DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation

Abstract

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. Modified piggyBac (PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of PB among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates PB transposition by binding to PB transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components Ku70, Ku80, and DNA-PKcs. Overexpression or knockdown of DNA-PK components enhances or suppresses PB transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of PB is significantly reduced in Ku80 heterozygous mutant mice, confirming the role of DNA-PK in facilitating PB transposition in vivo. Fused dimer PBase can efficiently promote transposition. FRET experiments with tagged dimer PBase molecules indicated that DNA-PK promotes the paired-end complex formation of the PB transposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitating PB transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the PB ends. Consistent with this, deletions shortening the distance between the two PB ends, such as PB vectors with closer ends (PB-CE vectors), have a profound effect on transposition efficiency. Taken together, our study indicates that in addition to regulating DNA repair fidelity during transposition, DNA-PK also affects transposition efficiency by promoting paired-end complex formation. The approach of CE vectors provides a simple practical solution for designing efficient transposon vectors.piggyBac | DNA-PK | paired-end complex formation | transposition efficiency | CE transposon vectors T ransposition allows DNA transposons to serve as powerful genetic manipulation tools. During transposition, DNA transposons follow a "cut-and-paste" manner. A paired-end complex (PEC) is first formed by aligning both ends of the transposon, which is then excised and inserted into new loci (1, 2). Although the transposon-encoded transposase is suggested to be sufficient for PEC formation, it is unlikely to be the only protein involved in transposition. In fact, many DNA transposons are nonfunctional outside their natural hosts, suggesting the effects of host factors (3).piggyBac (PB) is a DNA transposon originally identified from the cabbage looper moth (4). Modified PB is highly active in mouse and human cells (5). Because of its potent transposition efficiency and ability to carry large insertions, PB provides unique opportunities for mutagenesis and transgenesis in mammalian systems (6-10). Although the PB transposon has a wide host range, including mammals, its transposition efficiency varies in cultured cells from different mammalian species (7,9). Thus, host factors may still be involved in the proces...

Six domesticated PiggyBac transposases together carry out programmed DNA elimination in Paramecium

“… This figure summarizes the observed effects of PGML KDs on Pgm-mediated IES excision. In line with previously published data ( Dubois et al, 2017 ) and known properties of the T. ni PiggyBac transposase ( Jin et al, 2017 ), the catalytically active form of Pgm is assumed to be a dimer. In the absence of information about the stoichiometry of the complex, one Pgm homodimer (active catalytic site drawn as a star) is represented at each IES boundary, with a large bridging structure formed by all PgmLs.…”

“…The T. ni PB transposase forms a dimer in solution and probably works as a higher-order oligomer during assembly of the transposition complex ( Jin et al, 2017 ). Previous work in Paramecium established that Pgm multimerizes in cell extracts and several Pgm subunits are required to complete IES excision in vivo ( Dubois et al, 2017 ).…”

“…Biochemical and structural studies of DD(D/E) transposases and retroviral integrases bound to their cognate DNA substrates have revealed that assembly of a productive transpososome often involves more transposase subunits than those actually engaged in catalysis, the supernumerary subunits playing an architectural role within the transposition complex ( Montaño et al, 2012 ; Hickman et al, 2014 ); reviewed in Hickman and Dyda, 2015 ). In particular, recent studies have shown that the T. ni PB transposase dimerizes in solution ( Jin et al, 2017 ) and higher-order complexes were proposed to assemble during piggyBac transposition ( Morellet et al, 2018 ). Within a transpososome, all transposase subunits are generally identical because they are encoded by a single gene carried by the mobile element itself.…”

Functional diversification of Paramecium Ku80 paralogs safeguards genome integrity during precise programmed DNA elimination

“…Thus, because most of~10,000 T. thermophila IESs are located in intergenic or other non-coding regions [45], the pressure for precise excision may not be as strong as for P. tetraurelia IESs. Tpb2 likely interacts with Ku70/Ku80, because this appears to be a general property of PiggyBac transposases, be they domesticated or not (this work and [26,46]), but this interaction would simply direct the loading of Ku onto broken ends once DSBs have been introduced, which would represent a minimal form of coupling. Apart from Tpb2-dependent IESs, 12 intragenic piggyBac-derived IESs were described in T. thermophila [47,48].…”

CAR T Cell Generation by piggyBac Transposition from Linear Doggybone DNA Vectors Requires Transposon DNA-Flanking Regions

“…In plasmid vectors, it has been demonstrated that a shorter amount of DNA external to the transposon ITRs leads to more efficient transposition with a variety of transposases, including piggyBac. [42][43][44] However, the effect of this configuration in circular plasmid is to physically bring the ITRs closer together, thereby facilitating paired-end complex formation, the initial step in transposition where the transposase aligns both ends of the transposon. 43 The benefit of this transposon ITR configuration in circular vectors is not applicable to linear vectors, where different factors appear to be important.…”