Figure 1.

The combined clamp/polymerase activities of HIV-1 RT. Reactions were conducted with the indicated template/primer sets, as described in the ‘Materials and Methods’ section. Each oligonucleotide is indicated by the designation that appears in Supplementary Table S1 along with its length in nucleotides (in parenthesis). Asterisks indicate the 5′-end ³²P label. The ends of the primer (I1) and template (I3) are indicated by capital letters and those of the functional templates (either I3 or I8) are indicated by lower case letters. Nicks between two closely-adjacent templates are indicated by arrowheads. (A) A schematic description of the clamp/polymerase activity. This scheme is drawn for the forward synthesis with set #1 (steps 1 and 2) and for the strand displacement synthesis with set #4. See the text for further explanations. (B) Effects of template/primers (T/Ps) molar ratios on HIV-1 RT activity with substrate set #1. 1, T/P only; 2, T/P+RT; 3–9, T/P+RT+dNTPs, at the indicated molar ratios of template over primer. (C) Effects of T/Ps molar ratios on HIV-1 RT activity with substrate set #2. 1, T/P (I3/I1) only; 2, T/P+RT; 3–8, RT+dNTPs+T/P+I8. In all lanes, the molar ratios of I3 to I1 were 1:1 and the ratios of I8 over (I3/I1) are indicated. In lane 8, I8 was 4-fold higher than I1, with no I3 present. (D) The strand displacement synthesis (SD) with set #4. The molar ratios of I1:I3:I8 were 4:4:1, respectively. 1, T/P only; 2, T/P+RT; 3, T/P+RT+dNTPs; 4, a control reaction of RT+dNTPs+set #2 [as shown above in (C)].