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Originally published In Press as doi:10.1074/jbc.M001291200 on March 16, 2000

J. Biol. Chem., Vol. 275, Issue 21, 16316-16322, May 26, 2000
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Kinetic Analysis of Nucleotide Incorporation by Mammalian DNA Polymerase delta *

Heidi J. EinolfDagger and F. Peter Guengerich§

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

The kinetics of nucleotide incorporation into 24/36-mer primer/template DNA by purified fetal calf thymus DNA polymerase (pol) delta  was examined using steady-state and pre-steady-state kinetics. The role of the pol delta  accessory protein, proliferating cell nuclear antigen (PCNA), on DNA replication by pol delta  was also examined by kinetic analysis. The steady-state parameter kcat was similar for pol delta  in the presence and absence of PCNA (0.36 and 0.30 min-1, respectively); however, the Km for dNTP was 20-fold higher in the absence of PCNA (0.067 versus 1.2 µM), decreasing the efficiency of nucleotide insertion. Pre-steady-state bursts of nucleotide incorporation were observed for pol delta  in the presence and absence of PCNA (rates of polymerization (kpol) of 1260 and 400 min-1, respectively). The reduction in polymerization rate in the absence of PCNA was also accompanied by a 2-fold decrease in burst amplitude. The steady-state exonuclease rate of pol delta  was 0.56 min-1 (no burst, 103-fold lower than the rate of polymerization). The small phosphorothioate effect of 2 for correct nucleotide incorporation into DNA by pol delta ·PCNA indicated that the rate-limiting step in the polymerization cycle occurs prior to phosphodiester bond formation. A KddNTP value of 0.93 µM for poldelta ·dNTP binding was determined by pre-steady-state kinetics. A 5-fold increase in KdDNA for the pol delta ·DNA complex was measured in the absence of PCNA. We conclude that the major replicative mammalian polymerase, pol delta , exhibits kinetic behavior generally similar to that observed for several prokaryotic model polymerases, particularly a rate-limiting step following product formation in the steady state (dissociation of oligonucleotides) and a rate-limiting step (probably conformational change) preceding phosphodiester bond formation. PCNA appears to affect pol delta  replication in this model mainly by decreasing the dissociation of the polymerase from the DNA.

* This work was supported in part by United States Public Health Grants R35 CA44353 and P30 ES00267.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported in part by United States Public Health Service Grant F32 CA75731. Current address: Dept. of Drug Metabolism and Pharmacokinetics, Novartis Pharmaceuticals, Bldg. 405, Rm. 462A, 59 Rte. 10, East Hanover, NJ 07936-1080. Tel.: 973-781-3119; Fax: 973-781-5023; E-mail: heidi.einolf@pharma.novartis.com.

§ To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638B Medical Research Bldg. I, 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: guengerich@toxicology.mc.vanderbilt.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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