PCNA accelerates the nucleotide incorporation rate by DNA polymerase

Abstract

DNA polymerase delta (Pol ) is responsible for the elongation and maturation of Okazaki fragments in eukaryotic cells. Proliferating cell nuclear antigen (PCNA) recruits Pol to the DNA and serves as a processivity factor. Here, we show that PCNA also stimulates the catalytic rate of Saccharomyces cerevisiae Pol by >10-fold. We determined template/primer DNA binding affinities and stoichiometries by Pol in the absence of PCNA, using electrophoretic mobility shift assays, fluorescence intensity changes and fluorescence anisotropy binding titrations. We provide evidence that Pol forms higher ordered complexes upon binding to DNA. The Pol catalytic rates in the absence and presence of PCNA were determined at millisecond time resolution using quench flow kinetic measurements. The observed rate for single nucleotide incorporation by a preformed DNA-Pol complex in the absence of PCNA was 40 s−1. PCNA enhanced the nucleotide incorporation rate by >10 fold. Compared to wild-type, a growth-defective yeast PCNA mutant (DD41,42AA) showed substantially less stimulation of the Pol nucleotide incorporation rate, identifying the face of PCNA that is important for the acceleration of catalysis.