REPLICATION ERRORS DURING IN-VIVO TY1 TRANSPOSITION ARE LINKED TO HETEROGENEOUS RNASE-H CLEAVAGE SITES

We previously identified a mutational hotspot upstream of the Ty1 US-p rimer binding site (PBS) border and proposed a novel mechanism to acco unt for this phenomenon during Ty1 replication. In this report, we ver ify key points of our model and show that in vivo RNase H cleavage of Ty1 RNA during minus-strand strong-stop synthesis creates heterogeneou s 5' RNA ends. The preferred cleavage sites closest to the PBS are 6 a nd 3 bases upstream of the US-PBS border. Minus-strand cDNA synthesis terminates at multiple sites determined by RNase H cleavage, and DNA i ntermediates frequently contain 3'-terminal sequence changes at or nea r their template ends. These data indicate that nontemplated terminal base addition during reverse transcription is a real in vivo phenomeno n and suggest that this mechanism is a major source of sequence variab ility among retrotransposed genetic elements.