Tipping the balance between replicative and simple transposition

The bacterial insertion sequence IS903 has the unusual ability to transpose both replicatively and non-replicativeiy. The majority of products are sim ple insertions, while co-integrates, the product of replicative transpositi on, occur at a low frequency (<0.1% of simple insertions), In order to defi ne the critical steps that determine the outcome of IS903 transposition, we have isolated mutants that specifically increase the rate of replicative t ransposition, Here we show that the nucleotide immediately flanking the tra nsposon influences both overall transposition frequency and co-integrate fo rmation. In particular, when the 3 ' -flanking nucleotide is A, co-integrat es are increased 500-fold compared with a 3 ' C. In addition, we have isola ted five transposase mutants that increase replicative transposition. These residues are close to the catalytic residues and are thus likely to be par t of the active site. These are the first transposase mutations described t hat affect the product of transposition. Our results are consistent with th e hypothesis that a delay in cleavage of the 5 ' -flanking DNA will increas e the effective half-life of the 3 ' -nicked transposon intermediate and co nsequently enhance co-integrate formation.