Figure 4. Pi-dependent phosphorolysis catalyzed by HIV-1 RT.
A. Diagram showing that pyrophosphorolysis and Pi-dependent phosphorolysis are the reverse reaction of polymerization in which the substrate is a nucleotide triphosphate or diphosphate, respectively. B. Time course of phosphorolysis and pyrophosphorolysis reactions catalyzed by HIV-1 RT and KF exo-. Reactions were performed with 5mM PPi or K2HPO4, 10 nM enzyme and 5 nM template-primer. Aliquots of the reaction were stopped after 1, 2 and 5 minutes and analyzed by denaturing PAGE. Lane B contained a ‘no phosphate’ control in which template-primer was incubated with RT for 20 minutes. Full-length, 5′-end labeled primer is indicated ‘p’, phosphorolysis products shortened by a single nucleotide from the 3′ end are indicated ‘p-1’. C. Radiolabeled nucleotide products released from the primer 3′ terminus through a phosphorolysis reaction were separated by PEI-cellulose TLC. HIV-1 RT or KF exo- (5 nM) were incubated with primer template in the presence of 10 mM Pi or 150 µM PPi, and aliquots removed after 2.5, 5 and 15 minutes. Lanes B1 and B2 contained control reactions incubated for 15 minutes with RT and KF exo- respectively, in the absence of added phosphate. The position of TTP and TDP was determined by comparison to unlabeled standards.