DNA: REPLICATION REPAIR AND RECOMBINATION
Trapping HIV-1 Reverse Transcriptase Before and After Translocation on DNA*

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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 N 2amino 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.

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Published, JBC Papers in Press, January 28, 2003, DOI 10.1074/jbc.M212911200

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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.