Figure 4.

Strand transfer of a precut OE/IE^ME transposon is catalyzed in trans. The rationale for the experiment is shown in a. A linear precut transposon with one OE and one IE^ME, when incubated with Tnp EK/LP and Tnp sC7/EA326 and a target plasmid, facilitates single-ended insertion of the transposon into target DNA. To determine whether the reaction is inserting the transposon at IE^ME (in trans) or OE (in cis), the reaction products are digested with either ClaI or SnaBI. Strand transfer reactions shown in Fig. 3b were incubated for 3 h followed by heat treatment of transposase (68°C for 20′). Where indicated, aliquots of the reaction were digested with either ClaI or SnaBI restriction endonuclease. Undigested and digested samples were then treated with SDS and heat to remove transposase followed by electrophoresis on a 1.1% agarose gel. The results of the reaction reveal that the single-ended insertion [lane 7 (X)] is shifted to a position near the relaxed plasmid band after ClaI digestion [lane 8 (X^C)], whereas the SnaBI digestion leads to a small shift [lane 9 (X^S)]. These results indicate in trans catalysis during strand transfer.