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J. Biol. Chem., Vol. 279, Issue 18, 18535-18543, April 30, 2004

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Contribution of the 3'- to 5'-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis^*

Liping Song, Murari Chaudhuri, Charles W. Knopf, and Deborah S. Parris¶

From the Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, Ohio 43210 and Institute for Applied Tumor Virology, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120, Heidelberg, Germany

Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase catalytic subunit (pol) is less faithful than for most replicative DNA polymerases, despite the presence of an associated 3'- to 5'-exonuclease (exo) activity. To determine the aspects of fidelity affected by the exo activity, nucleotide incorporation and mismatch extension frequency for purified wild-type and an exo-deficient mutant (D368A) pol were compared using primer/templates that varied at only a single position. For both enzymes, nucleotide discrimination during incorporation occurred predominantly at the level of K_m for nucleotide and was the major contributor to fidelity. The contribution of the exo activity to reducing the efficiency of formation of half of all possible mispairs was 6-fold or less, and 30-fold when averaged for the formation of all possible mispairs. In steady-state reactions, mismatches imposed a significant kinetic barrier to extension independent of exo activity. However, during processive DNA synthesis in the presence of only three nucleotides, misincorporation and mismatch extension were efficient for both exo-deficient and wild-type pol catalytic subunits, although slower kinetics of mismatch extension by the exo-deficient pol were observed. The UL42 processivity factor decreased the extent of misincorporation by both the wild-type and the exo-deficient pol to similar levels, but mismatch extension by the wild-type pol·UL42 complex was much less efficient than by the mutant pol·UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol·UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by the kinetic barrier to extending a mismatch. The kinetic barrier also increases the probability that a mismatched primer terminus will be transferred to the exo site where it can be excised by the associated exo activity and subsequently extended with correct nucleotide.

Received for publication, September 4, 2003 , and in revised form, February 6, 2004.

^* This work was supported in part by Grant GM34930 from the National Institutes of Health (to D. S. P.) and core services provided by the Ohio State University Comprehensive Cancer Center Core Grant P30 CA16058. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 614-292-0735; Fax: 614-292-9805; E-mail: parris.1{at}osu.edu.

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