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J. Biol. Chem., Vol. 275, Issue 41, 32299-32309, October 13, 2000
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From the Department of Microbiology, School of Medicine, University
of Washington, Seattle, Washington 98195-7242
The ability of reverse transcriptase to generate,
extend, and remove the primer derived from the polypurine tract (PPT)
is vital for reverse transcription, since this process determines one
of the ends required for integration of the viral DNA. Based on the
ability of the RNase H activity of Moloney murine leukemia virus
reverse transcriptase to cleave a long RNA/DNA hybrid containing the
PPT, it appears that cleavages that could generate the plus-strand primer can occur by an internal cleavage mechanism without any positioning by an RNA 5'-end, and such cleavages may serve to minimize
cleavage events within the PPT itself. If the PPT were to be cleaved
inappropriately just upstream of the normal plus-strand origin site,
the resulting 3'-ends would not be extended by reverse transcriptase.
Extension of the PPT primer by at least 2 nucleotides is sufficient for
recognition and correct cleavage by RNase H at the RNA-DNA junction to
remove the primer. Specific removal of the PPT primer after polymerase
extension deviates from the general observation that primer removal
occurs by cleavage one nucleotide away from the RNA-DNA junction and
suggests that the same PPT specificity determinants responsible for
generation of the PPT primer also direct PPT primer removal. Once the
PPT primer has been extended and removed from the nascent plus-strand
DNA, reinitiation at the resulting plus-strand primer terminus does not
occur, providing a mechanism to prevent the repeated initiation of plus strands.
Analysis of Plus-strand Primer Selection, Removal, and
Reutilization by Retroviral Reverse Transcriptases*
*
This work was supported by National Institutes of Health
Grant R37 CA51605.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
Microbiology, Box 357242, University of Washington, Seattle, WA
98195-7242. Tel.: 206-543-8574; Fax: 206-543-8297; E-mail:
champoux@u.washington.edu.
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