Different DNA repair pathways support intact or truncated insertions by R2 retrotransposon protein

Non-long terminal repeat (non-LTR) retrotransposon proteins copy their RNA template into a genome through coordinated nicking and reverse transcriptase activities of target-primed reverse transcription. Mechanisms by which the first-strand complementary DNA (cDNA) becomes a stably inserted duplex, including requirements for junction formation at the cDNA 3' end and second-strand synthesis, are unknown. We screened for cellular factors that influence site-specific transgene synthesis into the human genome by an R2 retrotransposon protein. We discovered that insertion lengths and junction signatures differ based on alternative repair processes involving ATR-dependent polymerase θ end joining, 53BP1-directed shieldin and CST-polymerase α-primase fill-in synthesis, or limited strand annealing dependent on CtIP-MRN. These insights shed light on how genome-primed cDNA synthesis by a non-LTR retrotransposon protein can support stable new gene insertion, with major implications for native retrotransposon mobility and genome engineering.