PCNA accelerates the nucleotide incorporation rate by DNA polymerase δ.

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Mondol T,Stodola JL,Galletto R,Burgers PM

doi

10.1093/nar/gky1321

subject

Has Abstract

pub_date

2019-02-28 00:00:00

pages

1977-1986

issue

4

eissn

0305-1048

issn

1362-4962

pii

5272361

journal_volume

47

pub_type

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