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J. Biol. Chem., Vol. 278, Issue 34, 31536-31546, August 22, 2003
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Role of the Reverse Transcriptase, Nucleocapsid Protein, and Template Structure in the Two-step Transfer Mechanism in Retroviral Recombination*,
From the
Department of Biochemistry and
Biophysics, University of Rochester, Rochester, New York 14642,
¶Abteilung Physikalische Biochemie,
Max-Planck-Institut für Molekulare Physiologie, Otto-Hahn-Strasse 11,
D-44227 Dortmund, Germany, ||HIV Drug Resistance
Program and AIDS Vaccine Program,
Science Applications International Corporation, NCI Frederick, Frederick,
Maryland 21702, and the **Department de Pharmacochemie
Moleculaire et Structurale, INSERM U266, CNRS UMR 8600, 4 Ave. de
l'Observatoire, 75270 Paris Cedex 06, France
Template switching during reverse transcription promotes recombination in retroviruses. Efficient switches have been measured in vitro on hairpin-containing RNA templates by a two-step mechanism. Pausing of the reverse transcriptase (RT) at the hairpin base allowed enhanced cleavage of the initial donor RNA template, exposing regions of the cDNA and allowing the acceptor to base pair with the cDNA. This defines the first or docking step. The primer continued synthesis on the donor, transferring or locking in a second step. Here we determine the enzyme-dependent factors that influence template switching by comparing the RTs from human immunodeficiency virus, type 1 (HIV-1), and equine infectious anemia virus (EIAV). HIV-1 RT promoted transfers with higher efficiency than EIAV RT. We found that both RTs paused strongly at the base of the hairpin. While stalled, HIV-1 RT made closely spaced cuts, whereas EIAV RT made only a single cut. Docking occurred efficiently at the multiply cut but not at the singly cut site. HIV-1 nucleocapsid (NC) protein stimulated strand transfers. It improved RNase H activity of both RTs. It allowed the EIAV RT to make a distribution of cuts, greatly stimulating docking at the base of the hairpin. Most likely, it also promoted strand exchange, allowing transfers to be initiated from sites throughout the hairpin. Minor pause sites beyond the base of the hairpin correlated with the locking sites. The strand exchange properties of NC likely promote this step. We present a model that explains the roles of RNase H specificity, template structure, and properties of NC in the two-step transfer reaction.
Received for publication, May 2, 2003 , and in revised form, June 4, 2003.
* This work was supported by National Institutes of Health Grants GM49573 and T32-CA09363 (to M. N. H.) and also in part by NIH/NCI Contract N01-CO-12400 with SAIC-Frederick, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Trainee in the Medical Scientist Training Program funded by National
Institutes of Health Grant T32-GM07356 and is also supported by National
Institutes of Health Predoctoral Fellowship 5 F31 GM20911-02.
To whom correspondence should be addressed: Dept. of Biochemistry &
Biophysics, University of Rochester Medical Center, 601 Elmwood Ave., Box 712,
Rochester, NY 14642. Tel.: 585-275-2764; Fax: 585-271-2683; E-mail:
robert_bambara{at}urmc.rochester.edu.
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