Replication Errors during In Vivo Ty1 Transposition Are Linked to Heterogeneous RNase H Cleavage Sites
Author(s) -
Emilie H. Mules,
Ozcan Uzun,
Abram Gabriel
Publication year - 1998
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.18.2.1094
Subject(s) - biology , cleavage (geology) , rnase h , rnase p , rna , reverse transcriptase , dna , genetics , complementary dna , microbiology and biotechnology , gene , paleontology , fracture (geology)
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.