DNA-PK facilitates piggyBac transposition by promoting paired-end complex formation
DNA-PK facilitates piggyBac transposition by promoting paired-end complex formation
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DNA-PK 通过促进配对末端复合物形成促进 PiggyBac 转座
DOI：
10.1073/pnas.1612980114
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发表时间：
2017
影响因子：
11.1
通讯作者：
Xu Tian
中科院分区：
文献类型：
--
作者：
Jin Yan;Chen Yaohui;Zhao Shimin;Guan Kun Liang;Zhuang Yuan;Zhou Wenhao;Wu Xiaohui;Xu Tian
宿主因子的参与对于我们理解转座的潜在机制和基于转座子的技术的应用至关重要。 ModifiedpiggyBac(PB) 是哺乳动物中最有效的转座子系统之一。然而，PB在不同细胞系中的转座效率不同，限制了其应用。我们发现 DNA-PK 复合物通过与 PB 转座酶 (PBase) 结合并促进配对末端复合物形成来促进 PB 转座。质谱分析和免疫共沉淀揭示了 PBase 与 DNA-PK 成分 Ku70、Ku80 和 DNA-PKcs 之间的物理相互作用。 DNA-PK 成分的过表达或敲低分别增强或抑制组织培养细胞中的 PB 转座。此外，在Ku80杂合突变小鼠中，PBis的种系转座效率显着降低，证实了DNA-PK在促进体内PB转座中的作用。融合二聚体PBase可以有效促进转座。使用标记二聚体 PBase 分子的 FRET 实验表明 DNA-PK 促进 PB 转座子配对末端复合物的形成。这些数据为 DNA-PK 在促进 PB 转座中的作用提供了机制解释，并提出了通过增强 PBends 相互作用来促进转座的操作。与此一致的是，缩短两个PB端之间距离的删除，例如具有较近末端的PB载体（PB-CE载体），对转座效率具有深远的影响。综上所述，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 还通过促进配对末端复合物的形成来影响转座效率。 CE载体的方法为设计高效的转座子载体提供了一种简单实用的解决方案。
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-PKcs. Overexpression 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.