Evidence against a Simple Tethering Model for Enhancement of Herpes Simplex Virus DNA Polymerase Processivity by Accessory Protein UL42

Abstract

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...

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

“…The pol proteins eluted between 0.25 and 0.34 M KCl and were pooled, concentrated by ultrafiltration using Centricon-100 units (Amicon, Bedford, MA), diluted with an equal volume of buffer E (200 mM KCl, 20 mM Tris, pH 8.0, 1 mM EDTA, and 60% glycerol), flash-frozen in liquid nitrogen, and stored at Ϫ80°C. The wild-type and mutant pol proteins were also purified as a complex with UL42 as described previously (24). Protein concentrations were determined by the Bradford protein assay (Bio-Rad) using bovine serum albumin as a standard.…”

“…Protein Purification-Cells infected with recombinant baculovirus (10 plaque-forming units/cell) were harvested at 40-h postinfection and lysed, and soluble extracts were prepared as described previously (24). Protein fractionation was conducted with the aid of a fast protein liquid chromatography system (Amersham Biosciences) at room temperature as described below, and fractions were collected on ice.…”

Functional Interaction between the Herpes Simplex Virus Type 1 Polymerase Processivity Factor and Origin-Binding Proteins: Enhancement of UL9 Helicase Activity

“…UL42 protein was purified as described previously (12), except that the Blue Sepharose column was replaced with two linked 5-ml prepacked HiTrap Blue columns (Amersham Biosciences, Piscataway, N.J.). UL42 eluted from the HiTrap Blue column between 1.5 and 2 M KCl.…”

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

“…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.…”