In Vivo Ty1 Reverse Transcription Can Generate Replication Intermediates with Untidy Ends

Abstract

ABSTRACT Ty1 retrotransposition, like retroviral replication, is a complex series of events requiring reverse transcription of an RNA intermediate, RNA-primed minus- and plus-strand DNA synthesis, multiple strand transfers, and precise cleavages of the template and primers by RNase H. In this report, we examine the structure of in vivo Ty1 replication intermediates, specifically with regard to the behavior of reverse transcriptase upon reaching template ends and to the precision with which RNase H might generate these ends. While the expected 3′ termini were always identified, terminal nontemplated bases were also observed at all of the RNA and DNA template ends examined. Nontemplated A residues were most common at all 3′ ends, although C residues were preferentially added to minus-strand termini paused at the 5′ end of capped Ty1 RNA. In 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 from normal Ty1 template ends to various tRNA templates, with continued synthesis to specific modified bases. A similar pattern was obtained for Ty2, indicating that template ends offer unique opportunities for these two related reverse transcriptases to generate errors.