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 (ϳ16 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.The HSV-1 1 DNA polymerase holoenzyme is a stable heterodimer composed of a large, 134-kDa catalytic subunit (Pol) and a smaller, 51-kDa subunit, UL42 (1-4). Although Pol possesses inherent 5Ј to 3Ј polymerizing activity (5-7), its processivity is greatly enhanced by UL42 (3,8). Both subunits are absolutely essential for origin-dependent DNA synthesis and for productive viral replication (9 -13), indicating the importance of processive DNA synthesis to viral replication. However, the precise mechanism by which UL42 increases Pol processivity is not known and may differ from that utilized by ring-shaped processivity factors such as proliferating cell nuclear antigen and the ␤ subunit of Escherichia coli pol III (14).HSV-1 Pol, similar to pol ␦, possesses an inherent 3Ј to 5Ј exo activity that imparts proofreading ability to Pol (7, 15). Although the proofreading function of the HSV-1 DNA polymerase can reduce misincorporation frequency in vitro and in vivo (16 -18), the impact of UL42 on the exo activity of Pol and its effect on the fidelity of DNA synthesis have not been well studied. In fact, there has been much disagreement regarding the general role of processivity factors on the fidelity of their cognate polymerases. For example, proliferating cell nuclear antigen has been shown to increase base substitution errors and translesion synthesis by pol ␦ (19, 20). However, exodeficient T7 bacteriophage DNA polymerase has a decreased frequency of base substitutions but an increased frequency of frameshift mutations at reiterated sequences ...

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 DNA polymerase holoenzyme of herpes simplex virus type 1 (HSV-1) is a stable heterodimer consisting of a catalytic subunit (Pol) and a processivity factor (UL42). HSV-1 UL42 differs from most DNA polymerase processivity factors in possessing an inherent ability to bind to double-stranded DNA. It has been proposed that UL42 increases the processivity of Pol by directly tethering it to the primer and template (P/T). To test this hypothesis, we took advantage of the different sensitivities of Pol and Pol/UL42 activities to ionic strength. Although the activity of Pol is inhibited by salt concentrations in excess of 50 mM KCl, the activity of the holoenzyme is relatively refractory to changes in ionic strength from 50 to 125 mM KCl. We used nitrocellulose filter-binding assays and real-time biosensor technology to measure binding affinities and dissociation rate constants of the individual subunits and holoenzyme for a short model P/T as a function of the ionic strength of the buffer. We found that as observed for activity, the binding affinity and dissociation rate constant of the Pol/UL42 holoenzyme for P/T were not altered substantially in high-versus low-ionic-strength buffer. In 50 mM KCl, the apparent affinity with which UL42 bound the P/T did not differ by more than twofold compared to that observed for Pol or Pol/UL42 in the same low-ionic-strength buffer. However, increasing the ionic strength dramatically decreased the affinity of UL42 for P/T, such that it was reduced more than 3 orders of magnitude from that of Pol/UL42 in 125 mM KCl. Real-time binding kinetics revealed that much of the reduced affinity could be attributable to an extremely rapid dissociation of UL42 from the P/T in high-ionic-strength buffer. The resistance of the activity, binding affinity, and stability of the holoenzyme for the model P/T to increases in ionic strength, despite the low apparent affinity and poor stability with which UL42 binds the model P/T in high concentrations of salt, suggests that UL42 does not simply tether the Pol to DNA. Instead, it is likely that conformational alterations induced by interaction of UL42 with Pol allow for high-affinity and high-stability binding of the holoenzyme to the P/T even under high-ionic-strength conditions.The DNA polymerase of herpes simplex virus type 1 (HSV-1) is a heterodimer composed of a catalytic subunit (Pol), encoded by the UL30 gene, and a double-stranded (ds) DNA binding protein, encoded by the UL42 gene (6,13,15,31). The UL42 protein stimulates the activity of Pol on nicked and/or gapped DNA templates in high-salt buffer and increases the processivity of Pol on singly primed single-stranded (ss) DNA templates (12,15,17). The results of biochemical and genetic analyses have revealed that both Pol and UL42 protein are required for extensive leading and lagging strand DNA synthesis in cell-free (in vitro) assays (10,25,35,36) and for viral DNA replication in infected cells (in vivo) (20,22,27,33). The carboxyl-terminal (C-terminal) 227-amino-acid domain of Pol is suffici...

Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.

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