Mechanism of DNA targeting by human LINE-1

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.