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

Lynne M Dieckman; Robert E Johnson; Satya Prakash; M Todd Washington

doi:10.1021/bi100556m

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

Journal article

Peer reviewed

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

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

Biochemistry (Easton), Vol.49(34), pp.7344-7350

08/31/2010

DOI: 10.1021/bi100556m

PMCID: PMC2941984

PMID: 20666462

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Abstract

Eukaryotic DNA polymerase delta (pol delta) is a member of the B family of polymerases and synthesizes most of the lagging strand during DNA replication. Yeast pol delta is a heterotrimer comprised of three subunits: the catalytic subunit (Pol3) and two accessory subunits (Pol31 and Pol32). Although pol delta 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 delta. We found that pol delta 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(d)(dNTP) and k(pol)). Somewhat surprisingly, we found that pol delta synthesizes DNA at a slow rate with a k(pol) of approximately 1 s(-1). We suggest that, unlike its prokaryotic counterparts, pol delta requires replication accessory factors like proliferating cell nuclear antigen to achieve rapid rates of nucleotide incorporation.

DNA Replication

DNA Polymerase III - genetics

Saccharomyces cerevisiae - genetics

Catalytic Domain - genetics

Nucleotides - genetics

DNA - metabolism

Nucleotides - metabolism

Pressure

DNA - genetics

Saccharomyces cerevisiae - metabolism

Yeasts - genetics

Proliferating Cell Nuclear Antigen - genetics

Eukaryota

Nucleotidyltransferases - genetics

Kinetics

Nucleotidyltransferases - metabolism

DNA Polymerase III - metabolism

Proliferating Cell Nuclear Antigen - metabolism

Yeasts - metabolism

Details

Title: Subtitle: Pre-steady state kinetic studies of the fidelity of nucleotide incorporation by yeast DNA polymerase delta
Creators: Lynne M Dieckman - Department of Biochemistry, University of Iowa College of Medicine, Iowa City, Iowa 52242-1109, USA
Robert E Johnson
Satya Prakash
M Todd Washington
Resource Type: Journal article
Publication Details: Biochemistry (Easton), Vol.49(34), pp.7344-7350
DOI: 10.1021/bi100556m
PMID: 20666462
PMCID: PMC2941984
NLM abbreviation: Biochemistry
ISSN: 0006-2960
eISSN: 1520-4995
Publisher: United States
Grant note: GM081433 / NIGMS NIH HHS CA138546 / NCI NIH HHS R01 CA138546 / NCI NIH HHS R01 GM081433-03 / NIGMS NIH HHS R01 GM081433 / NIGMS NIH HHS
Language: English
Date published: 08/31/2010
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024531302771

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