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J. Biol. Chem., Vol. 277, Issue 32, 28400-28410, August 9, 2002
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From the During and after minus-strand DNA synthesis,
human immunodeficiency virus 1 (HIV-1) reverse transcriptase (RT)
degrades the RNA genome. To remove RNA left after polymerization, the
RT aligns to cut 18 nucleotides in from the 5' RNA end. The enzyme then repositions, making a secondary cut 8 nucleotides from the RNA 5' end.
Transfer of the minus strong stop DNA during viral replication requires
cleavage of template RNA. Removal of the terminal RNA segment is a
special case because the RNA-DNA hybrid forms a blunt end, shown
previously to resist cleavage when tested in vitro. We show
here that the structure of the substrate extending beyond the RNA 5'
end is an important determinant of cleavage efficiency. A short
single-stranded DNA extension greatly stimulated the secondary cleavage. Annealing an RNA segment to the DNA extension was even more stimulatory. Recessing the DNA from a blunt end by even one nucleotide caused the RT to reorient its binding, preventing secondary cleavage. The presence of the cap at the 5' end of the viral RNA did
not improve the efficiency of secondary cleavage. However, NC protein
greatly facilitated the secondary cut on the blunt-ended substrate,
suggesting that NC compensates for the unfavorable substrate structure.
Substrate Requirements for Secondary Cleavage by HIV-1 Reverse
Transcriptase RNase H*
§¶,§,,
,, and§§
Department of Biochemistry and Biophysics
and the Cancer Center, University of
Rochester, Rochester, New York 14642 and ** 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.
Present address: 800 Centennial Ave., Piscataway, NJ 08855.
§§
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.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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