Targeted conservative formation of cointegrates between two DNA molecules containing IS26 occurs via strand exchange at either IS end

We recently proposed a model for targeted, conservative cointegrate formation between DNA molecules each containing a copy of IS26, that involves Tnp26‐catalyzed strand exchange occurring at either the two left ends or the two right ends of the IS. Here, this model was validated by altering the bases at the outer left terminus, right terminus or both termini of one IS26. The correct bases at both ends were required in the untargeted replicative mode. However, when only one end was altered in one participating IS the frequency of targeted, conservative cointegrate formation was not reduced. The distribution of the altered bases in the cointegrates confirmed that the reaction occurred at the end where the terminal bases of both IS were correct, and cointegrates were not formed when both ends of the same IS were altered. The terminal bases of the active IS26 were also required to support deletion of the aphA1a translocatable unit (TU) from Tn4352B. The choices made by an incoming TU with a wild‐type IS26 when the target plasmid included one wild‐type IS26 and one with a frameshift in tnp26 demonstrated that Tnp26 exhibits a strong preference for cis action.