Rapid DNA cleavage by the LINE-1 endonuclease proximal to DNA ends and at mismatches

Long interspersed element 1 (LINE-1, L1) is a eukaryotic retrotransposon that propagates through an RNA intermediate. Its mutagenic insertion mechanism, target-primed reverse transcription (TPRT), requires coordinated activities of the encoded ORF2 protein (ORF2p) endonuclease (EN) and reverse transcriptase (RT) domains. EN initiates TPRT by nicking target genomic DNA, creating a 3'-OH that primes ORF2p RT for complementary DNA synthesis using the bound L1 RNA template. L1 insertions occur preferentially at 5'-TTTTT↓AA consensus motifs; this bias could reflect site-specific EN cleavage or sequence requirements in the subsequent RT priming step, in which the cut genomic DNA flap must base pair with the poly(A) RNA template. We find that, in vitro, EN is promiscuous, cutting linear DNA oligonucleotides and plasmids at many non-consensus sites. We discovered a cleavage activity on a mismatched substrate that was nicked ∼40-fold faster than duplex DNA containing the consensus site and identify three features promoting rapid cutting. First, EN cleaves two nucleotides downstream of mismatches, favoring A-G mismatches or T•G/U•G wobble pairs. Second, both mismatch and consensus sequences are cleaved >2-fold faster when proximal to a DNA end. Third, end-proximal EN cutting depends on end composition: 5' overhangs cut fastest, followed by 3' overhangs, followed by blunt ends. Together, these results indicate that EN cleavage is based primarily on DNA structure rather than sequence, that many L1 insertion attempts likely fail after cleavage at the priming step , and that mismatches and possibly other DNA conformational alterations promote EN cleavage, broadening our understanding of the genomic impact of L1.