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J. Biol. Chem., Vol. 279, Issue 46, 47446-47454, November 12, 2004

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Mechanistic Analysis of Pause Site-dependent and -independent Recombinogenic Strand Transfer from Structurally Diverse Regions of the HIV Genome^*

Suchitra S. Derebail and Jeffrey J. DeStefano

From the Department of Cell Biology and Molecular Genetics, University of Maryland College Park, College Park, Maryland 20742

Retroviral recombinants are generated by strand transfers occurring within internal regions of the viral genome and are a major source of genetic variability. Strand transfer has been linked to "pausing" occurring at secondary structures during synthesis by reverse transcriptase. Yet, weakly structured templates lacking strong pause sites also undergo efficient transfer. In this report, transfer crossover sites on high and low structured templates from the gag-pol frameshift region (GagPol) and the env (Env) regions, respectively, were determined by using a reconstituted in vitro strand transfer assay. The assay tested transfers occurring between a donor and acceptor template over a 150-nucleotide homologous region. The majority of crossovers were in a small 23-nucleotide region near a major pause site on GagPol, clearly indicating a pause-driven mechanism. In contrast, on Env, transfers were more dispersed clustering toward the end of the homologous region. Slowing down polymerization on Env by decreasing the dNTP concentration resulted in crossovers shifting toward the beginning of the homologous region. Removal of a small 38-nucleotide region at the 3'-end of the Env acceptor had a large effect on the level of strand transfer despite very few crossovers mapping to this region. This implicated this part of the acceptor in transfers occurring at downstream positions. For Env the results support a mechanism where the acceptor rapidly binds nascent DNA, then "zippers" downstream catching up with the donor-DNA hybrid and displacing the donor. Such a mechanism may be important to recombination in low structure regions of the HIV genome.

Received for publication, August 4, 2004 , and in revised form, August 31, 2004.

^* This work was supported by NIGM, National Institutes of Health Grant GM051140-10A2. 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.

To whom correspondence should be addressed: Dept. of Cell Biology and Molecular Genetics, University of Maryland, Building 231, College Park, MD 20742. Tel.: 301-405-5449; Fax: 301-314-9489; E-mail: jdestefa{at}umd.edu.

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