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

Abstract

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...

Mechanisms by which herpes simplex virus DNA polymerase limits translesion synthesis through abasic sites

“…Further experiments will be required to identify the actual rate-limiting step(s) for extension by the exo -pol beyond the AP site. However, it is interesting to note that there also was an absence of a burst and a very slow rate for extension of mismatches during processive synthesis by the exo -pol [9]. As suggested in that study, we think it likely that the P/T is held in the exo site for an extended period of time due to the inability of the pol to cleave the last nucleotide from the primer.…”

“…The WT and exo -mutant (D368A) HSV-1 pol genes (UL30) were expressed by infecting Spodoptera frugiperda 9 (Sf9) insect cells with recombinant baculoviruses containing the respective genes [9,29]. The baculoviruses containing the WT and D368A HSV-1 pol genes were gifts from Bob Lehman (Stanford University) and Charles Knopf (University of Heidelberg), respectively.…”

“…The baculoviruses containing the WT and D368A HSV-1 pol genes were gifts from Bob Lehman (Stanford University) and Charles Knopf (University of Heidelberg), respectively. Enzymes were purified to >95% purity as detailed previously [9,29] except that whole cell extracts were used.…”

“…The 1235-amino-acid HSV-1 DNA polymerase catalytic subunit (pol), a B family polymerase, is encoded by the UL30 gene and contains conserved 5' to 3' polymerizing and 3' to 5' exonuclease (exo) domains [2][3][4][5][6]. Functional analysis of proteins containing mutations in the exo domain indicates that the polymerizing and exo activities can function independently [7][8][9]. The region encoding the C-terminal domain of HSV-1 pol is poorly conserved.…”

Contributions of Nucleotide Excision Repair, DNA Polymerase η, and Homologous Recombination to Replication of UV-irradiated Herpes Simplex Virus Type 1

“…It appears that the overall replication fidelity is high, and existing genetic variability may be created by recombination between a limited number of strains rather than random replication errors (12)(13)(14). The contribution of the DNA polymerase to replication fidelity has been thoroughly examined, but cellular mechanisms contributing to the genetic stability of herpesviruses have, with few exceptions, not been extensively looked at (15)(16)(17)(18). It has been noted that several cellular repair proteins co-localize with viral replication proteins in a limited number of replication foci (19 -21).…”