An Alternative DNA Endonuclease Activity is Associated with the LINE-1 ORF2-encoded Protein

Long INterspersed Element-1 (L1) retrotransposons use activities contained within the L1 open reading frame 2-encoded protein (ORF2p) to mobilize throughout the genome via target-site primed reverse transcription (TPRT). The ORF2p endonuclease domain (EN) cleaves genomic DNA to liberate a 3'-hydroxyl (3'-OH) group that is used by the ORF2p reverse transcriptase domain (RT) to synthesize a cDNA copy of its bound L1 RNA template. L1 also can move by EN-independent retrotransposition (ENi), where a 3'-OH group at genomic DNA lesions, dysfunctional telomeres, or stalled replication forks is proposed to prime L1 reverse transcription in the absence of L1 EN cleavage. We previously reported that ribonucleoprotein (RNP) preparations from cells transfected with a human wild-type (WT) L1 or L1 EN-mutant, but not an L1 RT-mutant, can initiate reverse transcription from a DNA oligonucleotide primer/L1 RNA template complex. The WT and EN-deficient L1 RNP preparations also are associated with a nuclease activity that can process a 3' end modification that precludes DNA synthesis from an oligonucleotide prior to priming the L1 RT reaction. Here, we purified recombinant full-length WT, L1 EN-, and L1 RT-mutant human L1 ORF2p from insect cells. We report that the WT and L1 EN-mutant, but not the L1 RT-mutant, contain an alternative endonuclease activity (alt-EN). Alt-EN activity also is detected in a bacterially expressed L1 ORF2p protein that lacks the L1 EN and ORF2p cysteine-rich domains and a thermostable group II intron-encoded protein. Processing of diverse modified primers demonstrates endonucleolytic cleavage that is eliminated by mutations in the RT active site. We propose that alt-EN is an evolutionarily conserved activity within the RT fold that promoted ENi retrotransposition of primordial retrotransposons prior to the acquisition of an EN domain.