Figure 5.

Proofreading of PfRecJ on 3′-mismatched ribonucleotides during RNA primer extension by Pfu DNA polymerase. (A) Proofreading of 3′-mismatched ribonucleotides by PfRecJ during primer extension catalyzed by primase and Pfu DNA polymerase. A pair of recessed RNA/DNA hybrids (3′-matched/mismatched recess) was used to confirm the proofreading function of PfRecJ in a polymerization reaction catalyzed by P. furiosus PolB and primase. The substrates (50 nM) were incubated with 50 nM of PolB and primase in the presence/absence of PfRecJ (400 nM) at 50°C for 30 min in a buffer consisting of 20 mM HEPES (pH 7.5), 30 mM NaCl, 10 mM KCl, 5 mM MnCl₂, 100 μM dNTPs, 4 U Rnsin and 100 ng/μl BSA. (B) Effect of RPA and PCNA on the proofreading of 3′-mismatched ribonucleotides by PfRecJ. Proofreading of the 3′-mismatched ribonucleotide during extension by PolB was performed in the presence of RPA and PCNA using a RNA/DNA hybrid carrying a 3′-mismatched ribonucleotide as a substrate. The RNA/DNA hybrid (50 nM) was incubated with PfRecJ (100 nM), PolB (50 nM), RPA (1 μM), PCNA (1 μM) or enzyme mixtures for 30 min at 50°C in a buffer consisting of 20 mM HEPES (pH 7.5), 30 mM NaCl, 10 mM KCl, 2 mM MnCl₂, 100 ng/μl BSA, 4 U Rnsin and 100 μM dNTPs. Lowercase and uppercase denote RNA and DNA, respectively.