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DNA-PK facilitatespiggyBactransposition by promoting paired-end complex formation.

Proceedings of the National Academy of Sciences of the United States of America (2017-06-25)
Yan Jin, Yaohui Chen, Shimin Zhao, Kun-Liang Guan, Yuan Zhuang, Wenhao Zhou, Xiaohui Wu, Tian Xu
ABSTRACT

The involvement of host factors is critical to our understanding of underlying mechanisms of transposition and the applications of transposon-based technologies. ModifiedpiggyBac(PB) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies ofPBamong different cell lines have restricted its application. We discovered that the DNA-PK complex facilitatesPBtransposition by binding toPBtransposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK componentsKu70,Ku80, andDNA-PKcsOverexpression or knockdown of DNA-PK components enhances or suppressesPBtransposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency ofPBis significantly reduced inKu80heterozygous mutant mice, confirming the role of DNA-PK in facilitatingPBtransposition 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 thePBtransposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitatingPBtransposition and suggest a transposition-promoting manipulation by enhancing the interaction of thePBends. Consistent with this, deletions shortening the distance between the twoPBends, such asPBvectors 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.

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