A cell-free system of Tn3 transposition and transposition immunity

Background: Tn 3 is a bacterial transposon, which encodes transposase required for its transposition. Tn 3 has terminal inverted repeat (IR) sequences of 38 bp in length, whose inner region, called the B domain, is bound by transposase. Tn 3 confers transposition immunity, a phenomenon in which Tn 3 transposes to a target replicon with Tn 3 much less frequently than to a target replicon with no Tn 3 .
Results: To understand transposition and transposition immunity at the molecular level, we constructed a cell-free system using a plasmid as the target. Transpositional recombination occurred in a cell extract containing transposase between the target and a donor plasmid carrying mini-Tn 3 at a high frequency. The reaction required ATP, Mg²⁺, dNTPs and 2% polyvinyl alcohol, and was inhibited by inhibitors for DNA synthesis and DNA gyrase. In this system, when a plasmid with the IR sequence was used as the target, the frequency of transposition was significantly decreased, demonstrating that the transposition immunity conferred by Tn 3 is reproduced in vitro . Preincubation of the target in the cell extract increased the level of transposition immunity. On the other hand, mutations within the B domain in the IR sequence of the target abolished transposition immunity.
Conclusions: Transposition of Tn 3 and transposition immunity could be reproduced in vitro . The results demonstrate that the binding of transposase to domain B of the IR sequence in the target replicon is responsible for transposition immunity. We propose that the transposition immunity results from conversion of the normal synaptic complex formed between the donor and target molecules to another complex which is inactive for transposition, due to the interaction between transposases binding to the IR sequences in the donor and target molecules.