IN-VIVO TY1 REVERSE TRANSCRIPTION CAN GENERATE REPLICATION INTERMEDIATES WITH UNTIDY ENDS

Ty1 retrotransposition, like retroviral replication, is a complex seri es of events requiring reverse transcription of an RNA intermediate, R NA-primed minus- and plus-strand DNA synthesis, multiple strand transf ers, and precise cleavages of the template and primers by RNase H. In this report, we examine the structure of in vivo Ty1 replication inter mediates, specifically with regard to the behavior of reverse transcri ptase upon reaching template ends and to the precision with which RNas e H might generate these ends. While the expected 3' termini were alwa ys identified, terminal nontemplated bases were also observed at all o f the RNA and DNA template ends examined. Nontemplated A residues were most common at all 3' ends, although C residues were preferentially a dded to minus-strand termini paused at the 5' end of capped Ty1 RNA. I n addition, we observed that RNase H removal of the tRNA primer and of the polypurine tract was not always precise or efficient. Finally, we noted numerous instances of Ty1 reverse transcriptase transferring fr om normal Ty1 template ends to various tRNA templates, with continued synthesis to specific modified bases. A similar pattern was obtained f or Ty2, indicating that template ends offer unique opportunities for t hese two related reverse transcriptases to generate errors.