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A more recent version of this article appeared on September 12, 2003
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M304262200v1
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Papers In Press, published online ahead of print June 20, 2003
J. Biol. Chem, 10.1074/jbc.M304262200
Submitted on April 23, 2003
Revised on June 18, 2003
Accepted on June 19, 2003

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 Gotte

Departments of Medicine, Microbiology and Immunology, McGill University, Lady Davis Institute - Jewish General Hospital, Montreal, Quebec H3T 1E2

Corresponding Author: mgoette{at}ldi.jgh.mcgill.ca

Resistance to nucleoside analogue inhibitors of the reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) often involves phosphorolytic excision of the incorporated chain-terminator. Previous crystallographic and modelling 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 templat e 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 c hain-terminator, cause 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-translocati onal 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 ch ain-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.i


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