DNA-PK facilitates <i>piggyBac</i> 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 <i>piggyBac</i> (<i>PB</i>) is one of the most potent transposon systems in mammals. However, varying transposition efficiencies of <i>PB</i> among different cell lines have restricted its application. We discovered that the DNA-PK complex facilitates <i>PB</i> transposition by binding to <i>PB</i> transposase (PBase) and promoting paired-end complex formation. Mass spectrometry analysis and coimmunoprecipitation revealed physical interaction between PBase and the DNA-PK components <i>Ku70</i>, <i>Ku80</i>, and <i>DNA-PKcs</i> Overexpression or knockdown of DNA-PK components enhances or suppresses <i>PB</i> transposition in tissue culture cells, respectively. Furthermore, germ-line transposition efficiency of <i>PB</i> is significantly reduced in <i>Ku80</i> heterozygous mutant mice, confirming the role of DNA-PK in facilitating <i>PB</i> 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 <i>PB</i> transposon. These data provide a mechanistic explanation for the role of DNA-PK in facilitating <i>PB</i> transposition and suggest a transposition-promoting manipulation by enhancing the interaction of the <i>PB</i> ends. Consistent with this, deletions shortening the distance between the two <i>PB</i> ends, such as <i>PB</i> vectors with closer ends (<i>PB</i>-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.