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J. Biol. Chem., Vol. 278, Issue 37, 35362-35372, September 12, 2003

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Site-specific Footprinting Reveals Differences in the Translocation Status of HIV-1 Reverse Transcriptase

IMPLICATIONS FOR POLYMERASE TRANSLOCATION AND DRUG RESISTANCE^*

Bruno Marchand   and Matthias Götte   ¶ ||

From the McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montréal, Québec H3T 1E2 and the Departments of Microbiology and Immunology and ^¶Experimental Medicine, McGill University, Montréal, Québec H3A 2T5, Canada

Resistance to nucleoside analogue inhibitors of the reverse transcriptase of the HIV-1 often involves phosphorolytic excision of the incorporated chain terminator. Previous crystallographic and modeling studies suggested that this reaction could only occur when the enzyme resides in a pre-translocational stage. Here we studied mechanisms of polymerase translocation using novel site-specific footprinting techniques. Classical footprinting approaches, based on the detection of protected nucleic acid residues, are not sensitive enough to visualize subtle structural differences at single nucleotide resolution. Thus, we developed chemical footprinting techniques that give rise to hyperreactive cleavage on the template strand mediated through specific contacts with the enzyme. Two specific cuts served as markers that defined the position of the polymerase and RNase H domain, respectively. We show that the presence of the next correct dNTP, following the incorporated chain terminator, caused a shift in the position of the two cuts a single nucleotide further downstream. The footprints point to monotonic sliding motions and provide compelling evidence for the existence of an equilibrium between pre- and post-translocational stages. Our data show that enzyme translocation is reversible and uncoupled from nucleotide incorporation and the release of pyrophosphate. This translocational equilibrium ensures access to the pre-translocational stage after incorporation of the chain terminator. The efficiency of excision correlates with an increase in the population of complexes that exist in the pre-translocational stage, and we show that the latter configuration is preferred with an enzyme that contains mutations associated with resistance to nucleoside analogue inhibitors.

Received for publication, April 23, 2003 , and in revised form, June 18, 2003.

^* This work was supported by grants from the Canadian Institutes of Health Research (to M. G.). The costs of publication of this article were defrayed in part by the payment of page charges. This 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: McGill University AIDS Centre (226), Lady Davis Institute-Jewish General Hospital, 3755, Chemin Côte-Ste-Catherine, Montréal, Québec H3T 1E2, Canada. Tel.: 514-340-8222 (ext. 3299); Fax: 514-340-7502; E-mail: mgoette{at}ldi.jgh.mcgill.ca.

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