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piggyBac can bypass DNA synthesis during cut and paste transposition

piggyBac can bypass DNA synthesis during cut and paste transposition The EMBO Journal (2008) 27, 1097–1109 & 2008 European Molecular Biology Organization All Rights Reserved 0261-4189/08 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL piggyBac can bypass DNA synthesis during cut and paste transposition Rupak Mitra, Jennifer Fain-Thornton and at the target site. Compared to transposon excision, only rarely is the donor site restored to its pre-transposon state, and Nancy L Craig* that is, only rarely does ‘precise excision’ occur. One strategy Department of Molecular Biology and Genetics, Howard Hughes for donor site repair is by end-joining, a pathway that leaves Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA ‘footprints’ in the donor site reflecting joining of target sequences that are duplicated upon transposon insertion DNA synthesis is considered a defining feature in the and remain attached to the donor site after element excision movement of transposable elements. In determining the (Coen et al, 1986; Weil and Kunze, 2000). The other pathway mechanism of piggyBac transposition, an insect transpo- for donor site repair is homology-dependent gene conversion using a sister chromatid or homologue as a template (Engels son that is being increasingly used for genome manipula- et al, 1990; Plasterk, 1991). Although homology-dependent tion in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during repair using a homologous site that lacks a copy of the piggyBac transposition, both at the donor site following element can result in precise excision, this is apparently an transposon excision and at the insertion site following infrequently used mechanism (Perkins-Balding et al, 1999). transposon integration. We demonstrate that piggyBac Imprecise excision may actually be a useful strategy as it can transposon excision occurs through the formation of tran- introduce variation into the host genome (Kidwell and Lisch, 2000). sient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3 OH With DNA cut and paste elements, the single-strand gaps transposon ends on to the target DNA. This is the same that flank the transposon in the new insertion site resulting strategy for target joining used by the members of DDE from the joining of transposon ends to staggered positions on superfamily of transposases and retroviral integrases. the target DNA must be repaired. It is this strategy of joining Analysis of mutant piggyBac transposases in vitro and in to staggered positions on the target DNA followed by DNA synthesis mediated by host repair proteins that results in the vivo using a piggyBac transposition system we have estab- lished in Saccharomyces cerevisiae suggests that piggyBac target site duplications, the hallmark of transposon insertion transposase is a member of the DDE superfamily of re- (Mizuuchi, 1983). This same strategy for target joining and combinases, an unanticipated result because of the lack of gap repair also accounts for the target site duplications that sequence similarity between piggyBac and DDE family of flank integrated retroviral-like elements (Brown et al, 1987). recombinases. DNA synthesis is also involved in the synthesis of the integrated DNA copies of non-LTR elements (Luan et al, The EMBO Journal (2008) 27, 1097–1109. doi:10.1038/ 1993), rolling-circle transposons such as IS91 (del Pilar emboj.2008.41; Published online 20 March 2008 Subject Categories: genome stability & dynamics Garcillan-Barcia et al, 2001) and helitrons (Kapitonov and Keywords: piggyBac;precise excision;transposase;transposition Jurka, 2001). Thus, DNA synthesis has been considered a defining feature of transposable elements in contrast to the breakage and joining of element phage lambda, which is mediated by sequence-specific topoisomerases that use covalent protein– Introduction DNA intermediates and do not involve DNA synthesis Transposable elements (transposons) are mobile DNA seg- (Grindley et al, 2006). ments present in the genomes of all organisms that can move Intriguingly, the DNA cut and paste transposon piggyBac between many different positions in the genome. They have from cabbage looper moth Trichoplusia ni (T.ni) consistently considerable influence on genome structure and function and shows precise excision upon element transposition (Cary thus are natural agents of genome evolution (Kazazian, et al, 1989; Fraser et al, 1995). Also unique for piggyBac 2004). Transposable elements are also extensively used as transposition is the exclusive use of TTAA target sites (Fraser laboratory tools for genome manipulation by insertional et al, 1995). Moreover, there is little obvious sequence mutagenesis and transgenesis (Boeke, 2002). similarity between transposases of the piggyBac and other A problem common to the movement of all transposable transposon superfamilies (Sarkar et al, 2003). The mobility of element that undergo cut and paste transposition is the piggyBac in various insects, mammalian cells including regeneration of intact duplex DNA both at the donor site human cell lines (Wilson et al, 2007), mice (Ding et al, 2005) and a number of heterologous systems including *Corresponding author. Department of Molecular Biology and Genetics, planarian Girardia tigrina (Gonzalez-Estevez et al, 2003), Howard Hughes Medical Institute, Johns Hopkins University School of the human pathogens Plasmodium falciparum (Balu et al, Medicine, 725 N Wolfe Street, Rm 502 PCTB, Baltimore, MD 21205-2185, USA. Tel.: þ 1 410 955 3933; Fax: þ 1 410 955 0831; 2005) and Schistosoma mansoni (Morales et al, 2007) has E-mail: [email protected] made piggyBac an attractive genetic tool. Here, we describe an in vitro system using piggyBac Received: 8 October 2007; accepted: 7 February 2008; published transposase purified from Escherichia coli and establish the online: 20 March 2008 &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1097 | | ∗ piggyBac in cut and paste transposition R Mitra et al 2 þ mechanism of the DNA breakage and joining reactions that for catalytically essential Mg ions (Rice and Baker, 2001; underline piggyBac transposition. We found that the pattern Zhou et al, 2004; Richardson et al, 2006). We show, using of cleavage during piggyBac excision results in complemen- secondary structure prediction and mutational analysis in tary TTAA overhangs on the ends of the donor DNA, allowing vitro and in vivo in genetically tractable Saccharomyces the simple ligation of these ends to restore the donor site to its cerevisiae, that piggyBac elements have catalytically essential pre-transposon sequence, accounting for the precise excision DDD acidic residues that apparently lie on a partial RNaseH of piggyBac. This transposon excision occurs through a hair- fold and thus are likely members of the DDE superfamily. Our pin intermediate on the transposon ends and leaves TTAA studies have thus revealed that the piggyBac system is not overhangs on the 5 ends of the excised linear transposon. only related to but also has unique aspects distinct from other The central step in piggyBac transposition, that is, the studied transposon systems. joining of the excised transposon to the target DNA, occurs by the direct attack of the 3 OH ends of the transposon to staggered positions at the 5 ends of a TTAA target sequence. Results This is the same strategy used by the widespread DDE family of bacterial and eukaryotic transposases and retroviral inte- piggyBac transposase promotes double-strand breaks grases (Rice and Baker, 2001). This strategy of target joining to excise piggyBac from the flanking donor DNA means that the TTAA overhangs on the 5 ends of the The ends of piggyBac are closely related; both contain a 13 bp transposon can base-pair with the 5 TTAA single-strand terminal inverted repeat and a 19 bp internal inverted repeat gaps on the target DNA that flank the positions of transposon (Cary et al, 1989) (Figure 1A). These repeat segments are, joining. These target gaps can then be sealed simply by however, separated by a 3 bp spacer on the left end and a ligation rather than by DNA synthesis. 31 bp spacer on the right end. In all the experiments reported Thus, the target site exclusivity for TTAA sites and the below, we have used DNA segments containing 70 and 72 bp strategy for excision from the donor site with 5 TTAA over- of the piggyBac left and right ends, respectively. These hangs means that piggyBac need not involve DNA synthesis, segments contain the 35 bp L-TIR (L-TIR) and 63 bp 13-3-19 a defining feature of all other characterized transposition R-TIR (R-TIR) sequences that are efficient substrates 13-31-19 reactions. for excision and interplasmid transposition in vivo (Elick In addition to a common mechanism, the DDE recombi- et al, 1997). These end segments, however, lack several nases share a common catalytic core of particular protein other internal repeats necessary for in vivo integration into folds that juxtapose acidic residues that provide binding sites the host genome (Li et al, 2005). A BC ∗ 136 DSB ∗ 103 103 5 ′ transposon R-TIR Donor strand flank Time (min) 0 15 2 10 20 Time (min) 0 1 2 5 10 20 ∗ L-TIR SEJ 3 bp 13 bp 19 bp 3.4 DEJ 2.7 R -TIR 136 Donor 123 flank 31 bp 19 bp 13 bp ~ ∗ 5′ transposon Linear strand 2.8Kb 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. C 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 þ EcoRI-digested l DNA in the agarose gels). 1098 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | ∗ piggyBac in cut and paste transposition R Mitra et al We cloned, expressed and purified T. ni piggyBac transpo- complex in which the transposon ends pair and interact with sase as a His-tagged derivative, from E. coli (see Materials and the target DNA. methods). Using band shift assays, we found that purified The majority of the SEJ and DEJ products appear at late transposase binds specifically to piggyBac L-TIR and R-TIR times (10–20 min) when the transposon ends have already end fragments that are flanked by donor DNA undergone DSBs separating them from the flanking donor (Supplementary Figure 1) and also to the end fragments DNA. Little target joining is observed at early times (2 min) lacking flanking donor DNA (data not shown). when only cleavage at the 3 end of the transposon has piggyBac excises precisely from a donor DNA and inserts occurred, suggesting that target joining can occur only after into a TTAA target site in vivo (Cary et al, 1989). To probe the the complete excision of the transposon end from the flanking mechanism of these reactions, we incubated piggyBac trans- donor DNA. We show below that DSBs are rapidly formed posase with DNA fragments containing the piggyBac R-TIR once nicking at the 3 transposon ends occurs, suggesting that (Figure 1) or L-TIR (Supplementary Figure 2) flanked by the nicking step can be a rate-limiting step in piggyBac 2þ donor DNA, a plasmid target DNA and Mg , which is an transposition. 2 þ essential cofactor (data not shown). Mn was a much less With the R-TIR substrate, we observed both SEJ and DEJ effective cofactor in all the assays performed here (data not products (Figure 1C); with the L-TIR substrate, we observed shown). only the SEJ product (Supplementary Figure 2B). The greater The 3 -end-labelled piggyBac R-TIR substrate was a 103 bp amount of target joined products with the R-TIR substrate is segment containing the 72 bp R-TIR sequence flanked by consistent with the greater amount of R-TIR end cleavage. 136 bp of donor DNA. When the R-TIR reaction products were displayed on a denaturing gel (Figure 1A), we observed piggyBac DSBs proceed through a hairpin intermediate two prominent new species reflecting a double-strand break on the transposon end (DSB) separating the transposon end from the flanking donor The above experiments suggested that nicking at the 3 DNA. The size of the slower migrating species of the two transposon end initiates piggyBac transposition. Thus, we most prominent new species is consistent with a nick at the 3 analysed the transposition reaction using a 35 bp piggyBac end of the transposon that would liberate the end-labelled L-TIR substrate flanked by donor DNA where the 3 OH 13-3-19 136 nt flanking donor top strand. The size of the faster end of the transposon is already exposed, that is, a ‘pre- migrating species of the two new species is consistent with nicked’ substrate. Analysis of the reaction products on a a break between the 5 transposon end and the donor DNA denaturing gel revealed the rapid accumulation of a new that releases the end-labelled 103 nt bottom strand of the species about 74 nt in length (Figure 2A). The size of this piggyBac R-TIR from the flanking donor DNA. Determination product is consistent with the formation of a hairpin on the of the exact positions of these cleavages is described below. transposon end that includes several nucleotides of the 0 0 It is notable that cleavage at the 3 and 5 transposon ends flanking donor DNA; its exact sequence is considered below. 0 0 did not occur simultaneously. The cleavage at the 3 transpo- Hairpin formation reflects cleavage at the 5 end of the son end that generated the slower migrating species occurred transposon and concomitant liberation of the transposon 0 0 before cleavage at the 5 end that generated the faster- from the flanking donor DNA. The 5 transposon end clea- migrating species. Similar DSB reaction products were ob- vage is much faster with the pre-nicked substrate than with tained using piggyBac L-TIR DNA (Supplementary Figure intact ends; some hairpin is evident at 1 min with the pre- 2A), although at lower efficiency compared to piggyBac R- nicked substrate (Figure 2A), whereas only 3 end nicking is TIR. Thus, nicking at the 3 transposon end appears to initiate evident by 5 min with the intact end with the flanking donor transposition. substrate (Figure 1B). With the pre-nicked substrate, a high Also faintly visible is a very slow migrating species marked level of hairpin product is present until 10–20 min and the ‘c’. As discussed in detail below, this species is a hairpin that amount decreases thereafter, suggesting that the hairpin 0 0 includes both the 5 and 3 strands of the piggyBac R-TIR end species can be resolved by the transposase (Figure 2A). and is a transposition intermediate. DSBs resulting from hairpin formation on the transposon end are already known to occur with the prokaryotic ele- ments Tn10 (Kennedy et al, 1998) and Tn5 (Bhasin et al, piggyBac transposase joins the ends of the transposon 1999); piggyBac is the first example of a eukaryotic element to the target DNA that uses this mechanism. When the products of the above reactions were displayed on To test whether piggyBac hairpin formation, leading to a native agarose gel, we observed joining of the cleaved R-TIR transposon excision, requires an exposed 3 OH transposon (Figure 1C) and L-TIR (Supplementary Figure 2B) fragments end, we compared hairpin formation of pre-nicked piggyBac 0 0 to a circular plasmid target DNA to form two different L-TIR substrates that had either a 3 deoxyguanosine or a 3 products. The joining of one transposon end to one strand dideoxyguanosine at their 3 ends (Figure 2B). If hairpin of the target DNA forms a single-end join (SEJ) in which one formation requires a 3 OH transposon end, the dideoxy- target strand is broken by the covalent joining of one trans- containing substrate should be unable to support hairpin poson end and the other plasmid strand is intact, resulting in formation and hence a DSB. In contrast to the pre-nicked 3 a nicked circular plasmid. Concerted joining of two transpo- deoxyguanosine substrate, which efficiently formed the 74 nt son ends to separate strands of the same target DNA forms a hairpin species with the concomitant release of the top 20 nt double-end join (DEJ) in which each strand of the target DNA flanking donor DNA (Figure 2B, lanes 2 and 3), the 3 is covalently linked to one transposon end, forming a linear, dideoxyguanosine substrate failed to form the hairpin and double-stranded DNA molecule. Formation of such coupled undergo DSB (Figure 2B, lanes 4 and 5). Thus, hairpin DEJ products is consistent with formation of a transpososome formation is an essential step in piggyBac excision. &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1099 | | OH OH OH piggyBac in cut and paste transposition R Mitra et al A B Hairpin Hairpin 59 59 20 DSBs * resolution + + formation * ∗ 35 24 35 ∗ ∗ ∗ 35 ∗ 24 35 74 Pre-nicked L-TIR Top flank Hairpin Tr ansposon Pre-nicked L-TIR intermediate bottom dG ddG Time (min) 02 5 0025 0 Time (min) 76 12 5 102030 60 67 74 ∗ 67 ∗ 35 ∗ 35 ∗ M 12 35 4 6 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 0 0 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 0 0 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. When the products of transposition reactions (Figure 2A) of these single-stranded products in reactions using a using the pre-nicked piggyBac L-TIR substrate are displayed piggyBac L-TIR in which the strand containing the 3 terminal on a native agarose gel (Figure 3A), joining of the excised transposon end is labelled at its 5 internal end reveals the transposon ends to the target plasmid forming the SEJ and chemistry of target joining: the 3 OH terminal end of the DEJ products is observed. Significant levels of target joining piggyBac L-TIR joins covalently to the target DNA. In a species are seen late (5–10 min) in the time course of the separate experiment, we have shown directly that the 5 reactions, although hairpin formation on the transposon end ends of piggyBac do not join to target DNA (Supplementary is evident early (1 min). Such timing suggests that the trans- Figure 3). position reaction proceeds through end nicking and hairpin formation, leading to DSBs, that is, transposon excision, A 4-nt TTAA hairpin is formed on the piggyBac end which is followed by hairpin resolution to expose the trans- To define the sequence identity of the transposon end hairpin poson ends, after which target joining occurs. The much at the nucleotide level, we isolated the hairpin species higher levels of end cleavage and target joining observed generated from a transposition reaction using the pre-nicked with the pre-nicked substrate (Figure 3A) as compared to the piggyBac L-TIR substrate and then determined its sequence intact substrate (Figure 1C and Supplementary Figure 2B) using the Maxim–Gilbert G-reaction (Figure 4A). The G suggests that nicking at the 3 transposon end is a key sequence of the hairpin was identical to that of the bottom determinant of the rate of transposition. strand of the transposon DNA flanked by the donor DNA with respect to 3 terminal end of piggyBac at positions G ,G and 1 2 The 3 OH end of piggyBac joins to the target DNA G . However, the positions of the following G’s in the hairpin To determine directly which strand of a piggyBac end joins to and an intact transposon end differed. Whereas the G’s in the target DNA, we examined the products formed between a the intact transposon end when the flanking donor DNA pre-nicked piggyBac L-TIR fragment labelled at its internal 5 is attached to the transposon were at positions G ,G 12/S 17/S end, that is, at the end of the transposon strand containing a and G , the next G in the hairpin was at position G , 19/S 13/H 3 OH end, and a plasmid target DNA, on a denaturing agarose followed by G ,G ,G and G , reflecting the 17/H 21/H 25/H 27/H gel (Figure 3B). Whereas target joining leads to the formation sequence of the top strand of the transposon DNA of two products, that is, SEJs and DEJs, on a native gel (Figure 4A). (Figure 3A), only a single product was evident on a denatur- This pattern is consistent with a hairpin that includes the 3 ing agarose gel, consistent with the joining of one piggyBac end of the piggyBac L-TIR DNA including the G at the 3 L-TIR segment to a single target strand (Figure 3B). Detection terminus of the transposon, 4 nt from the flanking donor 1100 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | OH OH OH piggyBac in cut and paste transposition R Mitra et al Time (min) 0 12 5 10 20 30 60 Time (min) 0 12 5 10 20 30 60 AB SEJ 3.4 2.7 3.4 Single strand DEJ linear 2.7 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. Hairpin Hairpin formation resolution ∗ 74 74 ∗ ∗ 35 35 Pre-nicked L-TIR 12 3 ctcaaatcttttttaaCCCTAGAAAGATAGTCTGCGTAAAA gagtttagaaaaaattGGGATCTTTCTATCAGACGCATTTT 19 17 12 3 2 1 S S S Hairpin G27/H G25/H G19/S HHHH H G21/H 13 17 21 25 27 G17/S G17/H aaCCCTAGAAAGATAGTCTGCGTAAAA ttGGGATCTTTCTATCAGACGCATTTT G13/H 3 2 1 G12/S 35 nt G3 G3 G2 G2 G1 G1 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 oligonucleotide containing a TTAA hairpin for 20 min. Lane 1, the 3 strand of L-TIR as a 13-3-19 13-3-19 marker; lane 2, hairpin DNA without piggyBac transposase incubation; lane 3, hairpin DNA with piggyBac transposase incubation. DNA—T, T, A, A—joined to the C at the 5 end of the transposon piggyBac transposase can resolve a hairpin and the rest of the 5 strand of the piggyBac L-TIR. Thus, four transposon end nucleotides of flanking donor DNA are included in the hairpin. We also directly analysed the ability of piggyBac transposase As piggyBac always inserts into TTAA target sites, the element to resolve a pre-formed hairpin. Using a self-complementary will always be flanked by TTAA in the donor site and the hairpin oligonucleotide, we generated a piggyBac hairpin L-TIR will always include the TTAA sequence. We explore the effects species containing a 4 nt TTAA loop. In the presence of the of changing this sequence below. transposase, the hairpin DNA is resolved to generate a 35 nt &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1101 | | Intact substrate hairpin G - substrate G - hairpin ∗ piggyBac in cut and paste transposition R Mitra et al DNA fragment corresponding to cleavage at the 3 end of We examined the ability of piggyBac transposase to pro- piggyBac on the bottom strand of the piggyBac L-TIR mote DSBs at piggyBac L-TIRs flanked by GCGC, a sequence (Figure 4B, lane 3). Thus, the lack of hairpin accumulation different at all positions from the standard TTAA sequence. in Figure 1B likely results from rapid resolution of the hairpin Display of the reaction products on a denaturing gel reveals intermediate. that no nicks or DSBs occur with the GCGC flank (Figure 6A). Furthermore, no hairpinning is observed with a pre-nicked GCGC flank L-TIR (Figure 6B) nor does resolution of a GCGC- piggyBac correctly inserts into TTAA target sites in vitro containing hairpin L-TIR occur (Supplementary Figure 5A). piggyBac inserts exclusively into TTAA target sites in vivo and Thus, with a flanking GCGC, there is a defect in end proces- thus TTAA target sequence duplications flank a newly in- sing at every step, that is nicking, hairpin formation and serted transposon (Cary et al, 1989). To determine the fidelity hairpin resolution, revealing that the TTAA flanking sequence of piggyBac target joining in vitro, we used PCR to generate a has a key role in transposon excision. mini-piggyBac element in which a gene for kanamycin resis- It is also notable that little (o10% of wild type) target tance is flanked on both ends by piggyBac L-TIR 13-3-19 joining is observed with a pre-nicked GCGC L-TIR substrate sequences with their 3 OH already exposed; this element (Figure 6C), indicating that although the 3 OH at this trans- lacked the TTAA extensions present on the 5 ends of an poson end is already exposed, it is a poor substrate for target authentic excised piggyBac element. This mini-piggyBac ele- joining. ment was used as a substrate in an in vitro transposition We also analysed donor cleavage with piggyBac L-TIR reaction containing pUC19 plasmid DNA as a target. transposon ends in which the standard TTAA flanking se- Transposition products were recovered by selection for kana- quence was changed at three positions (GCGA), two positions mycin-resistant E. coli after transformation and the transpo- (GCAA) and one position (GTAA) (Figure 6A). Nicking and son–plasmid junctions were sequenced. Four independent DSBs were detected only with the GTAA substrate that transposition reactions were performed and five products differed at only one position from the 5 end from the from each reaction were recovered and analysed. In all standard TTAA flank (Figure 6A). Accumulation of the hair- cases, insertion occurred at a TTAA site and the element pin intermediate was also observed with this GTAA flanking was always flanked by a 4 bp TTAA target sequence duplica- sequence substrate, suggesting that hairpin resolution is also tion; insertions into 8 of the 13 different TTAA sites on the defective when the 3 end of the transposon is linked to a G pUC19 plasmid were recovered (Supplementary Figure 4). rather than the natural T (Supplementary Figure 5B). These Thus, insertion into TTAA target sites is an intrinsic property experiments demonstrate that the efficiency of end cleavage of the piggyBac transposase and the TTAA extensions present is highly influenced by the flanking donor DNA. on the 5 ends of an excised piggyBac transposon are not We have also analysed the influence on target joining of required for TTAA target site selection. the sequence of the 4 nt extension on the 5 ends of excised Thus, our in vitro transposition system directs insertion of piggyBac (Supplementary Figure 6). An L-TIR end lacking any piggyBac into its preferred TTAA target sequence, which is a extension on the 5 transposon end was a better substrate true representation of piggyBac transposition in vivo. than an end having a 5 TTAA extension. Target joining by an end with the usual 5 TTAA extension was much better than There are four steps in piggyBac transposition 0 that with a 5 GCGC extension. Thus, although piggyBac We conclude that piggyBac transposition involves four dis- transposase can join transposons with flush ends to target tinct chemical steps (Figure 5). Step 1: a DSB initiates with a DNA, the usual TTAA overhangs on the 5 transposon ends nick at the junction of the 3 end of the transposon and the efficiently couple excision and integration. flanking donor DNA, exposing a reactive 3 OH on the trans- poson end. Step 2: the 3 OH then acts as a nucleophile and Identification of the catalytic core of piggyBac attacks 4 nt into the flanking donor DNA on the complemen- transposase tary strand, generating a hairpin on the transposon end and The above experiments revealed that the key chemical steps simultaneously releasing the transposon DNA from the donor 2 þ in piggyBac transposition are the Mg -dependent excision of backbone. The flanking donor DNA contains complementary the transposon to yield a transposon with 3 OH ends, fol- 5 TTAA extensions that can rejoin to repair the donor gap to 0 lowed by the direct nucleophilic attack of these 3 OH ends on give a precise excision. Step 3: The hairpin on the transposon the target DNA. The chemistry of these steps is identical to end is resolved by the transposase, leaving a 4 nt overhang at that of the DDE recombinase family, which contains many the 5 end of the excised linear transposon and re-exposing transposases and retroviral integrases (Rice and Baker, 2001; 0 0 the 3 OH at the transposon end. Step 4: the 3 OH at the Zhou et al, 2004; Richardson et al, 2006). In these recombi- transposon end then covalently joins to the target DNA. nases, highly conserved acidic amino acids (DDE or DDD) are closely juxtaposed on an RNaseH-like fold and coordinate piggyBac excision is influenced by the flanking donor essential metal ions. There is, however, little primary DNA sequence sequence homology between piggyBac and the DDE/DDD A newly inserted piggyBac element is flanked by the TTAA recombinases (Sarkar et al, 2003). target sequence duplications resulting from the staggered Secondary structure prediction of T. ni piggyBac by attack on the TTAA target sequence. After excision, the PSIPRED (Jones, 1999b) suggests that a portion of piggyBac TTAA nucleotides from the flanking donor DNA remain likely has part of the RNaseH-like fold that is conserved in attached to the 5 ends of piggyBac. Does the identity of the DDE recombinases such as HIV1 integrase (Dyda et al, 1994) flanking donor site sequences influence piggyBac excision (Supplementary Figure 7). Included within this region of and target joining? piggyBac are D268 and D346, which are invariant among a 1102 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | OH OH TTAA AATT TTAA TTAA AATT AATT TTAA AATT piggyBac in cut and paste transposition R Mitra et al TTAA CCC GGG TTAA AATT GGG CCC AATT Nick CCC GGG GGG CCC Hairpin formation AACCC GGG TT TTAA TTGGG CCC AA AATT Donor flank repair Hairpin resolution TTAA AATT CCC GGG GGG CCC TTAA Target joining AATT TTAA CCC GGG GGG CCC AATT Target site repair TTAA CCC GGG TTAA AATT AATT GGG CCC Figure 5 Schematic representation of the piggyBac cut and paste transposition. piggyBac transposition initiates with nicks at the 3 ends of the 0 0 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 0 0 0 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. 2 þ number of piggyBac-like ORFs that are associated with TIRs through their interactions with Mg . Moreover, the fact that from a variety of insects and animals (Supplementary Figure a single mutation can block these multiple catalytic steps 8) (Sarkar et al, 2003; Arkhipova and Meselson, 2005). The suggests that a single active site can mediate all the chemical positions of these invariant D’s on the putative piggyBac steps of recombination. RNaseH-like fold are equivalent to those of the two D’s on There are several conserved D’s outside of the RNaseH-like the RNaseH-like DDE transposases and retroviral integrases domain that are conserved among the piggyBac transposases: (Supplementary Figure 7). D227, D228, D239, D447 and D450 (Supplementary Figure 8). To probe the involvement of these conserved acidic amino We also made alanine substitutions at these positions and acids in piggyBac transposition, we purified piggyBac trans- tested the activities of the mutant proteins. Notably, D447A posases with alanine substitutions at D268 and D346 and was still capable of binding to the TIRs (Supplementary evaluated their ability to promote specific binding to the TIRs Figure 9) but was defective in all the catalytic steps of and the catalytic steps of transposition. transposition, suggesting that it also lies within the catalytic Although still capable of binding specifically to piggyBac core (Figure 7A–C and Supplementary Figure 10). L-TIR and R-TIR (Supplementary Figure 9), the D268A and D227A, D228A, D239A and D450A were all catalytically D346A mutants were defective in generating the DSBs that active in vitro; thus, these positions do not appear to be part separate the transposon ends from the flanking donor DNA of the catalytic core of the transposase (data not shown). (Figure 7A), resolution of a pre-formed hairpin to expose the 3 OH transposon (Figure 7B) and joining of a substrate TIR Other functional determinants of piggyBac transposase with an exposed 3 OH to a target DNA (Figure 7C). These Another highly conserved residue among piggyBac transpo- findings suggest that these conserved acidic amino acids have sases is W465 (Supplementary Figure 8). Planar interactions critical roles in all the catalytic steps of transposition, likely between such an aromatic amino acid and DNA bases can &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1103 | | OH OH OH OH piggyBac in cut and paste transposition R Mitra et al SEJ Hairpin Hairpin ∗ target A ∗ BC DSB formation formation joining ∗ ∗ Hairpin Donor 5′ transposon ∗ ∗ ∗ DEJ L-TIR flank resolution strand Pre-nicked L-TIR Pre-nicked L-TIR TTAA GCGC GCGA GCAA GTAA TTAA GCGC TTAA GCGC AATT CGCG CGCT CGTT CATT AATT CGCG AATT CGCG Tnsp Tnsp Tnsp –+ –+ –+ –+ –+ –+ – + – + – + Hairpin on transposon 67 end Hairpin on transposon end SEJ 3.4 DEJ 2.7 Donor flank 5′ transposon strand 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. have an important role in base-flipping steps, which can be The actin intron can be efficiently spliced from mRNA of this integral to DNA distortions necessary for the formation and gene, so that a strain carrying the URA3Hactin intron is a resolution of the altered DNA structures such as hairpins, as uracil prototroph. However, if a large DNA segment such as a demonstrated with Tn5 and Tn10 (Davies et al, 2000; several kilobase transposon is introduced into the actin Bischerour and Chalmers, 2007), Hermes (Zhou et al, 2004) intron, the resulting intron is too large to be spliced from and RAG recombinase (Lu et al, 2006). It has been suggested mRNA, making the strain a uracil auxotroph. Thus, excision (Arkhipova and Meselson, 2005) that piggyBac W465 is of the transposon and restoration of the donor site to the involved in DNA hairpinning. W465A, however, has much parental URA3Hactin intron configuration can be followed by reduced nicking activity and is compromised in all subse- assaying for reversion of uracil auxotrophy to uracil proto- quent catalytic steps of transposition although it still binds trophy (Figure 8A). The restoration of the gapped donor site specifically to the piggyBac TIRs (Supplementary Figures 9 in yeast could occur by end-joining or gene conversion using and 11). Thus, W465 cannot have a role only in DNA hairpin the chromosomal actin gene intron as a template (Paques and formation and resolution. Haber, 1999). Another conserved feature of piggyBac transposases is the In our yeast two-plasmid piggyBac system, a transposon C-terminal C C CHC motif. Analysis of this region by donor plasmid contains a mini-piggyBac transposon com- 2 2 2 GenTHREADER (Jones, 1999a) suggests that this region posed of 328 bp of the piggyBac left end and 361 bp of the 2 þ forms a Zn -binding PHD domain (Supplementary Figure piggyBac right end flanking a kanamycin resistance gene in 8); PHD domains bind to chromatin (Bienz, 2006). The C- the URA3Hactin intron cassette. The transposase is supplied terminus is, however, dispensable for piggyBac recombina- by a second plasmid containing the piggyBac transposase tion in vitro: piggyBac , which lacks the C C CHC motif, gene under the galactose-inducible control of the GALS 1–558 2 2 2 is as active in vitro as the wild-type piggyBac (data not promoter (Mumberg et al, 1994). 1–594 shown), perhaps because we have used naked DNA rather In the absence of transposase, the frequency of URA3 than chromatin as a target substrate. reversion was very low, about 10 (Figure 8B). Upon galac- tose induction of the piggyBac transposase gene, however, the piggyBac can transpose in S. cerevisiae frequency of URA3 reversion was very much higher, about To establish a simple genetic assay for the excision of 10 , indicating a high level of transposon excision piggyBac, we adapted a modified version of the yeast URA3 (Figure 8B). Considerable excision, that is, uracil prototrophy, was observed even without galactose induction, indicating gene as a transposon donor (Yu and Gabriel, 1999). In this that the low level of transposase present because of leakiness modified URA3 gene, the yeast actin intron has been intro- of the GALS promoter can promote transposition. duced into the URA3 gene to form a URA3Hactin intron gene. 1104 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | + piggyBac in cut and paste transposition R Mitra et al AB C Hairpin DSB resolution Donor 5′ transposon L-TIR 74 flank strand L-TIR SEJ 3.4 DEJ 2.7 Donor 2.8 kb flank 5′ transposon strand 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. N indicates nucleoprotein complexes formed by transposase binding to the labelled piggyBac L-TIRs. We demonstrated above that certain highly conserved have been identified in Xenopus (Hikosaka et al, 2007). We acidic amino acids are necessary for piggyBac activity in have developed a faithful in vitro transposition system that vitro. Are they also essential for piggyBac activity in vivo? defines the chemical steps by which piggyBac undergoes cut We evaluated transposition in yeast promoted by mutant and paste transposition. Our findings account for several transposases substituted with alanines at D268, D346, D447 distinctive features of piggyBac transposition and particularly and W465. In all cases, the frequency of uracil prototrophy notable is that piggyBac can transpose without DNA synth- promoted by these mutant transposases was more than five esis, a process involved in all other characterized transposi- orders of magnitude less than that observed with wild type tion reactions. The systems we have described here provide and not significantly different from that observed in the valuable tools for further development of piggyBac-based absence of transposase, that is, about 10 (Figure 8B). systems for insertional mutagenesis and transgenesis as To demonstrate that the mutant proteins are stably pro- well as for dissection of piggyBac transposition at the mole- duced, we have shown in a yeast transposon integration cular level. assay (Supplementary data) that the presence of the mutant transposases inhibits transposition by wild-type transposase, piggyBac excision involves the formation and resolution that is, the mutants are dominant negatives and are thus of hairpins on the transposon ends stably produced (Supplementary Figure 12). The yeast sys- To undergo cut and paste transposition, piggyBac transposase tem thus supports the hypothesis based on in vitro data that induces DSBs that separate the transposon ends from the these amino acids are part of the piggyBac active site. flanking donor DNA. piggyBac DSB initiates with a nick at the 0 0 3 end of the transposon. The free 3 OH then attacks the complementary strand to form a hairpin on the transposon Discussion end, concomitantly releasing the transposon from the flank- piggyBac: a widespread eukaryotic DNA transposon ing donor DNA. As we find that more hairpin intermediate is Cabbage looper moth piggyBac is a DNA transposable ele- formed at earlier times with a pre-nicked substrate compared ment and a member of the widespread piggyBac family of to an intact one in which nicking must first occur, introduc- transposons (Sarkar et al, 2003) with recently active elements tion of the nick may be a rate-limiting step in transposition. &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1105 | | D268A D346A D447A WT 35 nt (marker) WT D268A D346A D447A WT D268A D346A D447A piggyBac in cut and paste transposition R Mitra et al Actin intron XhoI can splice URA URA+ URA Actin intron XhoI XhoI Actin intron cannot splice URA mini-piggyBac URA– URA Transposon excision Actin intron XhoI can splice URA+ URA URA Actin intron Transposase plasmid Excision frequency Glucose Galactose induction induction URA colonies –7 +– pGALS- Tnsp <1.0 X 10 –5 pGALS+ Tnsp WT +– 3.2 X 10 –7 pGALS – Tnsp –+ <1.0 X 10 –2 pGALS+ Tnsp WT –+ 2.4 X 10 –7 pGALS+ Tnsp D268A ND + <1.0 X 10 –7 pGALS+ Tnsp D346A ND + <1.0 X 10 –7 pGALS+ Tnsp D447A ND + <1.0 X 10 –7 pGALS+ Tnsp W465A ND + <1.0 X 10 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. The slow nicking step could, however, reflect the slow TTAA target sites. Indeed we have shown that the presence of assembly of an active transpososome on an intact substrate non-TTAA flanking base pairs can greatly inhibit piggyBac compared to rapid assembly of an active transpososome on a excision. Despite being 4 bp away, the phosphodiester bond pre-nicked substrate rather than a limitation in the chemical on the complementary strand that is attacked by the 3 OH on steps. It is also notable that little difference in hairpin forma- the transposon end is actually brought relatively near the 3 tion is observed between pre-nicked left and right ends or end of the transposon by the twist of the helix. The require- between pre-cleaved left and right ends whereas an intact left ment for the TTAA flank may reflect a requirement for DNA end is a much less efficient substrate than an intact right end. distortability to juxtapose the 3 OH transposon end and its These observations support the view that the formation of an position of attack on the complementary strand. Similar active transpososome is more stringently regulated on a DNA distortions have been shown by phasing analysis of the end with a transposon end still flanked than with cleaved DNAs sequences upon binding of Tn5 transposase (York and that are recombination intermediates. These results also Reznikoff, 1997; Ason and Reznikoff, 2004). The hairpin suggest that transpososome assembly occurs differently on species is then resolved by cleavage at the 3 end of the each end. transposon. 0 0 The transposase then opens the hairpin to expose the 3 OH Thus, the 5 transposon ends of the excised transposon are transposon end that can then attack the target DNA. A similar flanked by 4 nt, TTAA overhang, that derive from the flanking ‘hairpin on the transposon ends’ mechanism has been ob- donor site DNA, unlike the hairpin formed flush at the 5 served with the prokaryotic elements Tn5 and Tn10 (Kennedy transposon end with no flanking DNA in the Tn5 and Tn10 et al, 1998; Bhasin et al, 1999) and contrasts with the ‘hairpin transposons (Kennedy et al, 1998; Bhasin et al, 1999). on the donor DNA’ observed with the hAT transposon Hermes (Zhou et al, 2004) and RAG recombinase (van Gent et al, piggyBac transposition is accompanied by precise 1996). DSBs through hairpins on the transposon ends in excision at the donor site piggyBac transposition are the first to be reported in a After transposon excision, repair of the broken donor chro- eukaryotic transposon system. mosome that contains a gap at the position from which the The intramolecular hairpin reaction occurs by the attack of element was excised must occur. With most eukaryotic the transposon end 3 OH on the donor phosphodiester bond transposons, the original target sequence is not reformed 0 0 that is 5 to the TTAA sequences that always flank the 5 ends during donor site rejoining but rather a ‘footprint’ remains of the transposon. These flanking TTAAs are present in all that reflects the joining of the flanking donor ends that piggyBac donor sites because piggyBac inserts specifically at contain the duplicated target sequence (Coen et al, 1986; 1106 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | piggyBac in cut and paste transposition R Mitra et al Weil and Kunze, 2000). A distinctive feature of piggyBac The ability of piggyBac to repair both the broken donor transposition is that transposition is always followed by backbone and the target site by ligation of TTAA sequences reconstitution of a single TTAA sequence at the donor site, flanking the gapped donor sites and the transposon ends that is, precise excision occurs (Cary et al, 1989). We have rather than DNA synthesis could help make piggyBac inde- shown that piggyBac excises by a mechanism that cleanly pendent of the host repair machinery and contribute to its frees the 3 ends of the transposon and results in 4 nt TTAA functionality in a wide variety of organisms. overhangs on the 5 ends of the transposon. Consequently, there are also TTAA overhangs on both 5 ends of the broken piggyBac transposase is likely a DDE recombinase donor DNA. As these overhangs are present on complemen- piggyBac transposase binds specifically to both the left and 2 þ tary strands, they can readily pair and be ligated by the host right ends of piggyBac transposon and promotes the Mg - repair machinery, yielding a TTAA at the donor site equiva- dependent joining of a 3 OH transposon end to a target DNA. lent to the original target site (Figure 5). Thus, gene conver- Thus, piggyBac uses the same target joining mechanism, that sion using a homologue as a template need not be invoked to is, direct nucleophilic attack, as do the DDE recombinases account for precise excision in the case of piggyBac transposi- (Rice and Baker, 2001). The catalytic cores of DDE recombi- tion (Paques and Haber, 1999). nases share a common structure: an RNaseH-like fold on It should also be noted that target site-specific insertion which the conserved acidic DDE/DDD amino acids are juxta- 2þ into a palindromic sequence is not sufficient to ensure precise posed to coordinate Mg ions that are essential cofactors for excision. Tc1 mariner elements always insert at TA sites. the chemical steps of transposition (Rice and Baker, 2001; However, cleavage of the 5 transposon ends actually occurs Hickman et al, 2005; Richardson et al, 2006). However, there inside the element, leaving non-complementary overhangs is no obvious sequence similarity between piggyBac and DDE that cannot be simply ligated together such that footprints are recombinases (Sarkar et al, 2003). seen most frequently upon element excision (Plasterk, 1991). From the results of secondary structure prediction and mutational analysis, we suggest that part of the piggyBac The sequence TTAA coordinates piggyBac excision and active site has a section of an RNaseH-like fold on which the target site insertion D residues D268 and D346 that are essential for DNA break- Previous in vivo experiments (Fraser et al, 1995) and our in age and joining are located in positions comparable to those vitro experiments have shown that piggyBac inserts preferen- of the N-terminal D residues of the DDE recombinases. We tially into a TTAA target site such that the newly inserted have also identified another essential C-terminal D, D447, transposon is flanked by TTAAs. Although excised piggyBac that we suggest performs the same function as the C-terminal is flanked by TTAA on the 5 transposon ends, these flanking E in the DDE recombinases. Further analysis of these mutants nucleotides are not essential for correct target joining: a will be required to establish if these amino acids have a role piggyBac element with flush 5 ends still chooses TTAA as a in transpososome formation as has been reported in the case target. We have found that the flanking TTAA sequence is of Mu transposase (Kim et al, 1995). also critical to transposon excision; changing the flanking These results provide evidence that piggyBac is a member TTAA can block nicking at the 3 transposon end, hairpin of the DDE recombinase superfamily, extending the range of formation and hairpin resolution. Flanking donor sequence protein sequences that can form this essential catalytic core. also influences the excision of other elements (Wu and Tn5 and Tn10 transposase carries out four distinct chemi- Chaconas, 1992; Williams et al, 1999). cal reactions in Tn5 and Tn10 transposition: nicking at the This requirement for the same sequence for target integra- 3 OH transposon end, hairpin formation, hairpin resolution tion and DSB formation to promote excision suggests that this and target joining. The available evidence argues that a single TTAA sequence is recognized by the transposase during both active site contributed from a single transposase monomer at excision and integration. each transposon end is used repeatedly to catalyse all four steps (Bolland and Kleckner, 1996; Reznikoff, 2003). The use Target joining and the generation of intact DNA at the of a single active site to carry out alternating hydrolysis and target site transesterification has been supported by studies on other We have found that piggyBac joins to the target DNA by the proteins that use two metal ions in their active site that are held in place by acidic amino acids (Nowotny et al, 2005). As direct attack of the 3 OH ends of the transposon to staggered piggyBac transposase performs similar chemical reactions positions on to the TTAA target DNA sequence. This direct nucleophilic attack is the hallmark of target joining by the and mutations in the catalytic DDD residues block all of DDE superfamily that contains many prokaryotic and eukar- these steps, we are attracted to the view that piggyBac yotic transposases and retroviral integrase (Rice and Baker, transposition similarly involves the activity of a single active 2001; Hickman et al, 2005; Richardson et al, 2006). As the 5 site at each transposon end. ends of the newly integrated transposon are flanked by 5 TTAA, the generation of intact duplex DNA at the site of Genetic analysis of piggyBac transposition in insertion can be accomplished by pairing and ligation of the S. cerevisiae complementary TTAA sequences on the 5 transposon ends We have developed a piggyBac transposition system in the and at the gaps that flank the newly inserted transposon. This highly genetically tractable S. cerevisiae. In this system, we pairing and ligation strategy will result in TTAA base pairs can directly follow the excision of a mini-piggyBac element flanking the newly inserted transposon. This strategy is from a Ura derivative of the URA3 gene containing the distinct from the repair of target gaps by host polymerase transposon by measuring the frequency of uracil prototrophy and ligase as occurs in other transposon systems (Mizuuchi, in the presence of piggyBac transposase. piggyBac excision 1984). can occur at high frequency, that is, about 1/100 cells are &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1107 | | piggyBac in cut and paste transposition R Mitra et al 2 þ equilibrated Ni Sepharose column (GE Healthcare). The column uracil prototrophs in a single colony grown on media indu- was washed with 10 column volumes of TSG buffer followed by 6 cing the expression of the transposase. We have used this column volumes of TSG þ 50 mM imidazole buffer. piggyBac–Myc– system to show that amino acids that we identified as being His fusion protein was eluted with TSGþ 200 mM imidazole buffer, part of the catalytic site in vitro are also necessary for activity dialysed against storage buffer (20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 25% v/v glycerol) and stored at 801C. in vivo. In addition to facilitating the analysis of piggyBac transpo- piggyBac DSB and target joining reactions sition in vivo, including the isolation of hyperactive mutants 150 nM of piggyBac transposase was incubated with 1.5 nM of that will aid insertional mutagensis in mammalian cells, this radiolabelled piggyBac L-TIR or R-TIR DNA (see Supplementary data) in 25 mM HEPES (pH 8.0), 3 mM Tris (pH 8.0), 75 mM NaCl, piggyBac system is also well suited for the in vivo manipula- 2 mM DTT, 10 mM MgCl , 0.01% BSA, 3.75% glycerol and 10 nM tion of the yeast genome. pUC19 in a final volume of 20 mlat301C for different time intervals. Reactions were stopped by incubation in 1% SDS and 20 mM EDTA Domesticated piggyBac transposases for 30 min at 651C and displayed on a 1% native agarose–1 TAE gel. For analysis of DNA nicking and hairpin formation, the reaction Proteins related to piggyBac transposases are present in many products were phenol/chloroform extracted, ethanol precipitated eukaryotic genomes, including insects, fish and mammals. In and displayed on a 5% acrylamide–7 M urea–1  TBE denaturing the human genome, there are five piggyBac-derived genes, acrylamide gel. All gels described here were dried and exposed to PGBD1–5 (Sarkar et al, 2003). Our work shows that PGBD1–3 phosphoimager plates and analysed by Imagequant software (GE Healthcare). and 5 are very unlikely to have transposase activity as at least one of the active site amino acids we have identified in T. ni Analysis of hairpin formation using a pre-nicked piggyBac piggyBac is mutated in each of these proteins. In PGBD4, left end however, the catalytically essential amino acids are intact, 0 A 35 bp oligonucleotide corresponding to the 3 end of piggyBac L- although this provides no proof that PGB4 is actually a TIR was radiolabelled at its 5 end with [g- P]ATP (GE 13-3-19 Healthcare) and T4 polynucleotide kinase. After purification on a transposase. Expressed cellular genes that are significantly G25 column, the end-labelled oligonucleotide was annealed with related to transposases but are not flanked by terminal equimolar amounts of a 59 bp oligonucleotide containing the 5 end inverted repeats or target site duplications have been ob- of piggyBac L-TIR flanked by 24 nt flanking donor DNA and a 13-3-19 served for many DNA transposons, some of which are 24 nt oligonucleotide corresponding to the bottom strand of flanking donor DNA. The annealed oligonucleotide mixture was ‘domesticated’ transposases co-opted for by the cell for then directly used as the substrate in DSB and target joining processes that do not involve DNA breakage and joining reactions as described above. The reaction products were displayed such as transcriptional regulation (Volff, 2006). on native agarose, denaturing acrylamide and 1% agarose–50 mM NaOH gels. Materials and methods Supplementary data Supplementary data are available at The EMBO Journal Online piggyBac transposase expression and purification (http://www.embojournal.org). The piggyBac transposase ORF (594 amino acids) was PCR amplified from the plasmid pBhelper (Lobo et al, 1999) and cloned between the NcoI and KpnI sites of plasmid pBAD Myc-HisC Acknowledgements (Invitrogen) to generate a pBAD piggyBac–Myc–His6 fusion construct (p-transposase). E. coli Top10 (Invitrogen) cells contain- We thank David O’Brochta (University of Maryland, College Park) ing p-transposase were grown with shaking at 301C in LB medium for providing the piggyBac transposon plasmids, and Abram Gabriel containing 100 mg/ml ampicillin till an OD of 0.6. The culture was (Rutgers University) and Jef Boeke (JHU-SOM) for providing yeast then induced with 0.1% L-arabinose for 18 h at 161C. Following plasmids and strains. We also thank the Craig lab members for their induction, cells were lysed with a French Pressure cell (Fisher valuable insights into this project. This work was partly supported Scientific) in TSG buffer (20 mM Tris–HCl (pH 8.0), 500 mM NaCl, by National Institute of Health (NIH) grant NC90020064 to NLC. 10% v/v glycerol). The lysate was then loaded onto a pre- NLC is an Investigator of the Howard Hughes Medical Institute. 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piggyBac can bypass DNA synthesis during cut and paste transposition

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The EMBO Journal (2008) 27, 1097–1109 & 2008 European Molecular Biology Organization All Rights Reserved 0261-4189/08 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL piggyBac can bypass DNA synthesis during cut and paste transposition Rupak Mitra, Jennifer Fain-Thornton and at the target site. Compared to transposon excision, only rarely is the donor site restored to its pre-transposon state, and Nancy L Craig* that is, only rarely does ‘precise excision’ occur. One strategy Department of Molecular Biology and Genetics, Howard Hughes for donor site repair is by end-joining, a pathway that leaves Medical Institute, Johns Hopkins University School of Medicine, Baltimore, MD, USA ‘footprints’ in the donor site reflecting joining of target sequences that are duplicated upon transposon insertion DNA synthesis is considered a defining feature in the and remain attached to the donor site after element excision movement of transposable elements. In determining the (Coen et al, 1986; Weil and Kunze, 2000). The other pathway mechanism of piggyBac transposition, an insect transpo- for donor site repair is homology-dependent gene conversion using a sister chromatid or homologue as a template (Engels son that is being increasingly used for genome manipula- et al, 1990; Plasterk, 1991). Although homology-dependent tion in a variety of systems including mammalian cells, we have found that DNA synthesis can be avoided during repair using a homologous site that lacks a copy of the piggyBac transposition, both at the donor site following element can result in precise excision, this is apparently an transposon excision and at the insertion site following infrequently used mechanism (Perkins-Balding et al, 1999). transposon integration. We demonstrate that piggyBac Imprecise excision may actually be a useful strategy as it can transposon excision occurs through the formation of tran- introduce variation into the host genome (Kidwell and Lisch, 2000). sient hairpins on the transposon ends and that piggyBac target joining occurs by the direct attack of the 3 OH With DNA cut and paste elements, the single-strand gaps transposon ends on to the target DNA. This is the same that flank the transposon in the new insertion site resulting strategy for target joining used by the members of DDE from the joining of transposon ends to staggered positions on superfamily of transposases and retroviral integrases. the target DNA must be repaired. It is this strategy of joining Analysis of mutant piggyBac transposases in vitro and in to staggered positions on the target DNA followed by DNA synthesis mediated by host repair proteins that results in the vivo using a piggyBac transposition system we have estab- lished in Saccharomyces cerevisiae suggests that piggyBac target site duplications, the hallmark of transposon insertion transposase is a member of the DDE superfamily of re- (Mizuuchi, 1983). This same strategy for target joining and combinases, an unanticipated result because of the lack of gap repair also accounts for the target site duplications that sequence similarity between piggyBac and DDE family of flank integrated retroviral-like elements (Brown et al, 1987). recombinases. DNA synthesis is also involved in the synthesis of the integrated DNA copies of non-LTR elements (Luan et al, The EMBO Journal (2008) 27, 1097–1109. doi:10.1038/ 1993), rolling-circle transposons such as IS91 (del Pilar emboj.2008.41; Published online 20 March 2008 Subject Categories: genome stability & dynamics Garcillan-Barcia et al, 2001) and helitrons (Kapitonov and Keywords: piggyBac;precise excision;transposase;transposition Jurka, 2001). Thus, DNA synthesis has been considered a defining feature of transposable elements in contrast to the breakage and joining of element phage lambda, which is mediated by sequence-specific topoisomerases that use covalent protein– Introduction DNA intermediates and do not involve DNA synthesis Transposable elements (transposons) are mobile DNA seg- (Grindley et al, 2006). ments present in the genomes of all organisms that can move Intriguingly, the DNA cut and paste transposon piggyBac between many different positions in the genome. They have from cabbage looper moth Trichoplusia ni (T.ni) consistently considerable influence on genome structure and function and shows precise excision upon element transposition (Cary thus are natural agents of genome evolution (Kazazian, et al, 1989; Fraser et al, 1995). Also unique for piggyBac 2004). Transposable elements are also extensively used as transposition is the exclusive use of TTAA target sites (Fraser laboratory tools for genome manipulation by insertional et al, 1995). Moreover, there is little obvious sequence mutagenesis and transgenesis (Boeke, 2002). similarity between transposases of the piggyBac and other A problem common to the movement of all transposable transposon superfamilies (Sarkar et al, 2003). The mobility of element that undergo cut and paste transposition is the piggyBac in various insects, mammalian cells including regeneration of intact duplex DNA both at the donor site human cell lines (Wilson et al, 2007), mice (Ding et al, 2005) and a number of heterologous systems including *Corresponding author. Department of Molecular Biology and Genetics, planarian Girardia tigrina (Gonzalez-Estevez et al, 2003), Howard Hughes Medical Institute, Johns Hopkins University School of the human pathogens Plasmodium falciparum (Balu et al, Medicine, 725 N Wolfe Street, Rm 502 PCTB, Baltimore, MD 21205-2185, USA. Tel.: þ 1 410 955 3933; Fax: þ 1 410 955 0831; 2005) and Schistosoma mansoni (Morales et al, 2007) has E-mail: [email protected] made piggyBac an attractive genetic tool. Here, we describe an in vitro system using piggyBac Received: 8 October 2007; accepted: 7 February 2008; published transposase purified from Escherichia coli and establish the online: 20 March 2008 &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1097 | | ∗ piggyBac in cut and paste transposition R Mitra et al 2 þ mechanism of the DNA breakage and joining reactions that for catalytically essential Mg ions (Rice and Baker, 2001; underline piggyBac transposition. We found that the pattern Zhou et al, 2004; Richardson et al, 2006). We show, using of cleavage during piggyBac excision results in complemen- secondary structure prediction and mutational analysis in tary TTAA overhangs on the ends of the donor DNA, allowing vitro and in vivo in genetically tractable Saccharomyces the simple ligation of these ends to restore the donor site to its cerevisiae, that piggyBac elements have catalytically essential pre-transposon sequence, accounting for the precise excision DDD acidic residues that apparently lie on a partial RNaseH of piggyBac. This transposon excision occurs through a hair- fold and thus are likely members of the DDE superfamily. Our pin intermediate on the transposon ends and leaves TTAA studies have thus revealed that the piggyBac system is not overhangs on the 5 ends of the excised linear transposon. only related to but also has unique aspects distinct from other The central step in piggyBac transposition, that is, the studied transposon systems. joining of the excised transposon to the target DNA, occurs by the direct attack of the 3 OH ends of the transposon to staggered positions at the 5 ends of a TTAA target sequence. Results This is the same strategy used by the widespread DDE family of bacterial and eukaryotic transposases and retroviral inte- piggyBac transposase promotes double-strand breaks grases (Rice and Baker, 2001). This strategy of target joining to excise piggyBac from the flanking donor DNA means that the TTAA overhangs on the 5 ends of the The ends of piggyBac are closely related; both contain a 13 bp transposon can base-pair with the 5 TTAA single-strand terminal inverted repeat and a 19 bp internal inverted repeat gaps on the target DNA that flank the positions of transposon (Cary et al, 1989) (Figure 1A). These repeat segments are, joining. These target gaps can then be sealed simply by however, separated by a 3 bp spacer on the left end and a ligation rather than by DNA synthesis. 31 bp spacer on the right end. In all the experiments reported Thus, the target site exclusivity for TTAA sites and the below, we have used DNA segments containing 70 and 72 bp strategy for excision from the donor site with 5 TTAA over- of the piggyBac left and right ends, respectively. These hangs means that piggyBac need not involve DNA synthesis, segments contain the 35 bp L-TIR (L-TIR) and 63 bp 13-3-19 a defining feature of all other characterized transposition R-TIR (R-TIR) sequences that are efficient substrates 13-31-19 reactions. for excision and interplasmid transposition in vivo (Elick In addition to a common mechanism, the DDE recombi- et al, 1997). These end segments, however, lack several nases share a common catalytic core of particular protein other internal repeats necessary for in vivo integration into folds that juxtapose acidic residues that provide binding sites the host genome (Li et al, 2005). A BC ∗ 136 DSB ∗ 103 103 5 ′ transposon R-TIR Donor strand flank Time (min) 0 15 2 10 20 Time (min) 0 1 2 5 10 20 ∗ L-TIR SEJ 3 bp 13 bp 19 bp 3.4 DEJ 2.7 R -TIR 136 Donor 123 flank 31 bp 19 bp 13 bp ~ ∗ 5′ transposon Linear strand 2.8Kb 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. C 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 þ EcoRI-digested l DNA in the agarose gels). 1098 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | ∗ piggyBac in cut and paste transposition R Mitra et al We cloned, expressed and purified T. ni piggyBac transpo- complex in which the transposon ends pair and interact with sase as a His-tagged derivative, from E. coli (see Materials and the target DNA. methods). Using band shift assays, we found that purified The majority of the SEJ and DEJ products appear at late transposase binds specifically to piggyBac L-TIR and R-TIR times (10–20 min) when the transposon ends have already end fragments that are flanked by donor DNA undergone DSBs separating them from the flanking donor (Supplementary Figure 1) and also to the end fragments DNA. Little target joining is observed at early times (2 min) lacking flanking donor DNA (data not shown). when only cleavage at the 3 end of the transposon has piggyBac excises precisely from a donor DNA and inserts occurred, suggesting that target joining can occur only after into a TTAA target site in vivo (Cary et al, 1989). To probe the the complete excision of the transposon end from the flanking mechanism of these reactions, we incubated piggyBac trans- donor DNA. We show below that DSBs are rapidly formed posase with DNA fragments containing the piggyBac R-TIR once nicking at the 3 transposon ends occurs, suggesting that (Figure 1) or L-TIR (Supplementary Figure 2) flanked by the nicking step can be a rate-limiting step in piggyBac 2þ donor DNA, a plasmid target DNA and Mg , which is an transposition. 2 þ essential cofactor (data not shown). Mn was a much less With the R-TIR substrate, we observed both SEJ and DEJ effective cofactor in all the assays performed here (data not products (Figure 1C); with the L-TIR substrate, we observed shown). only the SEJ product (Supplementary Figure 2B). The greater The 3 -end-labelled piggyBac R-TIR substrate was a 103 bp amount of target joined products with the R-TIR substrate is segment containing the 72 bp R-TIR sequence flanked by consistent with the greater amount of R-TIR end cleavage. 136 bp of donor DNA. When the R-TIR reaction products were displayed on a denaturing gel (Figure 1A), we observed piggyBac DSBs proceed through a hairpin intermediate two prominent new species reflecting a double-strand break on the transposon end (DSB) separating the transposon end from the flanking donor The above experiments suggested that nicking at the 3 DNA. The size of the slower migrating species of the two transposon end initiates piggyBac transposition. Thus, we most prominent new species is consistent with a nick at the 3 analysed the transposition reaction using a 35 bp piggyBac end of the transposon that would liberate the end-labelled L-TIR substrate flanked by donor DNA where the 3 OH 13-3-19 136 nt flanking donor top strand. The size of the faster end of the transposon is already exposed, that is, a ‘pre- migrating species of the two new species is consistent with nicked’ substrate. Analysis of the reaction products on a a break between the 5 transposon end and the donor DNA denaturing gel revealed the rapid accumulation of a new that releases the end-labelled 103 nt bottom strand of the species about 74 nt in length (Figure 2A). The size of this piggyBac R-TIR from the flanking donor DNA. Determination product is consistent with the formation of a hairpin on the of the exact positions of these cleavages is described below. transposon end that includes several nucleotides of the 0 0 It is notable that cleavage at the 3 and 5 transposon ends flanking donor DNA; its exact sequence is considered below. 0 0 did not occur simultaneously. The cleavage at the 3 transpo- Hairpin formation reflects cleavage at the 5 end of the son end that generated the slower migrating species occurred transposon and concomitant liberation of the transposon 0 0 before cleavage at the 5 end that generated the faster- from the flanking donor DNA. The 5 transposon end clea- migrating species. Similar DSB reaction products were ob- vage is much faster with the pre-nicked substrate than with tained using piggyBac L-TIR DNA (Supplementary Figure intact ends; some hairpin is evident at 1 min with the pre- 2A), although at lower efficiency compared to piggyBac R- nicked substrate (Figure 2A), whereas only 3 end nicking is TIR. Thus, nicking at the 3 transposon end appears to initiate evident by 5 min with the intact end with the flanking donor transposition. substrate (Figure 1B). With the pre-nicked substrate, a high Also faintly visible is a very slow migrating species marked level of hairpin product is present until 10–20 min and the ‘c’. As discussed in detail below, this species is a hairpin that amount decreases thereafter, suggesting that the hairpin 0 0 includes both the 5 and 3 strands of the piggyBac R-TIR end species can be resolved by the transposase (Figure 2A). and is a transposition intermediate. DSBs resulting from hairpin formation on the transposon end are already known to occur with the prokaryotic ele- ments Tn10 (Kennedy et al, 1998) and Tn5 (Bhasin et al, piggyBac transposase joins the ends of the transposon 1999); piggyBac is the first example of a eukaryotic element to the target DNA that uses this mechanism. When the products of the above reactions were displayed on To test whether piggyBac hairpin formation, leading to a native agarose gel, we observed joining of the cleaved R-TIR transposon excision, requires an exposed 3 OH transposon (Figure 1C) and L-TIR (Supplementary Figure 2B) fragments end, we compared hairpin formation of pre-nicked piggyBac 0 0 to a circular plasmid target DNA to form two different L-TIR substrates that had either a 3 deoxyguanosine or a 3 products. The joining of one transposon end to one strand dideoxyguanosine at their 3 ends (Figure 2B). If hairpin of the target DNA forms a single-end join (SEJ) in which one formation requires a 3 OH transposon end, the dideoxy- target strand is broken by the covalent joining of one trans- containing substrate should be unable to support hairpin poson end and the other plasmid strand is intact, resulting in formation and hence a DSB. In contrast to the pre-nicked 3 a nicked circular plasmid. Concerted joining of two transpo- deoxyguanosine substrate, which efficiently formed the 74 nt son ends to separate strands of the same target DNA forms a hairpin species with the concomitant release of the top 20 nt double-end join (DEJ) in which each strand of the target DNA flanking donor DNA (Figure 2B, lanes 2 and 3), the 3 is covalently linked to one transposon end, forming a linear, dideoxyguanosine substrate failed to form the hairpin and double-stranded DNA molecule. Formation of such coupled undergo DSB (Figure 2B, lanes 4 and 5). Thus, hairpin DEJ products is consistent with formation of a transpososome formation is an essential step in piggyBac excision. &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1099 | | OH OH OH piggyBac in cut and paste transposition R Mitra et al A B Hairpin Hairpin 59 59 20 DSBs * resolution + + formation * ∗ 35 24 35 ∗ ∗ ∗ 35 ∗ 24 35 74 Pre-nicked L-TIR Top flank Hairpin Tr ansposon Pre-nicked L-TIR intermediate bottom dG ddG Time (min) 02 5 0025 0 Time (min) 76 12 5 102030 60 67 74 ∗ 67 ∗ 35 ∗ 35 ∗ M 12 35 4 6 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 0 0 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 0 0 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. When the products of transposition reactions (Figure 2A) of these single-stranded products in reactions using a using the pre-nicked piggyBac L-TIR substrate are displayed piggyBac L-TIR in which the strand containing the 3 terminal on a native agarose gel (Figure 3A), joining of the excised transposon end is labelled at its 5 internal end reveals the transposon ends to the target plasmid forming the SEJ and chemistry of target joining: the 3 OH terminal end of the DEJ products is observed. Significant levels of target joining piggyBac L-TIR joins covalently to the target DNA. In a species are seen late (5–10 min) in the time course of the separate experiment, we have shown directly that the 5 reactions, although hairpin formation on the transposon end ends of piggyBac do not join to target DNA (Supplementary is evident early (1 min). Such timing suggests that the trans- Figure 3). position reaction proceeds through end nicking and hairpin formation, leading to DSBs, that is, transposon excision, A 4-nt TTAA hairpin is formed on the piggyBac end which is followed by hairpin resolution to expose the trans- To define the sequence identity of the transposon end hairpin poson ends, after which target joining occurs. The much at the nucleotide level, we isolated the hairpin species higher levels of end cleavage and target joining observed generated from a transposition reaction using the pre-nicked with the pre-nicked substrate (Figure 3A) as compared to the piggyBac L-TIR substrate and then determined its sequence intact substrate (Figure 1C and Supplementary Figure 2B) using the Maxim–Gilbert G-reaction (Figure 4A). The G suggests that nicking at the 3 transposon end is a key sequence of the hairpin was identical to that of the bottom determinant of the rate of transposition. strand of the transposon DNA flanked by the donor DNA with respect to 3 terminal end of piggyBac at positions G ,G and 1 2 The 3 OH end of piggyBac joins to the target DNA G . However, the positions of the following G’s in the hairpin To determine directly which strand of a piggyBac end joins to and an intact transposon end differed. Whereas the G’s in the target DNA, we examined the products formed between a the intact transposon end when the flanking donor DNA pre-nicked piggyBac L-TIR fragment labelled at its internal 5 is attached to the transposon were at positions G ,G 12/S 17/S end, that is, at the end of the transposon strand containing a and G , the next G in the hairpin was at position G , 19/S 13/H 3 OH end, and a plasmid target DNA, on a denaturing agarose followed by G ,G ,G and G , reflecting the 17/H 21/H 25/H 27/H gel (Figure 3B). Whereas target joining leads to the formation sequence of the top strand of the transposon DNA of two products, that is, SEJs and DEJs, on a native gel (Figure 4A). (Figure 3A), only a single product was evident on a denatur- This pattern is consistent with a hairpin that includes the 3 ing agarose gel, consistent with the joining of one piggyBac end of the piggyBac L-TIR DNA including the G at the 3 L-TIR segment to a single target strand (Figure 3B). Detection terminus of the transposon, 4 nt from the flanking donor 1100 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | OH OH OH piggyBac in cut and paste transposition R Mitra et al Time (min) 0 12 5 10 20 30 60 Time (min) 0 12 5 10 20 30 60 AB SEJ 3.4 2.7 3.4 Single strand DEJ linear 2.7 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. Hairpin Hairpin formation resolution ∗ 74 74 ∗ ∗ 35 35 Pre-nicked L-TIR 12 3 ctcaaatcttttttaaCCCTAGAAAGATAGTCTGCGTAAAA gagtttagaaaaaattGGGATCTTTCTATCAGACGCATTTT 19 17 12 3 2 1 S S S Hairpin G27/H G25/H G19/S HHHH H G21/H 13 17 21 25 27 G17/S G17/H aaCCCTAGAAAGATAGTCTGCGTAAAA ttGGGATCTTTCTATCAGACGCATTTT G13/H 3 2 1 G12/S 35 nt G3 G3 G2 G2 G1 G1 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 oligonucleotide containing a TTAA hairpin for 20 min. Lane 1, the 3 strand of L-TIR as a 13-3-19 13-3-19 marker; lane 2, hairpin DNA without piggyBac transposase incubation; lane 3, hairpin DNA with piggyBac transposase incubation. DNA—T, T, A, A—joined to the C at the 5 end of the transposon piggyBac transposase can resolve a hairpin and the rest of the 5 strand of the piggyBac L-TIR. Thus, four transposon end nucleotides of flanking donor DNA are included in the hairpin. We also directly analysed the ability of piggyBac transposase As piggyBac always inserts into TTAA target sites, the element to resolve a pre-formed hairpin. Using a self-complementary will always be flanked by TTAA in the donor site and the hairpin oligonucleotide, we generated a piggyBac hairpin L-TIR will always include the TTAA sequence. We explore the effects species containing a 4 nt TTAA loop. In the presence of the of changing this sequence below. transposase, the hairpin DNA is resolved to generate a 35 nt &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1101 | | Intact substrate hairpin G - substrate G - hairpin ∗ piggyBac in cut and paste transposition R Mitra et al DNA fragment corresponding to cleavage at the 3 end of We examined the ability of piggyBac transposase to pro- piggyBac on the bottom strand of the piggyBac L-TIR mote DSBs at piggyBac L-TIRs flanked by GCGC, a sequence (Figure 4B, lane 3). Thus, the lack of hairpin accumulation different at all positions from the standard TTAA sequence. in Figure 1B likely results from rapid resolution of the hairpin Display of the reaction products on a denaturing gel reveals intermediate. that no nicks or DSBs occur with the GCGC flank (Figure 6A). Furthermore, no hairpinning is observed with a pre-nicked GCGC flank L-TIR (Figure 6B) nor does resolution of a GCGC- piggyBac correctly inserts into TTAA target sites in vitro containing hairpin L-TIR occur (Supplementary Figure 5A). piggyBac inserts exclusively into TTAA target sites in vivo and Thus, with a flanking GCGC, there is a defect in end proces- thus TTAA target sequence duplications flank a newly in- sing at every step, that is nicking, hairpin formation and serted transposon (Cary et al, 1989). To determine the fidelity hairpin resolution, revealing that the TTAA flanking sequence of piggyBac target joining in vitro, we used PCR to generate a has a key role in transposon excision. mini-piggyBac element in which a gene for kanamycin resis- It is also notable that little (o10% of wild type) target tance is flanked on both ends by piggyBac L-TIR 13-3-19 joining is observed with a pre-nicked GCGC L-TIR substrate sequences with their 3 OH already exposed; this element (Figure 6C), indicating that although the 3 OH at this trans- lacked the TTAA extensions present on the 5 ends of an poson end is already exposed, it is a poor substrate for target authentic excised piggyBac element. This mini-piggyBac ele- joining. ment was used as a substrate in an in vitro transposition We also analysed donor cleavage with piggyBac L-TIR reaction containing pUC19 plasmid DNA as a target. transposon ends in which the standard TTAA flanking se- Transposition products were recovered by selection for kana- quence was changed at three positions (GCGA), two positions mycin-resistant E. coli after transformation and the transpo- (GCAA) and one position (GTAA) (Figure 6A). Nicking and son–plasmid junctions were sequenced. Four independent DSBs were detected only with the GTAA substrate that transposition reactions were performed and five products differed at only one position from the 5 end from the from each reaction were recovered and analysed. In all standard TTAA flank (Figure 6A). Accumulation of the hair- cases, insertion occurred at a TTAA site and the element pin intermediate was also observed with this GTAA flanking was always flanked by a 4 bp TTAA target sequence duplica- sequence substrate, suggesting that hairpin resolution is also tion; insertions into 8 of the 13 different TTAA sites on the defective when the 3 end of the transposon is linked to a G pUC19 plasmid were recovered (Supplementary Figure 4). rather than the natural T (Supplementary Figure 5B). These Thus, insertion into TTAA target sites is an intrinsic property experiments demonstrate that the efficiency of end cleavage of the piggyBac transposase and the TTAA extensions present is highly influenced by the flanking donor DNA. on the 5 ends of an excised piggyBac transposon are not We have also analysed the influence on target joining of required for TTAA target site selection. the sequence of the 4 nt extension on the 5 ends of excised Thus, our in vitro transposition system directs insertion of piggyBac (Supplementary Figure 6). An L-TIR end lacking any piggyBac into its preferred TTAA target sequence, which is a extension on the 5 transposon end was a better substrate true representation of piggyBac transposition in vivo. than an end having a 5 TTAA extension. Target joining by an end with the usual 5 TTAA extension was much better than There are four steps in piggyBac transposition 0 that with a 5 GCGC extension. Thus, although piggyBac We conclude that piggyBac transposition involves four dis- transposase can join transposons with flush ends to target tinct chemical steps (Figure 5). Step 1: a DSB initiates with a DNA, the usual TTAA overhangs on the 5 transposon ends nick at the junction of the 3 end of the transposon and the efficiently couple excision and integration. flanking donor DNA, exposing a reactive 3 OH on the trans- poson end. Step 2: the 3 OH then acts as a nucleophile and Identification of the catalytic core of piggyBac attacks 4 nt into the flanking donor DNA on the complemen- transposase tary strand, generating a hairpin on the transposon end and The above experiments revealed that the key chemical steps simultaneously releasing the transposon DNA from the donor 2 þ in piggyBac transposition are the Mg -dependent excision of backbone. The flanking donor DNA contains complementary the transposon to yield a transposon with 3 OH ends, fol- 5 TTAA extensions that can rejoin to repair the donor gap to 0 lowed by the direct nucleophilic attack of these 3 OH ends on give a precise excision. Step 3: The hairpin on the transposon the target DNA. The chemistry of these steps is identical to end is resolved by the transposase, leaving a 4 nt overhang at that of the DDE recombinase family, which contains many the 5 end of the excised linear transposon and re-exposing transposases and retroviral integrases (Rice and Baker, 2001; 0 0 the 3 OH at the transposon end. Step 4: the 3 OH at the Zhou et al, 2004; Richardson et al, 2006). In these recombi- transposon end then covalently joins to the target DNA. nases, highly conserved acidic amino acids (DDE or DDD) are closely juxtaposed on an RNaseH-like fold and coordinate piggyBac excision is influenced by the flanking donor essential metal ions. There is, however, little primary DNA sequence sequence homology between piggyBac and the DDE/DDD A newly inserted piggyBac element is flanked by the TTAA recombinases (Sarkar et al, 2003). target sequence duplications resulting from the staggered Secondary structure prediction of T. ni piggyBac by attack on the TTAA target sequence. After excision, the PSIPRED (Jones, 1999b) suggests that a portion of piggyBac TTAA nucleotides from the flanking donor DNA remain likely has part of the RNaseH-like fold that is conserved in attached to the 5 ends of piggyBac. Does the identity of the DDE recombinases such as HIV1 integrase (Dyda et al, 1994) flanking donor site sequences influence piggyBac excision (Supplementary Figure 7). Included within this region of and target joining? piggyBac are D268 and D346, which are invariant among a 1102 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | OH OH TTAA AATT TTAA TTAA AATT AATT TTAA AATT piggyBac in cut and paste transposition R Mitra et al TTAA CCC GGG TTAA AATT GGG CCC AATT Nick CCC GGG GGG CCC Hairpin formation AACCC GGG TT TTAA TTGGG CCC AA AATT Donor flank repair Hairpin resolution TTAA AATT CCC GGG GGG CCC TTAA Target joining AATT TTAA CCC GGG GGG CCC AATT Target site repair TTAA CCC GGG TTAA AATT AATT GGG CCC Figure 5 Schematic representation of the piggyBac cut and paste transposition. piggyBac transposition initiates with nicks at the 3 ends of the 0 0 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 0 0 0 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. 2 þ number of piggyBac-like ORFs that are associated with TIRs through their interactions with Mg . Moreover, the fact that from a variety of insects and animals (Supplementary Figure a single mutation can block these multiple catalytic steps 8) (Sarkar et al, 2003; Arkhipova and Meselson, 2005). The suggests that a single active site can mediate all the chemical positions of these invariant D’s on the putative piggyBac steps of recombination. RNaseH-like fold are equivalent to those of the two D’s on There are several conserved D’s outside of the RNaseH-like the RNaseH-like DDE transposases and retroviral integrases domain that are conserved among the piggyBac transposases: (Supplementary Figure 7). D227, D228, D239, D447 and D450 (Supplementary Figure 8). To probe the involvement of these conserved acidic amino We also made alanine substitutions at these positions and acids in piggyBac transposition, we purified piggyBac trans- tested the activities of the mutant proteins. Notably, D447A posases with alanine substitutions at D268 and D346 and was still capable of binding to the TIRs (Supplementary evaluated their ability to promote specific binding to the TIRs Figure 9) but was defective in all the catalytic steps of and the catalytic steps of transposition. transposition, suggesting that it also lies within the catalytic Although still capable of binding specifically to piggyBac core (Figure 7A–C and Supplementary Figure 10). L-TIR and R-TIR (Supplementary Figure 9), the D268A and D227A, D228A, D239A and D450A were all catalytically D346A mutants were defective in generating the DSBs that active in vitro; thus, these positions do not appear to be part separate the transposon ends from the flanking donor DNA of the catalytic core of the transposase (data not shown). (Figure 7A), resolution of a pre-formed hairpin to expose the 3 OH transposon (Figure 7B) and joining of a substrate TIR Other functional determinants of piggyBac transposase with an exposed 3 OH to a target DNA (Figure 7C). These Another highly conserved residue among piggyBac transpo- findings suggest that these conserved acidic amino acids have sases is W465 (Supplementary Figure 8). Planar interactions critical roles in all the catalytic steps of transposition, likely between such an aromatic amino acid and DNA bases can &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1103 | | OH OH OH OH piggyBac in cut and paste transposition R Mitra et al SEJ Hairpin Hairpin ∗ target A ∗ BC DSB formation formation joining ∗ ∗ Hairpin Donor 5′ transposon ∗ ∗ ∗ DEJ L-TIR flank resolution strand Pre-nicked L-TIR Pre-nicked L-TIR TTAA GCGC GCGA GCAA GTAA TTAA GCGC TTAA GCGC AATT CGCG CGCT CGTT CATT AATT CGCG AATT CGCG Tnsp Tnsp Tnsp –+ –+ –+ –+ –+ –+ – + – + – + Hairpin on transposon 67 end Hairpin on transposon end SEJ 3.4 DEJ 2.7 Donor flank 5′ transposon strand 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. have an important role in base-flipping steps, which can be The actin intron can be efficiently spliced from mRNA of this integral to DNA distortions necessary for the formation and gene, so that a strain carrying the URA3Hactin intron is a resolution of the altered DNA structures such as hairpins, as uracil prototroph. However, if a large DNA segment such as a demonstrated with Tn5 and Tn10 (Davies et al, 2000; several kilobase transposon is introduced into the actin Bischerour and Chalmers, 2007), Hermes (Zhou et al, 2004) intron, the resulting intron is too large to be spliced from and RAG recombinase (Lu et al, 2006). It has been suggested mRNA, making the strain a uracil auxotroph. Thus, excision (Arkhipova and Meselson, 2005) that piggyBac W465 is of the transposon and restoration of the donor site to the involved in DNA hairpinning. W465A, however, has much parental URA3Hactin intron configuration can be followed by reduced nicking activity and is compromised in all subse- assaying for reversion of uracil auxotrophy to uracil proto- quent catalytic steps of transposition although it still binds trophy (Figure 8A). The restoration of the gapped donor site specifically to the piggyBac TIRs (Supplementary Figures 9 in yeast could occur by end-joining or gene conversion using and 11). Thus, W465 cannot have a role only in DNA hairpin the chromosomal actin gene intron as a template (Paques and formation and resolution. Haber, 1999). Another conserved feature of piggyBac transposases is the In our yeast two-plasmid piggyBac system, a transposon C-terminal C C CHC motif. Analysis of this region by donor plasmid contains a mini-piggyBac transposon com- 2 2 2 GenTHREADER (Jones, 1999a) suggests that this region posed of 328 bp of the piggyBac left end and 361 bp of the 2 þ forms a Zn -binding PHD domain (Supplementary Figure piggyBac right end flanking a kanamycin resistance gene in 8); PHD domains bind to chromatin (Bienz, 2006). The C- the URA3Hactin intron cassette. The transposase is supplied terminus is, however, dispensable for piggyBac recombina- by a second plasmid containing the piggyBac transposase tion in vitro: piggyBac , which lacks the C C CHC motif, gene under the galactose-inducible control of the GALS 1–558 2 2 2 is as active in vitro as the wild-type piggyBac (data not promoter (Mumberg et al, 1994). 1–594 shown), perhaps because we have used naked DNA rather In the absence of transposase, the frequency of URA3 than chromatin as a target substrate. reversion was very low, about 10 (Figure 8B). Upon galac- tose induction of the piggyBac transposase gene, however, the piggyBac can transpose in S. cerevisiae frequency of URA3 reversion was very much higher, about To establish a simple genetic assay for the excision of 10 , indicating a high level of transposon excision piggyBac, we adapted a modified version of the yeast URA3 (Figure 8B). Considerable excision, that is, uracil prototrophy, was observed even without galactose induction, indicating gene as a transposon donor (Yu and Gabriel, 1999). In this that the low level of transposase present because of leakiness modified URA3 gene, the yeast actin intron has been intro- of the GALS promoter can promote transposition. duced into the URA3 gene to form a URA3Hactin intron gene. 1104 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | + piggyBac in cut and paste transposition R Mitra et al AB C Hairpin DSB resolution Donor 5′ transposon L-TIR 74 flank strand L-TIR SEJ 3.4 DEJ 2.7 Donor 2.8 kb flank 5′ transposon strand 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. N indicates nucleoprotein complexes formed by transposase binding to the labelled piggyBac L-TIRs. We demonstrated above that certain highly conserved have been identified in Xenopus (Hikosaka et al, 2007). We acidic amino acids are necessary for piggyBac activity in have developed a faithful in vitro transposition system that vitro. Are they also essential for piggyBac activity in vivo? defines the chemical steps by which piggyBac undergoes cut We evaluated transposition in yeast promoted by mutant and paste transposition. Our findings account for several transposases substituted with alanines at D268, D346, D447 distinctive features of piggyBac transposition and particularly and W465. In all cases, the frequency of uracil prototrophy notable is that piggyBac can transpose without DNA synth- promoted by these mutant transposases was more than five esis, a process involved in all other characterized transposi- orders of magnitude less than that observed with wild type tion reactions. The systems we have described here provide and not significantly different from that observed in the valuable tools for further development of piggyBac-based absence of transposase, that is, about 10 (Figure 8B). systems for insertional mutagenesis and transgenesis as To demonstrate that the mutant proteins are stably pro- well as for dissection of piggyBac transposition at the mole- duced, we have shown in a yeast transposon integration cular level. assay (Supplementary data) that the presence of the mutant transposases inhibits transposition by wild-type transposase, piggyBac excision involves the formation and resolution that is, the mutants are dominant negatives and are thus of hairpins on the transposon ends stably produced (Supplementary Figure 12). The yeast sys- To undergo cut and paste transposition, piggyBac transposase tem thus supports the hypothesis based on in vitro data that induces DSBs that separate the transposon ends from the these amino acids are part of the piggyBac active site. flanking donor DNA. piggyBac DSB initiates with a nick at the 0 0 3 end of the transposon. The free 3 OH then attacks the complementary strand to form a hairpin on the transposon Discussion end, concomitantly releasing the transposon from the flank- piggyBac: a widespread eukaryotic DNA transposon ing donor DNA. As we find that more hairpin intermediate is Cabbage looper moth piggyBac is a DNA transposable ele- formed at earlier times with a pre-nicked substrate compared ment and a member of the widespread piggyBac family of to an intact one in which nicking must first occur, introduc- transposons (Sarkar et al, 2003) with recently active elements tion of the nick may be a rate-limiting step in transposition. &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1105 | | D268A D346A D447A WT 35 nt (marker) WT D268A D346A D447A WT D268A D346A D447A piggyBac in cut and paste transposition R Mitra et al Actin intron XhoI can splice URA URA+ URA Actin intron XhoI XhoI Actin intron cannot splice URA mini-piggyBac URA– URA Transposon excision Actin intron XhoI can splice URA+ URA URA Actin intron Transposase plasmid Excision frequency Glucose Galactose induction induction URA colonies –7 +– pGALS- Tnsp <1.0 X 10 –5 pGALS+ Tnsp WT +– 3.2 X 10 –7 pGALS – Tnsp –+ <1.0 X 10 –2 pGALS+ Tnsp WT –+ 2.4 X 10 –7 pGALS+ Tnsp D268A ND + <1.0 X 10 –7 pGALS+ Tnsp D346A ND + <1.0 X 10 –7 pGALS+ Tnsp D447A ND + <1.0 X 10 –7 pGALS+ Tnsp W465A ND + <1.0 X 10 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. The slow nicking step could, however, reflect the slow TTAA target sites. Indeed we have shown that the presence of assembly of an active transpososome on an intact substrate non-TTAA flanking base pairs can greatly inhibit piggyBac compared to rapid assembly of an active transpososome on a excision. Despite being 4 bp away, the phosphodiester bond pre-nicked substrate rather than a limitation in the chemical on the complementary strand that is attacked by the 3 OH on steps. It is also notable that little difference in hairpin forma- the transposon end is actually brought relatively near the 3 tion is observed between pre-nicked left and right ends or end of the transposon by the twist of the helix. The require- between pre-cleaved left and right ends whereas an intact left ment for the TTAA flank may reflect a requirement for DNA end is a much less efficient substrate than an intact right end. distortability to juxtapose the 3 OH transposon end and its These observations support the view that the formation of an position of attack on the complementary strand. Similar active transpososome is more stringently regulated on a DNA distortions have been shown by phasing analysis of the end with a transposon end still flanked than with cleaved DNAs sequences upon binding of Tn5 transposase (York and that are recombination intermediates. These results also Reznikoff, 1997; Ason and Reznikoff, 2004). The hairpin suggest that transpososome assembly occurs differently on species is then resolved by cleavage at the 3 end of the each end. transposon. 0 0 The transposase then opens the hairpin to expose the 3 OH Thus, the 5 transposon ends of the excised transposon are transposon end that can then attack the target DNA. A similar flanked by 4 nt, TTAA overhang, that derive from the flanking ‘hairpin on the transposon ends’ mechanism has been ob- donor site DNA, unlike the hairpin formed flush at the 5 served with the prokaryotic elements Tn5 and Tn10 (Kennedy transposon end with no flanking DNA in the Tn5 and Tn10 et al, 1998; Bhasin et al, 1999) and contrasts with the ‘hairpin transposons (Kennedy et al, 1998; Bhasin et al, 1999). on the donor DNA’ observed with the hAT transposon Hermes (Zhou et al, 2004) and RAG recombinase (van Gent et al, piggyBac transposition is accompanied by precise 1996). DSBs through hairpins on the transposon ends in excision at the donor site piggyBac transposition are the first to be reported in a After transposon excision, repair of the broken donor chro- eukaryotic transposon system. mosome that contains a gap at the position from which the The intramolecular hairpin reaction occurs by the attack of element was excised must occur. With most eukaryotic the transposon end 3 OH on the donor phosphodiester bond transposons, the original target sequence is not reformed 0 0 that is 5 to the TTAA sequences that always flank the 5 ends during donor site rejoining but rather a ‘footprint’ remains of the transposon. These flanking TTAAs are present in all that reflects the joining of the flanking donor ends that piggyBac donor sites because piggyBac inserts specifically at contain the duplicated target sequence (Coen et al, 1986; 1106 The EMBO Journal VOL 27 NO 7 2008 &2008 European Molecular Biology Organization | | piggyBac in cut and paste transposition R Mitra et al Weil and Kunze, 2000). A distinctive feature of piggyBac The ability of piggyBac to repair both the broken donor transposition is that transposition is always followed by backbone and the target site by ligation of TTAA sequences reconstitution of a single TTAA sequence at the donor site, flanking the gapped donor sites and the transposon ends that is, precise excision occurs (Cary et al, 1989). We have rather than DNA synthesis could help make piggyBac inde- shown that piggyBac excises by a mechanism that cleanly pendent of the host repair machinery and contribute to its frees the 3 ends of the transposon and results in 4 nt TTAA functionality in a wide variety of organisms. overhangs on the 5 ends of the transposon. Consequently, there are also TTAA overhangs on both 5 ends of the broken piggyBac transposase is likely a DDE recombinase donor DNA. As these overhangs are present on complemen- piggyBac transposase binds specifically to both the left and 2 þ tary strands, they can readily pair and be ligated by the host right ends of piggyBac transposon and promotes the Mg - repair machinery, yielding a TTAA at the donor site equiva- dependent joining of a 3 OH transposon end to a target DNA. lent to the original target site (Figure 5). Thus, gene conver- Thus, piggyBac uses the same target joining mechanism, that sion using a homologue as a template need not be invoked to is, direct nucleophilic attack, as do the DDE recombinases account for precise excision in the case of piggyBac transposi- (Rice and Baker, 2001). The catalytic cores of DDE recombi- tion (Paques and Haber, 1999). nases share a common structure: an RNaseH-like fold on It should also be noted that target site-specific insertion which the conserved acidic DDE/DDD amino acids are juxta- 2þ into a palindromic sequence is not sufficient to ensure precise posed to coordinate Mg ions that are essential cofactors for excision. Tc1 mariner elements always insert at TA sites. the chemical steps of transposition (Rice and Baker, 2001; However, cleavage of the 5 transposon ends actually occurs Hickman et al, 2005; Richardson et al, 2006). However, there inside the element, leaving non-complementary overhangs is no obvious sequence similarity between piggyBac and DDE that cannot be simply ligated together such that footprints are recombinases (Sarkar et al, 2003). seen most frequently upon element excision (Plasterk, 1991). From the results of secondary structure prediction and mutational analysis, we suggest that part of the piggyBac The sequence TTAA coordinates piggyBac excision and active site has a section of an RNaseH-like fold on which the target site insertion D residues D268 and D346 that are essential for DNA break- Previous in vivo experiments (Fraser et al, 1995) and our in age and joining are located in positions comparable to those vitro experiments have shown that piggyBac inserts preferen- of the N-terminal D residues of the DDE recombinases. We tially into a TTAA target site such that the newly inserted have also identified another essential C-terminal D, D447, transposon is flanked by TTAAs. Although excised piggyBac that we suggest performs the same function as the C-terminal is flanked by TTAA on the 5 transposon ends, these flanking E in the DDE recombinases. Further analysis of these mutants nucleotides are not essential for correct target joining: a will be required to establish if these amino acids have a role piggyBac element with flush 5 ends still chooses TTAA as a in transpososome formation as has been reported in the case target. We have found that the flanking TTAA sequence is of Mu transposase (Kim et al, 1995). also critical to transposon excision; changing the flanking These results provide evidence that piggyBac is a member TTAA can block nicking at the 3 transposon end, hairpin of the DDE recombinase superfamily, extending the range of formation and hairpin resolution. Flanking donor sequence protein sequences that can form this essential catalytic core. also influences the excision of other elements (Wu and Tn5 and Tn10 transposase carries out four distinct chemi- Chaconas, 1992; Williams et al, 1999). cal reactions in Tn5 and Tn10 transposition: nicking at the This requirement for the same sequence for target integra- 3 OH transposon end, hairpin formation, hairpin resolution tion and DSB formation to promote excision suggests that this and target joining. The available evidence argues that a single TTAA sequence is recognized by the transposase during both active site contributed from a single transposase monomer at excision and integration. each transposon end is used repeatedly to catalyse all four steps (Bolland and Kleckner, 1996; Reznikoff, 2003). The use Target joining and the generation of intact DNA at the of a single active site to carry out alternating hydrolysis and target site transesterification has been supported by studies on other We have found that piggyBac joins to the target DNA by the proteins that use two metal ions in their active site that are held in place by acidic amino acids (Nowotny et al, 2005). As direct attack of the 3 OH ends of the transposon to staggered piggyBac transposase performs similar chemical reactions positions on to the TTAA target DNA sequence. This direct nucleophilic attack is the hallmark of target joining by the and mutations in the catalytic DDD residues block all of DDE superfamily that contains many prokaryotic and eukar- these steps, we are attracted to the view that piggyBac yotic transposases and retroviral integrase (Rice and Baker, transposition similarly involves the activity of a single active 2001; Hickman et al, 2005; Richardson et al, 2006). As the 5 site at each transposon end. ends of the newly integrated transposon are flanked by 5 TTAA, the generation of intact duplex DNA at the site of Genetic analysis of piggyBac transposition in insertion can be accomplished by pairing and ligation of the S. cerevisiae complementary TTAA sequences on the 5 transposon ends We have developed a piggyBac transposition system in the and at the gaps that flank the newly inserted transposon. This highly genetically tractable S. cerevisiae. In this system, we pairing and ligation strategy will result in TTAA base pairs can directly follow the excision of a mini-piggyBac element flanking the newly inserted transposon. This strategy is from a Ura derivative of the URA3 gene containing the distinct from the repair of target gaps by host polymerase transposon by measuring the frequency of uracil prototrophy and ligase as occurs in other transposon systems (Mizuuchi, in the presence of piggyBac transposase. piggyBac excision 1984). can occur at high frequency, that is, about 1/100 cells are &2008 European Molecular Biology Organization The EMBO Journal VOL 27 NO 7 2008 1107 | | piggyBac in cut and paste transposition R Mitra et al 2 þ equilibrated Ni Sepharose column (GE Healthcare). The column uracil prototrophs in a single colony grown on media indu- was washed with 10 column volumes of TSG buffer followed by 6 cing the expression of the transposase. We have used this column volumes of TSG þ 50 mM imidazole buffer. piggyBac–Myc– system to show that amino acids that we identified as being His fusion protein was eluted with TSGþ 200 mM imidazole buffer, part of the catalytic site in vitro are also necessary for activity dialysed against storage buffer (20 mM Tris–HCl (pH 8.0), 500 mM NaCl, 25% v/v glycerol) and stored at 801C. in vivo. In addition to facilitating the analysis of piggyBac transpo- piggyBac DSB and target joining reactions sition in vivo, including the isolation of hyperactive mutants 150 nM of piggyBac transposase was incubated with 1.5 nM of that will aid insertional mutagensis in mammalian cells, this radiolabelled piggyBac L-TIR or R-TIR DNA (see Supplementary data) in 25 mM HEPES (pH 8.0), 3 mM Tris (pH 8.0), 75 mM NaCl, piggyBac system is also well suited for the in vivo manipula- 2 mM DTT, 10 mM MgCl , 0.01% BSA, 3.75% glycerol and 10 nM tion of the yeast genome. pUC19 in a final volume of 20 mlat301C for different time intervals. Reactions were stopped by incubation in 1% SDS and 20 mM EDTA Domesticated piggyBac transposases for 30 min at 651C and displayed on a 1% native agarose–1 TAE gel. For analysis of DNA nicking and hairpin formation, the reaction Proteins related to piggyBac transposases are present in many products were phenol/chloroform extracted, ethanol precipitated eukaryotic genomes, including insects, fish and mammals. In and displayed on a 5% acrylamide–7 M urea–1  TBE denaturing the human genome, there are five piggyBac-derived genes, acrylamide gel. All gels described here were dried and exposed to PGBD1–5 (Sarkar et al, 2003). Our work shows that PGBD1–3 phosphoimager plates and analysed by Imagequant software (GE Healthcare). and 5 are very unlikely to have transposase activity as at least one of the active site amino acids we have identified in T. ni Analysis of hairpin formation using a pre-nicked piggyBac piggyBac is mutated in each of these proteins. In PGBD4, left end however, the catalytically essential amino acids are intact, 0 A 35 bp oligonucleotide corresponding to the 3 end of piggyBac L- although this provides no proof that PGB4 is actually a TIR was radiolabelled at its 5 end with [g- P]ATP (GE 13-3-19 Healthcare) and T4 polynucleotide kinase. After purification on a transposase. Expressed cellular genes that are significantly G25 column, the end-labelled oligonucleotide was annealed with related to transposases but are not flanked by terminal equimolar amounts of a 59 bp oligonucleotide containing the 5 end inverted repeats or target site duplications have been ob- of piggyBac L-TIR flanked by 24 nt flanking donor DNA and a 13-3-19 served for many DNA transposons, some of which are 24 nt oligonucleotide corresponding to the bottom strand of flanking donor DNA. The annealed oligonucleotide mixture was ‘domesticated’ transposases co-opted for by the cell for then directly used as the substrate in DSB and target joining processes that do not involve DNA breakage and joining reactions as described above. The reaction products were displayed such as transcriptional regulation (Volff, 2006). on native agarose, denaturing acrylamide and 1% agarose–50 mM NaOH gels. Materials and methods Supplementary data Supplementary data are available at The EMBO Journal Online piggyBac transposase expression and purification (http://www.embojournal.org). The piggyBac transposase ORF (594 amino acids) was PCR amplified from the plasmid pBhelper (Lobo et al, 1999) and cloned between the NcoI and KpnI sites of plasmid pBAD Myc-HisC Acknowledgements (Invitrogen) to generate a pBAD piggyBac–Myc–His6 fusion construct (p-transposase). E. coli Top10 (Invitrogen) cells contain- We thank David O’Brochta (University of Maryland, College Park) ing p-transposase were grown with shaking at 301C in LB medium for providing the piggyBac transposon plasmids, and Abram Gabriel containing 100 mg/ml ampicillin till an OD of 0.6. The culture was (Rutgers University) and Jef Boeke (JHU-SOM) for providing yeast then induced with 0.1% L-arabinose for 18 h at 161C. Following plasmids and strains. We also thank the Craig lab members for their induction, cells were lysed with a French Pressure cell (Fisher valuable insights into this project. This work was partly supported Scientific) in TSG buffer (20 mM Tris–HCl (pH 8.0), 500 mM NaCl, by National Institute of Health (NIH) grant NC90020064 to NLC. 10% v/v glycerol). The lysate was then loaded onto a pre- NLC is an Investigator of the Howard Hughes Medical Institute. 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Journal

The EMBO Journal – Springer Journals

Published: Apr 9, 2008

Keywords: piggyBac; precise excision; transposase; transposition

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