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J Biol Chem, Vol. 275, Issue 1, 359-366, January 7, 2000
From the Studies of drug-resistant reverse transcriptases
(RTs) reveal the roles of specific structural elements and amino acids
in polymerase function. To characterize better the effects of
RT/template interactions on dNTP substrate recognition, we examined the
sensitivity of human immunodeficiency virus type 1 (HIV-1) RT
containing a new mutation in a "template grip" residue (P157S) to
the 5'-triphosphates of (
Site-specific Incorporation of Nucleoside Analogs by HIV-1
Reverse Transcriptase and the Template Grip Mutant P157S
TEMPLATE INTERACTIONS INFLUENCE SUBSTRATE RECOGNITION AT THE
POLYMERASE ACTIVE SITE*
,,
Departments of Biochemistry and Radiation
Oncology, Eccles Institute of Human Genetics and Huntsman Cancer
Institute, University of Utah, Salt Lake City, Utah 84112, the
§ Georgia Veterans Affairs Research Center for AIDS and HIV
Infections and Department of Pediatrics, Emory University School of
Medicine, Decatur, Georgia 30033, and the ¶ Center for
Comparative Medicine, University of California,
Davis, California 95616
)-
-2',3'-dideoxy-3'-thiacytidine (3TC),
()--2',3'-dideoxy-5-fluoro-3'-thiacytidine (FTC), and
3'-azido-3'-deoxythymidine (AZT). A primer extension assay was used to
monitor quantitatively drug monophosphate incorporation opposite each
of multiple target sites. Wild-type and P157S RTs had similar catalytic
activities and processivities on heteropolymeric RNA and DNA templates.
When averaged over multiple template sites, P157S RT was 2-7-fold
resistant to the 5'-triphosphates of 3TC, FTC, and AZT. Each drug
triphosphate inhibited polymerization more efficiently on the DNA
template compared with an RNA template of identical sequence. Moreover,
chain termination by 3TC and FTC was strongly influenced by template
sequence context. Incorporation of FTC and 3TC monophosphate varied up
to 10-fold opposite 7 different G residues in the DNA template, and the
P157S mutation altered this site specificity. In summary, these data
identify Pro157 as an important residue affecting
nucleoside analog resistance and suggest that interactions between RT
and the template strand influence dNTP substrate recognition at the RT
active site. Our findings are discussed within the context of the HIV-1
RT structure.
*
This work was supported by United States Public Health
Service Grants R01 AI34834, R01 AI38755, and P30 CA42014 (to
B. D. P.), R01 AI28189 (to T. W. N.), and F32 AI10139 (to
R. A. S.) from the National Institutes of Health and by the
Department of Veterans Affairs and the Georgia Research Center on AIDS
and HIV Infection (to R. F. S.).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: University of
Utah, Human Molecular Biology and Genetics, 15 N 2030 E, Rm. 2150, Salt
Lake City, UT 84112-5332. Tel.: 801-585-6342; Fax: 801-585-3501; E-mail: bpreston@hci.utah.edu
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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