Template and target-site recognition by human LINE-1 in retrotransposition

• 成果类型: Article
• 署名作者: Thawani, Akanksha; Ariza, Alfredo Jose Florez; Nogales, Eva; Collins, Kathleen
• 署名单位: University of California System; University of California Berkeley; University of California System; University of California Berkeley; Howard Hughes Medical Institute; United States Department of Energy (DOE); Lawrence Berkeley National Laboratory
• 刊物名称: Nature
• ISSN/ISSBN: 0028-6196
• DOI: 10.1038/s41586-023-06933-5
• 发表日期: 2024-02-01
• 关键词: reverse-transcriptase activity; crystal-structure; orf1 protein; dna; endonuclease; integration; ELEMENTS

摘要

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy. Human LINE-1 ORF2p relies on upstream single-stranded target DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break.