Fig. S1.

Characterization of human proteins used in this study. (A) Exonuclease activities of WT and exonuclease-deficient human pol δ. Assays were performed in duplicate. 5′-Labeled 29-mer primer alone (ssDNA) or annealed in the P29/Bio62 substrate was incubated with the indicated pol δ enzymes. Average activities and SDs from duplicate reactions are presented. *At the initial time point (1.5 min), all of the substrate was degraded, setting a lower limit for exonuclease activity of WT human pol δ at 200 nM/1.5 min = 133.33 nM/min. Thus, the D402A mutation reduces the exonuclease activity of human pol δ at least 14.4-fold on an optimal substrate for exonuclease activity. Exonuclease activity was not visible on the P29/Bio62 DNA substrate with the D402A mutant. (B) Activities of PCNA clamps in stimulating human RFC ATPase activity. The ATPase activity was determined at 25 °C in an assay solution containing 125 nM RFC, 125 nM PCNA (WT or monoubiquitinated, Ub), 125 nM P/T DNA (500 nMNeutravidin), 1 mM ATP, 3 mM phosphoenolpyruvate, 200 mM NADH, and 6–8 U phosphoenolpyruvate kinase-lactate dehydrogenase mix. For each condition, the initial rates of ATP hydrolysis are reported as an average of three independent experiments ± SD. (Ub)₃-PCNA stimulated the DNA-dependent ATPase activity of RFC to the same extent as WT PCNA, indicating a fully functional sliding clamp ring.