Abstract
The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.
Highlights
The piggyBac DNA transposon is used widely in genome engineering applications
The results here reveal that the distinguishing features of pB transposition, seamless excision and tetranucleotide targeting, are the consequences of a structural echo between flanking hairpin recognition and TTAA target recognition
The strand transfer complex (STC) structure suggests that the reason pB almost invariably integrates at a TTAA tetranucleotide[44,66] is a combination of structural features that include a dramatic target bend, a deformation that causes the TTAA to unpair, and a dense network of interactions that recognize TTAA in single-stranded form
Summary
The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates at TTAA tetranucleotides. PB has proved to be extremely versatile and the lack of a DNA footprint left behind after its transposition is a unique and valuable property[13,14,15,16] It is used in non-viral vectors for transgenesis[17,18], gene therapy[7,19], insertional mutagenesis[20], and genetic screens[21,22,23]. The reaction sequence differs from those of other eukaryotic DNA transposases, such as members of the Tc1/ mariner and hAT superfamilies, or the Transib precursor to RAG1 of the V(D)J recombination system[37,38,39,40], but is identical to the prokaryotic IS4 family of insertion sequences and transposons such as Tn541 Despite this shared reaction pathway, only pB precisely targets TTAA sequences[36]
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