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 wildtype 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 pol⅐UL42 complex was much less efficient than by the mutant pol⅐UL42. Thus, despite relatively frequent (1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol⅐UL42 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.Herpes simplex virus type 1 (HSV-1) 1 is the best characterized member of the large family of Herpesviridae pathogenic to humans, which also includes Epstein-Barr virus, varicella-zoster virus, human cytomegalovirus, and Kaposi sarcoma-associated herpes virus (reviewed in Ref. 1). Viruses in this family encode most of the proteins essential for and directly involved in DNA replication (2-4), including a well conserved DNA polymerase catalytic subunit (pol), which is a member of the polymerase B family (5, 6). HSV-1 pol possesses 5Ј-to 3Ј-polymerizing and 3Ј-to 5Ј-exonuclease (exo) activities (7,8), the latter of which is involved in the removal of incorrectly incorporated deoxyribonucleoside triphosphates (9 -12). The importance of this proofreading activity for maintaining fidelity of DNA replication was suggested by studies from our laboratory that demonstrated the relatively poor ability of HSV-1 pol to discriminate between the correct and incorrect nucleotide for incorporation in single turnover experiments (13). That study (13) reported that selectivity of correct o...

The exonucleolytic activities associated with herpes simplex virus type-1 (HSV-1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5' -3'-exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i. e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double-stranded DNA in a very processive manner and exclusively in the 5' -3' direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5' -3'-exonucleolytic activities of the DNase and the polymerase-associated 3' -5' exonuclease.The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N-terminal, central and C-terminal polypeptide domains of HSV-1 DNA polymerase. From the data presented it can be concluded that the cryptic 3'-5' exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.Herpes simplex virus (HSV) DNA polymerase is the major essential function of the viral DNA synthesis machinery [I]. The availability of well-characterized temperature-sensitive [l -31 and drug-resistant 14-61 DNA polymerase mutants and recent sequencing information [7 -91 render the viral polymerase an attractive model enzyme for studies of eukaryotic DNA polymerases. As a further powerful tool for the biochemical analysis we have recently established polyclonal antibodies directed against selected domains of the polymerase polypeptide. With these monospecific antibodies it was possible to demonstrate [lo], in agreement with the potential coding sequences and the mapping limits for the catalytic domain [9], that HSV type-1 (HSV-1) DNA

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