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Originally published In Press as doi:10.1074/jbc.M210023200 on January 8, 2003

J. Biol. Chem., Vol. 278, Issue 11, 8996-9004, March 14, 2003
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The Herpes Simplex Virus Type 1 DNA Polymerase Processivity Factor Increases Fidelity without Altering Pre-steady-state Rate Constants for Polymerization or Excision*

Murari Chaudhuri, Liping Song, and Deborah S. ParrisDagger

From the Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210

Pre-steady-state and steady-state kinetics of nucleotide incorporation and excision were used to assess potential mechanisms by which the fidelity of the herpes simplex virus type 1 DNA polymerase catalytic subunit (Pol) is enhanced by its processivity factor, UL42. UL42 had no effect on the pre-steady-state rate constant for correct nucleotide incorporation (150 s-1) nor on the primary rate-limiting conformational step. However, the equilibrium dissociation constant for the enzyme in a stable complex with primer-template was 44 nM for Pol and 7.0 nM for Pol/UL42. The catalytic subunit and holoenzyme both selected against incorrect nucleotide incorporation predominantly at the level of nucleotide affinity, although UL42 slowed by 4-fold the maximum rate of incorporation of incorrect, compared with correct, nucleotide. Pol, with or without UL42, cleaved matched termini at a slower rate than mismatched ones, but UL42 did not significantly alter the pre-steady-state rate constant for mismatch excision (~16 s-1). The steady-state rate constant for nucleotide addition was 0.09 s-1 and 0.03 s-1 for Pol and Pol/UL42, respectively, and enzyme dissociation was the rate-limiting step. The longer half-life for DNA complexes with Pol/UL42 (23 s) compared with that with Pol (8 s) affords a greater probability for excision when a misincorporation event does occur, accounting predominantly for the failure of Pol/UL42 to accumulate mismatched product at moderate nucleotide concentrations.

* This work was supported in part by National Institutes of Health Grant GM34940. Facilities, equipment, and core services were supported in part by the Ohio State University Department of Molecular Virology, Immunology, and Medical Genetics and by Comprehensive Cancer Center Core Grant P30 CA16058.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 To whom correspondence should be addressed: Dept. of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, 2198 Graves Hall, 333 W. 10th Ave., Columbus, OH 43210. Tel.: 614-292-0735; Fax: 614-292-9805; E-mail: parris.1@osu.edu.

Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
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