Figure 2.
Effect of PCNA, RFC, and RPA on hPolι activity. (A) DNA synthesis by hPolι in the presence of different combinations of PCNA, RFC, and RPA. The complete standard reaction mixture contained hPolι (10 nM), PCNA (100 ng), RFC (50 ng), and RPA (250 ng), and all four deoxynucleotides (100 μM each) along with a ssM13 DNA (2 nM) primed with a 5′ 32P-labeled 38-nt oligonucleotide. Various combinations of PCNA, RFC, and RPA were added to the reaction mixture as indicated. (B) Processivity of hPolι in the presence of PCNA, RFC, and RPA. hPolι (3 nM or 10 nM) alone (lanes 1 and 2, and 5 and 6), or in the presence of PCNA (100 ng), RFC (50 ng), and RPA (250 ng; lanes 3 and 4, and 7 and 8), was preincubated with circular ssM13 template DNA (10 nM) singly primed with a 5′ 32P-labeled oligonucleotide for 5 min at 37°C. Primer extension reactions were initiated by adding all four deoxynucleotides (500 μM each; lanes 1–4) or all four deoxynucleotides and excess sonicated herring sperm DNA (0.5 mg/ml) as a trap (lanes 5–8). After incubation for 10 min at 37°C, samples were quenched and run on a 10% polyacrylamide gel. To demonstrate the effectiveness of the trap, hPolι, along with PCNA, RFC, and RPA, was preincubated with the trap DNA together with the DNA substrate before addition of dNTPs (lane 9).