FIG 4.

ST activity of HIV-1 RT mutants. (A) Schematic description of the ST assay. After the extension of the 5′-³²P-end-labeled 20-nt DNA primer, which annealed to the 33-nt RNA template (step 1), the 30-nt products undergo the RT-directed RNase H cleavage to remove this RNA template; thus, the single-stranded (−) strong stop (SS) DNA is formed (step 2). Then, ST to the 35-nt acceptor DNA strand takes place (step 3). This is followed by further elongation of the nascent DNA to full-length 49-nt products (step 4). The products of this overall reaction were separated by urea-PAGE. (B) ST assay. Each reaction consists of equal DDDP activities of the HIV-1 RT mutants that were incubated for 30 min at 37°C. Reaction products were analyzed by urea-PAGE, followed by autoradiography. The strong-stop DNA transcript, that is, the products (30 nt) of the initial RDDP reactions, are marked as “P of RDDP.” The full-length (49-nt) DNA products, generated after ST had taken place, are marked as “P of ST.” S, 20-nt substrate.