DNA-PK facilitates transposition by promoting paired-end complex formation.

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