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

• Published in: The Journal of biological chemistry 2004-04, Vol.279 (18), p.18535
• Main Authors: Liping Song, Murari Chaudhuri, Charles W. Knopf, Deborah S. Parris
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M309848200

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.