Structures of vertebrate R2 retrotransposon complexes during target-primed reverse transcription and after second-strand nicking

作者：Akanksha Thawani, Anthony Rodríguez-Vargas, Briana Van Treeck, Nozhat T. Hassan, David L. Adelson, Eva Nogales, Kathleen Collins · 发表于：Science Advances · 年份：2025 · DOI：10.1126/sciadv.adu5533 · 被引用次数：7 · 研究领域：Chromosomal and Genetic Variations、CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques

R2 retrotransposons are site-specific eukaryotic non-long terminal repeat retrotransposons that copy and paste into gene loci encoding ribosomal RNAs. Recently, we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not well characterized. Here, we report high-resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins at the initiation of target-primed reverse transcription and after cDNA synthesis and second-strand nicking. Using biochemical and cellular assays, we illuminate the basis for high selectivity of template use and unique roles for each of the three zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif specific to vertebrate A-clade R2 proteins. Our work provides the first insights into A-clade R2 protein structure during gene insertion and may enable future improvement and adaptation of R2-based systems for precise transgene insertion.