FIGURE 4.

3′→5′ Exonucleolytic activity of Phi29 DNA polymerase on RNA and DNA substrates. The experiments were performed under the conditions described in “Materials and Methods,” using 5′-end-labeled 16-mer DNA (A) or RNA (B) oligonucleotides as substrates. Samples were incubated at 25°C for different time periods (5 s, 10 s, 10 s, 20 s, 40 s, 1 min, 2 min, 5 min, 10 min, 20 min, 30 min) and quenched by adding equal volume of 2× Loading dye solution. In the cases of Phi29 DNA polymerase exo− mutant (D12A/D66A), the samples were incubated for 30 min. Later the samples were analyzed by electrophoresis in 8% (w/v) denaturing polyacrylamide gel (29:1 [w/w] acrylamide/bisacrylamide, 7 M urea, 1× TBE buffer). The exonucleolytic degradation (C) was quantified by densitometry of autoradiographs and expressed as the rate constants of phosphodiesteric bond cleavage. The rates of cleavage were fit to single exponentials. The best fits (broken lines) gave rate constants of 4.01 ± 0.39 min⁻¹ for DNA and 0.394 ± 0.045 min⁻¹ for RNA.