piggyBac can bypass DNA synthesis during cut and paste transposition

Abstract

DNA synthesis is considered a defining feature in the movement of transposable elements. In determining the mechanism of piggyBac transposition, an insect transposon that is being increasingly used for genome manipulation in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during piggyBac transposition, both at the donor site following transposon excision and at the insertion site following transposon integration. We demonstrate that piggyBac transposon excision occurs through the formation of transient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3'OH transposon ends on to the target DNA. This is the same strategy for target joining used by the members of DDE superfamily of transposases and retroviral integrases. Analysis of mutant piggyBac transposases in vitro and in vivo using a piggyBac transposition system we have established in Saccharomyces cerevisiae suggests that piggyBac transposase is a member of the DDE superfamily of recombinases, an unanticipated result because of the lack of sequence similarity between piggyBac and DDE family of recombinases.

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).

Figure 2

piggyBac DSBs occur by means of a hairpin intermediate on the transposon end. (A) DNA hairpin formation is visualized using a pre-nicked end. A pre-nicked piggyBac L-TIR was incubated with piggyBac transposase in the presence of a target DNA for various times and then displayed on a denaturing acrylamide gel. (B) Hairpin formation requires a 3′OH transposon end. A pre-nicked piggyBac L-TIR with 3′G-OH (deoxy) or 3′G-H (dideoxy) was incubated with piggyBac transposase in the presence of a target DNA for 5 or 20 min and then displayed on a denaturing acrylamide gel. Lanes 1–3, piggyBac L-TIR with 3′G-OH (deoxy); lanes 3–5, piggyBac L-TIR with 3′G-H (dideoxy); lanes 1 and 3, ‘no protein' controls.

Figure 3

piggyBac joins the 3′OH of a pre-nicked transposon end substrate to the target DNA. (A) A pre-nicked end can join to target DNA. The products of the reactions described in Figure 2A are displayed on a native agarose gel. (B) The 3′OH transposon end joins to the target DNA. The products of the reactions described in Figure 2A are displayed on a denaturing agarose gel.

Figure 4

Sequence of the transposon end hairpin and resolution by the transposase. (A) Sequence of the transposon end hairpin. Maxim–Gilbert G-reaction of the hairpin intermediate formed from a pre-nicked piggyBac L-TIR substrate displayed on a denaturing acrylamide gel. G-substrate: G-reaction of the intact piggyBac L-TIR fragment with flanking DNA; G-hairpin: G-reaction of the piggyBac L-TIR hairpin; G/S: G-reaction of substrate; G/H: G-reaction of hairpin. The L-TIR is shown in uppercase and the flanking donor DNA in lowercase; the top and bottom strands of piggyBac L-TIR are joined by means of a 4 nt hairpin derived from the donor strand flanking the 5′ end of the transposon. Only part of L-TIR substrate and corresponding hairpin are shown. (B) piggyBac transposase can resolve a pre-formed transposon end hairpin. Transposase was incubated with L-TIR_13-3-19 oligonucleotide containing a TTAA hairpin for 20 min. Lane 1, the 3′ strand of L-TIR_13-3-19 as a marker; lane 2, hairpin DNA without piggyBac transposase incubation; lane 3, hairpin DNA with piggyBac transposase incubation.

Figure 5

Schematic representation of the piggyBac cut and paste transposition. piggyBac transposition initiates with nicks at the 3′ ends of the transposon, exposing 3′OHs. These 3′OHs then attack the complementary strand 4 nt into the flanking donor DNA, thereby forming hairpins on the transposon ends with the concomitant release of the transposon ends. Donor site repair can occur by ligation of the complementary 5′ TTAA overhangs on the flanking donor DNA ends, precisely reforming the TTAA target sequence. Transposon end hairpins are resolved by transposase, re-exposing the 3′OH transposon ends and generating 4 nt TTAA overhangs on the 5′ ends of the excised transposon. The 3′OH transposon ends join to the staggered positions at the 5′ T's of the TTAA/AATT target sequence. Repair of the single-strand gaps flanking the newly inserted transposon gives rise to the 4 bp TTAA target sequence duplication.

Figure 6

The flanking donor sequence influences transposon end processing. (A) Flanking sequence and 3′OH end nicking. piggyBac transposase was incubated with L-TIR fragments flanked by donor DNA of different sequences, as indicated, for 20 min and reactions displayed on a denaturing acrylamide gel. (B) Flanking donor DNA influences hairpin formation. Pre-nicked piggyBac L-TIRs with either a TTAA or GCGC flank were incubated with piggyBac transposase in the presence of a target plasmid and displayed on a denaturing gel. (C) Influence of flanking sequence on target joining. The reactions using the pre-nicked substrates were displayed on a native gel.

Figure 7

Mutation of conserved DDD amino acids blocks the catalytic activity of piggyBac transposase in vitro. (A) Mutation of the conserved D's blocks 3′OH nicking. Wild-type and mutant piggyBac transposases were incubated with a piggyBac L-TIR and a target plasmid for 20 min and then displayed on a denaturing acrylamide gel. (B) Mutation of the conserved D's blocks hairpin formation. Wild-type and mutant piggyBac transposases were incubated with a pre-formed piggyBac L-TIR hairpin oligonucleotide and then displayed on a denaturing gel. (C) Mutation of the conserved D's blocks target joining. Wild-type and mutant transposases were incubated with a pre-cleaved piggyBac L-TIR lacking the usual 4 nt overhangs at 5′ transposon end and a target plasmid and then displayed on a native agarose gel. ∞ indicates nucleoprotein complexes formed by transposase binding to the labelled piggyBac L-TIRs.

Figure 8

piggyBac can transpose efficiently in S. cerevisiae. (A) Schematic representation of the piggyBac transposition assay. piggyBac excision from a URA3 gene containing the actin intron in S. cerevisiae was evaluated by analysing the reversion of the donor site from uracil auxotrophy to uracil prototrophy after piggyBac excision. (B) Frequency of piggyBac excision.