Pre-steady state kinetic studies of the fidelity of nucleotide incorporation by yeast DNA polymerase δ

Lynne M. Dieckman, Robert E. Johnson, Satya Prakash, M. Todd Washington

Research output: Contribution to journalArticle

22 Scopus citations

Abstract

Eukaryotic DNA polymerase δ (pol δ) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol δ is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although pol δ is one of the major eukaryotic replicative polymerase, the mechanism by which it incorporates nucleotides is unknown. Here we report both steady state and pre-steady state kinetic studies of the fidelity of pol δ. We found that pol δ incorporates nucleotides with an error frequency of 10-4 to 10-5. Furthermore, we showed that for correct versus incorrect nucleotide incorporation, there are significant differences between both pre-steady state kinetic parameters (apparent K ddNTP and kpol). Somewhat surprisingly, we found that pol δ synthesizes DNA at a slow rate with a kpol of ∼1 s-1. We suggest that, unlike its prokaryotic counterparts, pol δ requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.

Original languageEnglish (US)
Pages (from-to)7344-7350
Number of pages7
JournalBiochemistry
Volume49
Issue number34
DOIs
StatePublished - Aug 31 2010

ASJC Scopus subject areas

  • Biochemistry

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