Hickman Alison B, Lannes Laurie, Furman Christopher M, Hong Christina, Franklin Lidiya, Ghirlando Rodolfo, Ghosh Arpita, Luo Wentian, Konstantinidou Parthena, Lorenzi Hernán A, Grove Anne, Haase Astrid D, Wilson Matthew H, Dyda Fred
Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.
Structural Motility, UMR 144 CNRS/Curie Institute, PSL Research University, Paris, cedex 05, France.
Nat Commun. 2025 Jan 7;16(1):458. doi: 10.1038/s41467-024-55784-9.
Members of the piggyBac superfamily of DNA transposons are widely distributed in host genomes ranging from insects to mammals. The human genome has retained five piggyBac-derived genes as domesticated elements although they are no longer mobile. Here, we have investigated the transposition properties of piggyBat from Myotis lucifugus, the only known active mammalian DNA transposon, and show that its low activity in human cells is due to subterminal inhibitory DNA sequences. Activity can be dramatically improved by their removal, suggesting the existence of a mechanism for the suppression of transposon activity. The cryo-electron microscopy structure of the piggyBat transposase pre-synaptic complex showed an unexpected mode of DNA binding and recognition using C-terminal domains that are topologically different from those of the piggyBac transposase. Here we show that structure-based rational re-engineering of the transposase through the removal of putative phosphorylation sites and a changed domain organization - in combination with truncated transposon ends - results in a transposition system that is at least 100-fold more active than wild-type piggyBat.
DNA转座子的piggyBac超家族成员广泛分布于从昆虫到哺乳动物的宿主基因组中。人类基因组保留了五个源自piggyBac的基因作为驯化元件，尽管它们已不再具有移动性。在这里，我们研究了来自小蝙蝠（Myotis lucifugus）的piggyBat的转座特性，它是唯一已知的活跃哺乳动物DNA转座子，并表明其在人类细胞中的低活性是由于亚末端抑制性DNA序列所致。去除这些序列可显著提高活性，这表明存在一种抑制转座子活性的机制。piggyBat转座酶突触前复合物的冷冻电子显微镜结构显示了一种意想不到的DNA结合和识别模式，该模式使用的C末端结构域在拓扑结构上与piggyBac转座酶的不同。在这里，我们表明，通过去除假定的磷酸化位点和改变结构域组织，结合截短的转座子末端，对转座酶进行基于结构的合理重新设计，可产生一种活性比野生型piggyBat至少高100倍的转座系统。
