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.
Highlights
In eukaryotes, three DNA polymerases are involved in accurate and efficient DNA replication, DNA polymerase alpha (Pol ␣), DNA polymerase delta (Pol ␦) and DNA polymerase epsilon (Pol ε) [1,2,3]
Pol ␣ which contains both polymerase and primase activities, synthesizes short RNA/DNA hybrid primers on the leading strand and initiates the synthesis of Okazaki fragments on the lagging strand
Pol ε is mainly responsible for the synthesis of leading strand [4], while Pol ␦ extends and matures Okazaki fragments on the lagging strand [3]
Summary
Three DNA polymerases are involved in accurate and efficient DNA replication, DNA polymerase alpha (Pol ␣), DNA polymerase delta (Pol ␦) and DNA polymerase epsilon (Pol ε) [1,2,3]. In order to ensure that our kinetic analysis would measure the rate of catalysis by a stable, preformed DNA-Pol ␦ complex, we first examined the stoichiometry of Pol ␦ to template/primer DNA using electrophoretic mobility shift assays, fluorescence anisotropy and fluorescence quenching methods.
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