PCNA accelerates the nucleotide incorporation rate by DNA polymerase δ

• Published in: Nucleic acids research Vol. 47; no. 4; pp. 1977 - 1986
• Main Authors: Mondol, Tanumoy, Stodola, Joseph L, Galletto, Roberto, Burgers, Peter M
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 28-02-2019
• Subjects: Catalysis; DNA - genetics; DNA Polymerase III - genetics; DNA Primers - genetics; DNA Replication - genetics; DNA-Binding Proteins - genetics; Nucleic Acid Enzymes; Proliferating Cell Nuclear Antigen - genetics; Protein Binding; Saccharomyces cerevisiae - genetics
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gky1321

Abstract
DNA polymerase delta (Pol δ) is responsible for the elongation and maturation of Okazaki fragments in eukaryotic cells. Proliferating cell nuclear antigen (PCNA) recruits Pol δ to the DNA and serves as a processivity factor. Here, we show that PCNA also stimulates the catalytic rate of Saccharomyces cerevisiae Pol δ by >10-fold. We determined template/primer DNA binding affinities and stoichiometries by Pol δ in the absence of PCNA, using electrophoretic mobility shift assays, fluorescence intensity changes and fluorescence anisotropy binding titrations. We provide evidence that Pol δ forms higher ordered complexes upon binding to DNA. The Pol δ 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 δ 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 δ nucleotide incorporation rate, identifying the face of PCNA that is important for the acceleration of catalysis.