Figure 2.

Influence of mutations in PCNA on the RFC-dependent synthesis of singly primed M13 DNA catalyzed by pol δ. (A) Reaction mixtures (10 μl) containing 40 mM Tris⋅HCl (pH 7.5), 0.5 mM DTT, 100 μg/ml BSA, 7 mM magnesium acetate, 100 μM dATP, dCTP and dGTP, 1 mM ATP, 20 mM [α³²P]dTTP (29,000 cpm/pmol), 6 fmol of singly primed M13 DNA, 0.25 μg of hSSB, 30 fmol of baculovirus cloned RFC, 0.21 unit of hpol δ, and PCNA as indicated were incubated at 37°C for 30 min. Aliquots were used to determine the amount of DNA synthesized. (B) Size of DNA products formed. Aliquots from reaction mixtures described in A were subjected to alkaline agarose gel electrophoresis. After drying, gels were subjected to autoradiography. (C) Size of DNA products formed in the presence of wild-type PCNA and various D41 PCNA mutants. Reaction mixtures as described in A, with higher levels of PCNA as indicated, were carried through the procedure described in B.