Figure 2

Efficiency of (-)ssDNA synthesis in cell-free assay. The efficiencies of the reactions with WT and mutant RTs were compared in time course experiments. (A) Graphic representation of the cell-free system (HIV-1 PBS RNA/tRNA^3Lys) used to monitor the synthesis of (-)ssDNA. (B) Synthesis of full-length DNA by WT and mutant enzymes. Reactions were initiated with 10 μM dNTPs and monitored by incorporation of [α-³²P]-dCTP. Full-length DNA product and pausing sites are shown on the left. (C) Graphic representation of the cell-free system (HIV-1 PBS RNA/tRNA^3Lys) used to monitor the efficiency of initiation of (-)ssDNA synthesis in the presence of the chain-terminator ddATP. (D) Initiation of (-) ssDNA synthesis by WT and mutant enzymes. Reactions were performed using 1 μM dNTPs, and ddATP was employed in place of dATP to give rise to a six-nucleotide initiation product. ddATP-terminated +6 product and +3 and +5 pausing position are shown on the left side. (E) Graphic representation of the gel-based assays shown in D.