Institute of Developmental Biology and Molecular Medicine & Children's Hospital, State Key Laboratory of Genetic Engineering, National Center for International Research of Development, Fudan University, Shanghai 200433, China.
Molecular and Cellular Biology Laboratory, Institutes of Biomedical Sciences, Fudan University, Shanghai 200032, China.
Proc Natl Acad Sci U S A. 2017 Jul 11;114(28):7408-7413. doi: 10.1073/pnas.1612980114. Epub 2017 Jun 23.
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.
宿主因素的参与对于我们理解转座的基本机制以及转座子技术的应用至关重要。改良的 () 是哺乳动物中最有效的转座子系统之一。然而，不同细胞系中转座效率的差异限制了其应用。我们发现 DNA-PK 复合物通过与转座酶（PBase）结合并促进末端配对复合物的形成来促进 转座。质谱分析和共免疫沉淀揭示了 PBase 与 DNA-PK 组件 、 、 和 的物理相互作用。DNA-PK 组件的过表达或敲低分别增强或抑制组织培养细胞中的 转座。此外， 杂合突变小鼠的 生殖系转座效率显著降低，证实了 DNA-PK 在体内促进 转座的作用。融合二聚体 PBase 可以有效地促进转座。带有标记二聚体 PBase 分子的 FRET 实验表明，DNA-PK 促进 转座子的末端配对复合物形成。这些数据为 DNA-PK 促进 转座提供了机制解释，并提示通过增强 末端的相互作用来促进转座。与之一致的是，缩短两个 末端之间距离的缺失，例如具有更近末端的 -CE 载体 (-CE 载体)，对转座效率有深远的影响。总之，我们的研究表明，除了在转座过程中调节 DNA 修复保真度外，DNA-PK 还通过促进末端配对复合物的形成来影响转座效率。CE 载体的方法为设计高效转座子载体提供了一种简单实用的解决方案。
