FIG. 4.

Initiation of (−) ssDNA synthesis by WT and mutant enzymes. The same system and conditions were used as for Fig. 3, except that ddATP was used instead of dATP in the dNTP mixture to restrict synthesis during initiation, with the addition of a stop nucleotide (nt) (ddATP) at position +6. A mixture of ddATP, dGTP, and dTTP was used at 10 μM, and dCTP was employed at 1 μM to allow incorporation of [α-³²P]dCTP, which was used as a tracer during the reaction. (A) Schematic representation of the reaction. Underlined letters indicate sites of chain termination. Dots define specific positions in the reaction, as indicated. (B) Data obtained with WT versus mutant RTs. (C) Quantification of the data shown in panel B. The graphs show the time-dependent release from pausing at position +3. (D) Limited DNA synthesis in reactions performed with a corresponding DNA primer.