FIGURE 4.

Rate of chain elongation reaction catalyzed by Pol δ and Pol ϵ. A, influence of the ratio of Pol ϵ to DNA on length of DNA products formed. Reaction mixtures (10 μl) containing 20 mm Tris-HCl, pH 7.5, 150 μg/ml BSA, 10 mm magnesium acetate, 1 mm DTT, 0.1 mm EDTA, 30 mm potassium glutamate, 1.5 mm ATP, 30 μm [α-³²P]dATP (23,230 cpm/pmol), 130 μm each of dCTP, dTTP, and dGTP, 0.73 nm singly primed M13, 70 nm E. coli SSB, 5.6 nm RFC, 50 nm PCNA, and either 7.3 or 2.19 nm hPol δ or the four-subunit hPol ϵ (as indicated) were incubated at 37 °C for the time specified. Reactions were halted with EDTA (10 mm final), and aliquots were removed to determine the level of nucleotide incorporation and size of DNA products following alkaline-agarose electrophoresis. B, influence of Pol/DNA ratio following preincubation of RFC/PCNA with DNA. Reactions were as described in A and contained 1 nm singly primed M13 DNA and the indicated levels of hPol δ and hPol ϵ. Reaction mixtures lacking Pols were preincubated for 3 min at 37 °C to preload RFC and PCNA onto DNA, after which the Pols were added. Mixtures were then incubated for the time indicated at 37 °C. C, influence of RFC levels on the rate and size of DNA synthesized with hPol ϵ and hPol δ. Reaction mixtures, as described in A, with the indicated levels of RFC and hPols, were incubated for 20 min at 37 °C.