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.

SUBMITTER: Mondol T 

PROVIDER: S-EPMC6393303 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature