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标题：DNA–PK facilitates piggyBac transposition by promoting paired-end complex formation
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作者：Yan Jin ; Yaohui Chen ; Shimin Zhao 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：28
页码：7408-7413
DOI：10.1073/pnas.1612980114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
关键词：piggyBac ; DNA–PK ; paired-end complex formation ; transposition efficiency ; CE transposon vectors