2025-10-19 中国科学院(CAS)
<関連情報>
- https://english.cas.cn/newsroom/research_news/life/202510/t20251016_1089550.shtml
- https://www.science.org/doi/10.1126/science.adu3433
ヒトLINE-1によるDNA標的化のメカニズム Mechanism of DNA targeting by human LINE-1
Wenxing Jin, Cong Yu, Yan Zhang, Changchang Cao, […] , and Rui-Ming Xu
Science Published:9 Oct 2025
DOI:https://doi.org/10.1126/science.adu3433
Editor’s summary
Long interspersed nuclear element-1 (LINE-1) mobilizes by inserting into new genomic sites through its open reading frame 2 protein (ORF2p), which has endonuclease (EN) and reverse transcriptase (RT) activities. Jin et al. determined ORF2p’s structure bound to an RNA-DNA hybrid in its RT site and a Y-shaped DNA on its surface. ORF2p binds double-stranded DNA without sequence specificity, and the single-stranded DNA path suggests dynamic EN positioning during cleavage. EN activity was strongly stimulated by forked or flap DNAs, indicating that ORF2p acts as a structure-selective endonuclease and explaining why LINE-1 mobilization predominantly occurs during DNA replication. —Di Jiang
Structured Abstract
INTRODUCTION
Retrotransposons copy and insert their coding sequences into new genomic locations via RNA intermediates, posing risks to genome integrity. In humans, the only autonomously active retrotransposon is the long interspersed nuclear element–1 (LINE-1), which invades the genome through target-primed reverse transcription (TPRT). This process is catalyzed by the LINE-1–encoded open reading frame 2 protein (ORF2p), which has endonuclease (EN) and reverse transcriptase (RT) activities. In this study, we investigated the molecular mechanisms governing LINE-1 genome targeting and TPRT execution by ORF2p.
RATIONALE
We produced human ORF2p in eukaryotic cells and purified a complex of ORF2p with endogenous nucleic acids. The structure and the composition of the complex were analyzed by cryo–electron microscopy (cryo-EM), deoxyribonuclease and ribonuclease digestions, and DNA and RNA sequencing (RNA-seq). Furthermore, we conducted in vitro EN activity assays to investigate the features of DNA substrates preferred by ORF2p for efficient cleavage.
RESULTS
Purification of LINE-1 ORF2p from either insect or human cells revealed tight binding between the protein and endogenous nucleic acids. Cryo-EM analysis identified two distinct pieces of copurified nucleic acids: an A-form RNA/DNA hybrid positioned in the RT active site pocket and a B-form double-stranded DNA (dsDNA) with a single-stranded tail bound to the ORF2p’s exterior surface, primarily through sequence-independent charge interactions. Structure comparison of the ORF2p and other non–long terminal repeat retrotransposon complexes revealed a generally similar DNA binding path, but ORF2p notably differed from others in lacking determinants for sequence-specific DNA binding and having a flexibly tethered EN domain located distant from the dsDNA binding region. Consistent with the biochemical and structural characterizations, sequencing of copurified DNA revealed prominent enrichment of repetitive satellite DNA elements. RNA-seq data indicated that ORF2p could bind and reverse transcribe a wide range of cellular RNAs.
Inspired by the structural finding of the binding of unwound DNA to ORF2p and the knowledge that LINE-1 integration occurred primarily during DNA replication, we investigated whether replication-intermediate DNA structures influence the EN activity of ORF2p. In vitro nuclease activity assays showed great stimulation of ORF2p activity toward DNA substrates having a fork or flap structure approximately 6 to 10 base pairs away in the 5′ direction of the strand carrying the TTTTT/AA consensus ORF2p cleavage motif. Cleavage of the opposite strand predominantly occurred between TA, TC, and TG dinucleotides 8 to 12 nucleotides (nts) away. As a result, nicking on both DNA strands by ORF2p resulted in a staggered flanking offset of 8 to 12 nts, which accounted for the generation of target site duplication from LINE-1 integration.
CONCLUSION
Our discovery of ORF2p’s binding mode to genomic DNA and its preferential nicking of forked and 5′-flap DNA, together with the ubiquitous presence of short nicking motifs, provides a clearer understanding of the TPRT process of LINE-1, in which ORF2p encounters DNA promiscuously and makes a productive cut only when its binding region coincides with the presence of suitably structured DNA, such as that generated during DNA replication. Stable binding of ORF2p to the dsDNA allows nicking of both strands of downstream DNA by the flexibly tethered EN domain in a stepwise manner.
DNA binding mode and target preference of LINE-1 ORF2p.
(Top, left to right) Depiction of a model of DNA targeting—bottom- and top-strand nicking by ORF2p during the TPRT process of LINE-1. The contour of the ORF2p structure is shown, with the DNA binding region colored in orange, the EN domain in green, and the rest of the structure in gray. The dashed magenta line followed by a stretch of “A’s” illustrates the RNA template with an adenine-rich 3′ tail, and the curved green arrow in the far-right panel indicates the movement of the EN domain for nicking distinct DNA strands. (Bottom) Panels show the preference of DNA substrates by ORF2p observed in in vitro nuclease assays.
Abstract
Long interspersed nuclear element–1 (LINE-1 or L1), the only autonomously active retrotransposon in humans today, constitutes a large proportion of the genome and continues to evolve the genome and impact fundamental biological processes. L1 retrotransposition critically depends on its endonuclease and reverse transcriptase subunit open reading frame 2 protein (ORF2p), which targets genomic loci and nicks DNA using an evolutionarily distinct yet not fully understood mechanism. Our structural and biochemical analyses revealed that ORF2p is a structure-dependent endonuclease. It binds a double-stranded DNA region upstream of the nicking site and recognizes a downstream forked or flap structure for efficient DNA nicking. This discovery suggests that L1 mobilization piggybacks on chromosomal processes with noncanonical DNA structure intermediates.
