Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, 20892, USA.
Department of Medicine, Division and Nephrology and Hypertension, Vanderbilt University Medical Center, Nashville, TN, USA.
Nat Commun. 2020 Jul 10;11(1):3446. doi: 10.1038/s41467-020-17128-1.
The piggyBac DNA transposon is used widely in genome engineering applications. Unlike other transposons, its excision site can be precisely repaired without leaving footprints and it integrates specifically at TTAA tetranucleotides. We present cryo-EM structures of piggyBac transpososomes: a synaptic complex with hairpin DNA intermediates and a strand transfer complex capturing the integration step. The results show that the excised TTAA hairpin intermediate and the TTAA target adopt essentially identical conformations, providing a mechanistic link connecting the two unique properties of piggyBac. The transposase forms an asymmetric dimer in which the two central domains synapse the ends while two C-terminal domains form a separate dimer that contacts only one transposon end. In the strand transfer structure, target DNA is severely bent and the TTAA target is unpaired. In-cell data suggest that asymmetry promotes synaptic complex formation, and modifying ends with additional transposase binding sites stimulates activity.
猪源 DNA 转座子广泛应用于基因组工程。与其他转座子不同，其切除位点可精确修复而不留痕迹，并特异性整合到 TTAA 四核苷酸上。我们展示了猪源转座酶复合物的 cryo-EM 结构：一个具有发夹 DNA 中间体的突触复合物和一个捕获整合步骤的链转移复合物。结果表明，切除的 TTAA 发夹中间体和 TTAA 靶标采用基本相同的构象，为连接猪源转座酶的两个独特特性提供了一个机制联系。转座酶形成一个不对称二聚体，其中两个中央结构域连接末端，而两个 C 端结构域形成一个单独的二聚体，仅与一个转座酶末端接触。在链转移结构中，靶 DNA 严重弯曲，TTAA 靶标未配对。细胞内数据表明，不对称性促进了突触复合物的形成，并且用额外的转座酶结合位点修饰末端会刺激其活性。
Zotero 插件
