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Mol Cell Biol, February 1998, p. 1094-1104, Vol. 18, No. 2
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Replication Errors during In Vivo Ty1 Transposition Are Linked to Heterogeneous RNase H Cleavage Sites

Emilie H. Mules,¹ Ozcan Uzun,^1,2 and Abram Gabriel^1,*

Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08855,¹ and Graduate Program in Biochemistry and Molecular Biology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, New Jersey 08854²

Received 14 August 1997/Returned for modification 16 October 1997/Accepted 6 November 1997

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.

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^* Corresponding author. Mailing address: Department of Molecular Biology and Biochemistry, Rutgers University, CABM 306, 679 Hoes Lane, Piscataway, NJ 08855. Phone: (732) 235-5097. Fax: (732) 235-4880. E-mail: gabriel{at}mbcl.rutgers.edu.

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