FIGURE 3.

Influence of hPCNA and hRFC on various hPol ϵ preparations. A, influence of PCNA. Reaction mixtures (10 μl) contained 20 mm Tris-HCl, pH 7.5, 0.2 mm DTT, 200 μg/μl BSA, 10 mm magnesium acetate, 35 μm [α-³²P]dATP (18,280 cpm/pmol), 2 mm ATP, 130 μm each of dCTP, dGTP, and dTTP, 0.2 m sodium glutamate, 1 nm singly primed M13, 350 nm E. coli SSB, 10 nm p261-N, 10.3 nm p261-FL, 11.2 nm p261 p59 complex or 11 nm four-subunit hPol ϵ (where indicated), 6 nm hRFC, and 100 nm PCNA. Mixtures were incubated for 30 min at 37 °C, and aliquots were used to measure nucleotide incorporation and size of DNA products following 1% alkaline-agarose gel separation and autoradiography. B, influence of PCNA levels on DNA synthesis using singly primed M13 DNA catalyzed by hPol δ, hPol ϵ, and p261-N. Reaction mixtures were as described in A (except that the level of glutamate was reduced to 30 mm) with the indicated levels of PCNA and either the four-subunit Pol ϵ, p261-N, or hPol δ preparation.