Mules E H, Uzun O, Gabriel A
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855, USA.
Mol Cell Biol. 1998 Feb;18(2):1094-104. doi: 10.1128/MCB.18.2.1094.
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.
我们之前在Ty1 U5-引物结合位点（PBS）边界上游鉴定出一个突变热点，并提出了一种新机制来解释Ty1复制过程中的这一现象。在本报告中，我们验证了模型的关键点，并表明在负链强终止合成过程中，体内Ty1 RNA的核糖核酸酶H切割会产生异质的5' RNA末端。最接近PBS的优先切割位点位于U5-PBS边界上游6个和3个碱基处。负链cDNA合成在由核糖核酸酶H切割确定的多个位点终止，并且DNA中间体在其模板末端或附近经常含有3'-末端序列变化。这些数据表明，逆转录过程中无模板的末端碱基添加是一种真实的体内现象，并表明这种机制是逆转座遗传元件间序列变异性的主要来源。
