Figure 1

Nucleotide-dependent primer modification by HIV-1 RT. [³²P]ddAMP-terminated L32 primer (5 nM), annealed to WL50 template, was incubated with excess HIV-1 RT (200 nM) at 37°C, and the products were separated by electrophoresis through a 20% denaturing polyacrylamide gel. The arrows indicate specific, nucleotide-dependent products formed during the primer modification reaction. The braces indicate ddATP, which migrates as multiple bands. “x” indicates an unidentified labeled product that was independent of added nucleotide. Panels show labeled products formed under the following conditions: (A) fifteen-min incubation in the presence of 3.2 mM of thermostable pyrophosphatase-treated (+PPase) or untreated (−PPase) dNTP, pyrophosphate (PPi), or NTP. A partial sequence of the primer and template is shown. ∗, radioactive ³²P. (B) Five-minute incubation with 3.2 mM PPase-treated GTP and indicated concentrations of PPase-treated dTTP. (C) Fifteen-minute incubation with 800 μM PPase-treated NTP, dNTP, or ddNTP, as indicated. (D) Five-minute incubation with 3.2 mM PPase-treated NTP, NDP, or NMP, as indicated. Lanes: 15, 5-min incubation with 3.2 mM PPi; 16, [³²P]ddATP as a reference. (E) Five-minute incubation with indicated amounts of PPase-treated GTP.