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J. Biol. Chem., Vol. 278, Issue 10, 8006-8017, March 7, 2003
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From the Retrovirus minus strand strong stop transfer
(minus strand transfer) requires reverse transcriptase-associated RNase
H, R sequence homology, and viral nucleocapsid protein. The minus
strand transfer mechanism in human immunodeficiency virus-1 was
examined in vitro with purified protein and substrates.
Blocking donor RNA 5'-end cleavage inhibited transfers when template
homology was 19 nucleotides (nt) or less. Cleavage of the donor 5'-end occurred prior to formation of transfer products. This suggests that
when template homology is short, transfer occurs through a primer
terminus switch-initiated mechanism, which requires cleavage of the
donor 5' terminus. On templates with 26-nt and longer homology, transfer occurred before cleavage of the donor 5' terminus. Transfer was unaffected when donor 5'-end cleavages were blocked but was reduced
when internal cleavages within the donor were restricted. Based on the
overall data, we conclude that in human immunodeficiency virus-1, which
contains a 97-nt R sequence, minus strand transfer occurs through an
acceptor invasion-initiated mechanism. Transfer is initiated at
internal regions of the homologous R sequence without requiring
cleavage at the donor 5'-end. The acceptor invades at gaps created by
reverse transcriptase-RNase H in the donor-cDNA hybrid. The
fragmented donor is eventually strand-displaced by the acceptor,
completing the transfer.
Mechanism of Minus Strand Strong Stop Transfer in HIV-1
Reverse Transcription*
,,, and¶
Department of Biochemistry and Biophysics
and the ¶ Cancer Center, University of Rochester, Rochester,
New York 14642 and the § Departement de Pharmacochimie
Moleculaire et Structurale, U266 INSERM, URA D1500 CNRS, UER des
Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire,
75270 Paris Cedex 06, France
*
This work was supported by National Institutes of Health
Grant 49573 (to R. A. B. and P. J. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The 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
Biochemistry and Biophysics, Box 712, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 585-275-2764; Fax:
585-271-2683; E-mail: robert_bambara@urmc.rochester.edu.
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