The R2 retrotransposon protein copies its RNA template into the genome through coordinated nicking and reverse transcriptase activity in the process of target-primed reverse transcription (TPRT).[2][3] The mechanisms by which the first cDNA strand becomes a stably inserted duplex, including the formation of a junction at the 3' end of the cDNA and the synthesis of the second strand, were unknown until now.[2][3] The researchers investigated the cellular factors influencing the site-specific synthesis of the transgene into the human genome by the R2 protein.[2][3] They found that the length of the insertions and the characteristics of the junctions differ according to alternative DNA repair pathways: ATR-dependent Polymerase θ end-joining, 53BP1-directed Shieldin/CST-Polα-primase fill-in synthesis, or CtIP-MRN-dependent limited strand annealing.[2][3] These pathways support either intact or truncated R2 insertions.[2] The results clarify how genome-primed cDNA synthesis by non-LTR retrotransposons promotes stable insertion of a new gene.[2][3]