Resistance to @entity2353 analogue inhibitors of the reverse transcriptase of the @entity2858 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 @entity377 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 @entity42636 , following the incorporated chain terminator, caused a shift in the position of the two cuts a single @entity377 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 @entity377 incorporation and the release of @entity9955 . 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 @entity2353 analogue inhibitors.

Site-specific footprinting reveals differences in the translocation status of XXXX reverse transcriptase. Implications for polymerase translocation and drug resistance.

@entity2353 :: ('CHEBI:33838', 'Chemical') :: ['nucleoside'] @entity2858 :: ('11676', 'Species') :: ['HIV-1'] @entity42636 :: ('CHEBI:27928', 'Chemical') :: ['dNTP'] @entity377 :: ('CHEBI:36976', 'Chemical') :: ['nucleotide'] @entity9955 :: ('MESH:C107241', 'Chemical') :: ['pyrophosphate']
@entity2858 :: (11676,Species) :: ['HIV-1']
