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J Virol, August 1998, p. 6490-6503, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
In Vivo Ty1 Reverse Transcription Can Generate
Replication Intermediates with Untidy Ends
Emilie H.
Mules,1
Ozcan
Uzun,1,2 and
Abram
Gabriel1,*
Department of Molecular Biology and
Biochemistry, Rutgers University, Piscataway, New Jersey
08855,1 and
Graduate Program in
Biochemistry and Molecular Biology, University of Medicine and
Dentistry of New Jersey, Robert Wood Johnson Medical School,
Piscataway, New Jersey 088542
Received 9 March 1998/Accepted 11 May 1998
Ty1 retrotransposition, like retroviral replication, is a complex
series of events requiring reverse transcription of an RNA intermediate, RNA-primed minus- and plus-strand DNA synthesis, multiple
strand transfers, and precise cleavages of the template and primers by
RNase H. In this report, we examine the structure of in vivo Ty1
replication intermediates, specifically with regard to the behavior of
reverse transcriptase upon reaching template ends and to the
precision with which RNase H might generate these ends. While the
expected 3' termini were always identified, terminal nontemplated bases
were also observed at all of the RNA and DNA template ends examined.
Nontemplated A residues were most common at all 3' ends, although C
residues were preferentially added to minus-strand termini paused at
the 5' end of capped Ty1 RNA. In addition, we observed that RNase H
removal of the tRNA primer and of the polypurine tract was not always
precise or efficient. Finally, we noted numerous instances
of Ty1 reverse transcriptase transferring from normal Ty1 template ends
to various tRNA templates, with continued synthesis to specific
modified bases. A similar pattern was obtained for Ty2, indicating that
template ends offer unique opportunities for these two related reverse
transcriptases to generate errors.
*
Corresponding author. Mailing address: Department of
Molecular Biology and Biochemistry, Rutgers University, CABM 306, 679 Hoes Lane, Piscataway, NJ 08855-1179. Phone: (732) 235-5097. Fax: (732)
235-4880. E-mail: gabriel{at}mbcl.rutgers.edu.
J Virol, August 1998, p. 6490-6503, Vol. 72, No. 8
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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