Endonuclease-independent insertion provides an alternative pathway for L1 retrotransposition in the human genome.

LINE-1 elements (L1s) are a family of highly successful retrotransposons comprising approximately 17% of the human genome, the majority of which have inserted through an endonuclease-dependent mechanism termed target-primed reverse transcription. Recent in vitro analyses suggest that in the absence of non-homologous end joining proteins, L1 elements may utilize an alternative, endonuclease-independent pathway for insertion. However, it remains unknown whether this pathway operates in vivo or in cell lines where all DNA repair mechanisms are functional. Here, we have analyzed the human genome to demonstrate that this alternative pathway for L1 insertion has been active in recent human evolution and characterized 21 loci where L1 elements have integrated without signs of endonuclease-related activity. The structural features of these loci suggest a role for this process in DNA double-strand break repair. We show that endonuclease-independent L1 insertions are structurally distinguishable from classical L1 insertion loci, and that they are associated with inter-chromosomal translocations and deletions of target genomic DNA.