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 U5-primer binding site (PBS) border and proposed a novel mechanism to account for this phenomenon during Ty1 replication. In this report, we verify key points of our model and show that in vivo RNase H cleavage of Ty1 RNA during minus-strand strong-stop synthesis creates heterogeneous 5' RNA ends. The preferred cleavage sites closest to the PBS are 6 and 3 bases upstream of the U5-PBS border. Minus-strand cDNA synthesis terminates at multiple sites determined by RNase H cleavage, and DNA intermediates frequently contain 3'-terminal sequence changes at or near their template ends. These data indicate that nontemplated terminal base addition during reverse transcription is a real in vivo phenomenon and suggest that this mechanism is a major source of sequence variability among retrotransposed genetic elements.
Mules
E H
EH
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855, USA.
Uzun
O
O
Gabriel
A
A
D004261:DNA Replication; D004251:DNA Transposable Elements; D004271:DNA, Fungal / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D016914:Ribonuclease H / Q000378:metabolism*; D012441:Saccharomyces cerevisiae; D017422:Sequence Analysis, DNA; D013698:Templates, Genetic
D004251:DNA Transposable Elements; D004271:DNA, Fungal; D016914:Ribonuclease H