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J. Biol. Chem., Vol. 278, Issue 18, 16280-16288, May 2, 2003
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From the A disulfide cross-linking strategy was used to
covalently trap as a stable complex (complex N) a short-lived, kinetic
intermediate in DNA polymerization. This intermediate corresponds to
the product of polymerization prior to translocation. We also prepared
the trapped complex that corresponds to the product of polymerization after translocation (complex P). The cross-linking method that we used
is a variation of a technique developed by the Verdine and
Harrison laboratories. It involves disulfide interchange between an
engineered sulfhydryl group of the protein (Q258C mutation) and a
disulfide-containing tether attached at the N2
amino group of a modified dG in either the template or the primer strand of the nucleic acid. We report here a highly efficient synthesis
of the precursor, bis(3-aminopropyl)disulfide dihydrochloride, used to
introduce this substituent into the oligonucleotide. Efficient cross-linking takes place when the base pair containing the substituent is positioned seven registers from the dNTP-binding site (N site) and
the N site is occupied. Complex N, but not complex P, is a substrate
for the ATP-based excision reaction that unblocks nucleoside reverse transcriptase inhibitor (NRTI)-terminated primers and causes resistance to several NRTIs, confirming predictions that the
excision reaction takes place only when the 3'-end of the primer is
bound at the N site. These techniques can be used for biochemical and
structural studies of the mechanism of DNA polymerization, translocation, and excision-based resistance of RT to NRTIs. They may
also be useful in studying other DNA or RNA polymerases or other enzymes.
Trapping HIV-1 Reverse Transcriptase Before and
After Translocation on DNA*
,,,,,,
Center for Advanced Biotechnology and
Medicine and Department of Chemistry and Chemical Biology, Rutgers
University, Piscataway, New Jersey 08854-5638, the
§ Laboratory of Bioorganic Chemistry, NIDDK, National
Institutes of Health (NIH), Bethesda, Maryland 20892-0820, and the
¶ HIV Drug Resistance Program, NCI-Frederick Cancer Research and
Development Center, NIH, Frederick, Maryland 21702-1201
*
This work was supported by National Institutes of Health
(NIH) Grants AI 27690 (MERIT award) and GM 56609 (to E. A.) and by NIGMS, NIH and NCI, NIH (to S. H. H.).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. Tel.:
732-235-5323; Fax: 732-235-5788; E-mail:
arnold@cabm.rutgers.edu.
Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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