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J. Biol. Chem., Vol. 280, Issue 49, 40534-40543, December 9, 2005

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Evidence That HIV-1 Reverse Transcriptase Employs the DNA 3' End-directed Primary/Secondary RNase H Cleavage Mechanism during Synthesis and Strand Transfer^*

Vandana Purohit1, Mini Balakrishnan, Baek Kim, and Robert A. Bambara¶2

From the Departments of Biochemistry and Biophysics and Microbiology and Immunology and the ^¶Cancer Center, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

We previously analyzed strand transfers catalyzed by human immunodeficiency virus, type 1 reverse transcriptase (RT) in a hairpin-containing RNA template system. In this system, RT produces a series of adjacent RNase H cuts before the hairpin base on the first, or donor template that clears a region of the donor, facilitating invasion by the second, or acceptor RNA. Here we analyze characteristics of the prominent cuts before the hairpin base and their role in strand transfers. Analysis of the template cleavage pattern during synthesis suggested that the RT performs DNA 3' end-directed primary and secondary cuts while paused at the hairpin base and that these cuts contribute to creation of the invasion site. RT catalyzed similar cleavages on a substrate representing a paused cDNA-template intermediate. DNA 3' end-directed secondary cuts, which require positioning of the polymerase active site downstream of the primer terminus, had previously not been specifically identified during synthesis. Our findings indicate that during synthesis DNA 3' end-directed primary and secondary cuts occur at pause sites. RT mutants with substitutions at the His⁵³⁹ residue in the RNase H active site were defective in secondary cleavages. Analysis of the template cleavage pattern generated by the His⁵³⁹ mutants during synthesis revealed inefficient cleavage at the invasion site, correlating with defects in strand transfer. Overall, results indicate RT can catalyze pause-associated DNA 3' end-directed primary and secondary cuts during synthesis and these cuts can contribute to strand transfer by creation of an invasion site.

Received for publication, July 19, 2005 , and in revised form, September 21, 2005.

^* This work was supported by National Institutes of Health Grants GM 49573. 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.

¹ Trainee supported by National Institutes of Health Grant T32-DA07232.

² To whom correspondence should be addressed: 601 Elmwood Ave., Box 712, Rochester, NY 14642. Tel.: 585-275-3269; Fax: 585-275-6007; E-mail: robert_bambara{at}urmc.rochester.edu.

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