one end (constant end) by IRR and at the other end by a GTTC or CTTG sequence present in the donor (variable end) in a way that usually results in multiple tandem insertions of the donor plasmid in the target site. These results are easily accommodated by a rolling-circle replicative transposition mechanism. This model also draws support from the finding that the IS91 transposase is related in sequence to the superfamily of rolling-circle replication proteins and the observation that IRR shows some conservation in sequence and secondary structure with the origins of replication of some rolling-circle replication plasmids.