Effect of K65R mutation on the translocation state of RT bound to T/P
. (A) The translocation state of HIV-1 RT after EFdA-MP incorporation was determined using site-specific Fe2+ footprinting. Td43/Pd30-EFdA-MP (100 nM) with 5'-Cy3-label on the DNA template was incubated with WT or K65R HIV-1 RT (600 nM) and various concentrations of the next incoming nucleotide (dTTP). The complexes were treated for 5 minutes with ammonium iron sulphate (1 mM) and resolved on a polyacrylamide 7 M urea gel. An excision at position −18 indicates a pre-translocation complex, while the one at position −17 represents a post-translocation complex. (B) The post-translocated complexes were determined from the gels and plotted using GraphPad Prism. Light blue indicates the physiological dNTP concentrations. (C) Schematic representation of the position of EFdA-MP-terminated primers at the pre- and post-translocated sites.