Figure 1.
piggyBac transposase catalyses DSBs and target joining. (A) Schematic representation of the piggyBac ends. The piggyBac left end (L-TIR) consists of a 13 bp terminal inverted repeat and a 19 bp internal inverted repeat separated by a 3 bp spacer; the right end (R-TIR) has a 31 bp spacer. The arrows indicate repeat sequences. (B) piggyBac transposase promotes DSBs. piggyBac transposase releases the transposon end from the flanking donor DNA by DSB, generating new products on a denaturing acrylamide gel. Ψ indicates hairpin intermediate. Here and in all other figures, * indicates the position of radiolabel. M indicates marker (radiolabelled MspI-digested pBR322 DNA in all denaturing acrylamide gels). Here and in all other figures, the solid line indicates the different portions of the same scan that were combined to form the relevant panel. (C) piggyBac transposase promotes target joining. piggyBac transposase joins the excised transposon to the target DNA generating products SEJ (nicked circular plasmid formed by joining of one transposon end to one plasmid strand) and DEJ (linearized plasmid formed by concerted joining of two transposon ends to two plasmid strands), which are displayed on a native agarose gel. Slower migrating species reflect joining to oligomeric plasmids. Here and in all other figures, M indicates marker (BglII+EcoRI-digested λ DNA in the agarose gels).