Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, CA 94720, USA.
Cell Rep. 2024 May 28;43(5):114239. doi: 10.1016/j.celrep.2024.114239. Epub 2024 May 15.
R2 non-long terminal repeat (non-LTR) retrotransposons are among the most extensively distributed mobile genetic elements in multicellular eukaryotes and show promise for applications in transgene supplementation of the human genome. They insert new gene copies into a conserved site in 28S ribosomal DNA with exquisite specificity. R2 clades are defined by the number of zinc fingers (ZFs) at the N terminus of the retrotransposon-encoded protein, postulated to additively confer DNA site specificity. Here, we illuminate general principles of DNA recognition by R2 N-terminal domains across and between clades, with extensive, specific recognition requiring only one or two compact domains. DNA-binding and protection assays demonstrate broadly shared as well as clade-specific DNA interactions. Gene insertion assays in cells identify the N-terminal domains sufficient for target-site insertion and reveal roles in second-strand cleavage or synthesis for clade-specific ZFs. Our results have implications for understanding evolutionary diversification of non-LTR retrotransposon insertion mechanisms and the design of retrotransposon-based gene therapies.
R2 非长末端重复（non-LTR）逆转录转座子是多细胞真核生物中分布最广泛的移动遗传元件之一，在人类基因组中转基因补充方面具有应用前景。它们以极高的特异性将新的基因拷贝插入到 28S 核糖体 DNA 的保守位点中。R2 进化枝是通过逆转录转座子编码蛋白 N 端的锌指（ZF）数量来定义的，据推测这些锌指可以累加赋予 DNA 位点特异性。在这里，我们阐明了 R2 N 端结构域在不同进化枝之间和之内的 DNA 识别的一般原则，广泛的特异性识别仅需要一个或两个紧凑的结构域。DNA 结合和保护实验证明了广泛共享的以及进化枝特异性的 DNA 相互作用。细胞中的基因插入实验确定了靶位点插入所必需的 N 端结构域，并揭示了对于特定进化枝的 ZF 的第二链切割或合成的作用。我们的结果对于理解非 LTR 逆转录转座子插入机制的进化多样化以及基于逆转录转座子的基因治疗的设计具有重要意义。
