Herpes Simplex Virus Processivity Factor UL42 Imparts Increased DNA-Binding Specificity to the Viral DNA Polymerase and Decreased Dissociation from Primer-Template without Reducing the Elongation Rate

Herpes simplex virus DNA polymerase consists of a catalytic subunit, Pol, and a processivity subunit, UL42, that, unlike other established processivity factors, binds DNA directly. We used gel retardation and filter-binding assays to investigate how UL42 affects the polymerase-DNA interaction. The Pol/UL42 heterodimer bound more tightly to DNA in a primer-template configuration than to single-stranded DNA (ssDNA), while Pol alone bound more tightly to ssDNA than to DNA in a primer-template configuration. The affinity of Pol/UL42 for ssDNA was reduced severalfold relative to that of Pol, while the affinity of Pol/UL42 for primer-template DNA was increased ~15-fold relative to that of Pol. The affinity of Pol/UL42 for circular double-stranded DNA (dsDNA) was reduced drastically relative to that of UL42, but the affinity of Pol/UL42 for short primer-templates was increased modestly relative to that of UL42. Pol/UL42 associated with primer-template DNA ~2-fold faster than did Pol and dissociated ~10-fold more slowly, resulting in a half-life of 2 h and a subnanomolar K_d. Despite such stable binding, rapid-quench analysis revealed that the rates of elongation of Pol/UL42 and Pol were essentially the same, ~30 nucleotides/s. Taken together, these studies indicate that (i) Pol/UL42 is more likely than its subunits to associate with DNA in a primer-template configuration rather than nonspecifically to either ssDNA or dsDNA, and (ii) UL42 reduces the rate of dissociation from primer-template DNA but not the rate of elongation. Two models of polymerase-DNA interactions during replication that may explain these findings are presented.     

Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity. To investigate these issues, we substituted alanine for each of four conserved arginine residues on the positively charged surface. Each single substitution decreased the DNA binding affinity of UL42, with 14- to 30-fold increases in apparent dissociation constants. The mutant proteins exhibited no meaningful change in affinity for binding to the C terminus of the catalytic subunit of the polymerase, indicating that the substitutions exert a specific effect on DNA binding. The substitutions decreased UL42-mediated long-chain DNA synthesis by the polymerase in the same rank order in which they affected DNA binding, consistent with a role for DNA binding in polymerase processivity. Combining these substitutions decreased DNA binding further and impaired the complementation of a UL42 null virus in transfected cells. Additionally, using a revised mathematical model to analyze rates of dissociation of UL42 from DNAs of various lengths, we found that dissociation from internal sites, which would be the most important for tethering the polymerase, was relatively slow, even at ionic strengths that permit processive DNA synthesis by the holoenzyme. These data provide evidence that the basic surface of UL42 interacts with DNA and support a model in which DNA binding by UL42 is important for processive DNA synthesis.     

The herpes simplex virus type 1 DNA polymerase processivity factor increases fidelity without altering pre-steady-state rate constants for polymerization or excision

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 ( approximately 16 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 extreme C terminus of herpes simplex virus DNA polymerase is crucial for functional interaction with processivity factor UL42 and for viral replication.

The herpes simplex virus DNA polymerase is composed of two subunits, a large catalytic subunit (Pol) and a smaller subunit (UL42) that increases the processivity of the holoenzyme. The interaction between the two polypeptides is of interest both for the mechanism by which it enables the enzyme to synthesize long stretches of DNA processively and as a possible target for the rational design of novel antiviral drugs. Here, we demonstrate through a combination of insertion and deletion mutagenesis that the carboxy-terminal 35 amino acids of Pol are crucial for binding UL42. The functional importance of the interaction was confirmed by the finding that a pol mutant defective for UL42 binding retained polymerase activity, but did not synthesize longer DNA products in the presence of UL42. Moreover, several association-incompetent mutants failed to complement the replication of a pol null mutant in a transient transfection assay, confirming that the Pol-UL42 interaction is necessary for virus replication in vivo and therefore a valid target for directed drug design.     

Identification of crucial hydrogen-bonding residues for the interaction of herpes simplex virus DNA polymerase subunits via peptide display, mutational, and calorimetric approaches

The catalytic subunit, Pol, of herpes simplex virus DNA polymerase interacts via its extreme C terminus with the processivity subunit, UL42. This interaction is critical for viral replication and thus a potential target for antiviral drug action. To investigate the Pol-binding region on UL42, we engineered UL42 mutations but also used random peptide display to identify artificial ligands of the Pol C terminus. The latter approach selected ligands with homology to residues 171 to 176 of UL42. Substitution of glutamine 171 with alanine greatly impaired binding to Pol and stimulation of long-chain DNA synthesis by Pol, identifying this residue as crucial for subunit interactions. To study these interactions quantitatively, we used isothermal titration calorimetry and wild-type and mutant forms of Pol-derived peptides and UL42. Each of three peptides corresponding to either the last 36, 27, or 18 residues of Pol bound specifically to UL42 in a 1:1 complex with a dissociation constant of 1 to 2 microM. Thus, the last 18 residues suffice for most of the binding energy, which was due mainly to a change in enthalpy. Substitutions at positions corresponding to Pol residue 1228 or 1229 or at UL42 residue 171 abolished or greatly reduced binding. These residues participate in hydrogen bonds observed in the crystal structure of the C terminus of Pol bound to UL42. Thus, interruption of these few bonds is sufficient to disrupt the interaction, suggesting that small molecules targeting the relevant side chains could interfere with Pol-UL42 binding.     

Interaction of herpes simplex virus type 1 DNA polymerase and the UL42 accessory protein with a model primer template.

Genetic and biochemical studies have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication. A number of studies have previously suggested that these two proteins specifically interact, and more recent studies have confirmed that the viral DNA polymerase from HSV-1-infected cells consists of a heterodimer of the UL30 (Pol; the catalytic subunit) and UL42 polypeptides. A comparison of the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells indicated that the Pol-UL42 complex is more highly processive than Pol alone on singly primed M13 single-stranded substrates. The results of these studies are consistent with the idea that the UL42 polypeptide is an accessory subunit of the HSV-1 DNA polymerase that acts to increase the processivity of polymerization. Preliminary experiments suggested that the increase in processivity was accompanied by an increase in the affinity of the polymerase for the ends of linear duplex DNA. We have further characterized the effect of the UL42 polypeptide on a defined hairpin primer template substrate. Gel shift and filter binding studies show that the affinity of the Pol catalytic subunit for the 3' terminus of the primer template increases 10-fold in the presence of UL42. DNase I footprinting experiments indicate that the Pol catalytic subunit binds to the primer template at a position that protects 14 bp of the 3' duplex region and an adjacent 18 bases of the single-stranded template. The presence of the UL42 polypeptide results in the additional protection of a contiguous 5 to 14 bp in the duplex region but does not affect the 5' position of the Pol subunit. Free UL42 protects the entire duplex region of the substrate but does not bind to the single-stranded region. Taken together, these results suggest that the increase in processivity in the presence of UL42 is related to the double-stranded DNA-binding activity of free UL42 and that the role of UL42 in the DNA polymerase complex is to act as a clamp, decreasing the probability that the polymerase will dissociate from the template after each cycle of catalysis.     

The positively charged surface of herpes simplex virus UL42 mediates DNA binding

Herpes simplex virus DNA polymerase is a heterodimer composed of UL30, a catalytic subunit, and UL42, a processivity subunit. Mutations that decrease DNA binding by UL42 decrease long chain DNA synthesis by the polymerase. The crystal structure of UL42 bound to the C terminus of UL30 revealed an extensive positively charged surface ("back face"). We tested two hypotheses, 1) the C terminus of UL30 affects DNA binding and 2) the positively charged back face mediates DNA binding. Addressing the first hypothesis, we found that the presence of a peptide corresponding to the UL30 C terminus did not result in altered binding of UL42 to DNA. Addressing the second hypothesis, previous work showed that substitution of four conserved arginine residues on the basic face with alanines resulted in decreased DNA affinity. We tested the affinities for DNA and the stimulation of long chain DNA synthesis of mutants in which the four conserved arginine residues were substituted individually or together with lysines and also a mutant in which a conserved glutamine residue was substituted with an arginine to increase positive charge on the back face. We also engineered cysteines onto this surface to permit disulfide cross-linking studies. Last, we assayed the effects of ionic strength on DNA binding by UL42 to estimate the number of ions released upon binding. Our results taken together strongly suggest that the basic back face of UL42 contacts DNA and that positive charge on this surface is important for this interaction.     

Binding parameters and thermodynamics of the interaction of the human cytomegalovirus DNA polymerase accessory protein, UL44, with DNA: implications for the processivity mechanism

The mechanisms of processivity factors of herpesvirus DNA polymerases remain poorly understood. The proposed processivity factor for human cytomegalovirus DNA polymerase is a DNA-binding protein, UL44. Previous findings, including the crystal structure of UL44, have led to the hypothesis that UL44 binds DNA as a dimer via lysine residues. To understand how UL44 interacts with DNA, we used filter-binding and electrophoretic mobility shift assays and isothermal titration calorimetry (ITC) analysis of binding to oligonucleotides. UL44 bound directly to double-stranded DNA as short as 12bp, with apparent dissociation constants in the nanomolar range for DNAs >18bp, suggesting a minimum DNA length for UL44 interaction. UL44 also bound single-stranded DNA, albeit with lower affinity, and for either single- or double-stranded DNA, there was no apparent sequence specificity. ITC analysis revealed that UL44 binds to duplex DNA as a dimer. Binding was endothermic, indicating an entropically driven process, likely due to release of bound ions. Consistent with this hypothesis, analysis of the relationship between binding and ionic strength indicated that, on average, 4±1 monovalent ions are released in the interaction of each monomer of UL44 with DNA. The results taken together reveal interesting implications for how UL44 may mediate processivity.     

The crystal structure of an unusual processivity factor, herpes simplex virus UL42, bound to the C terminus of its cognate polymerase

Herpes simplex virus DNA polymerase is a heterodimer composed of a catalytic subunit, Pol, and an unusual processivity subunit, UL42, which, unlike processivity factors such as PCNA, directly binds DNA. The crystal structure of a complex of the C-terminal 36 residues of Pol bound to residues 1-319 of UL42 reveals remarkable similarities between UL42 and PCNA despite contrasting biochemical properties and lack of sequence homology. Moreover, the Pol-UL42 interaction resembles the interaction between the cell cycle regulator p21 and PCNA. The structure and previous data suggest that the UL42 monomer interacts with DNA quite differently than does multimeric toroidal PCNA. The details of the structure lead to a model for the mechanism of UL42, provide the basis for drug design, and allow modeling of other proteins that lack sequence homology with UL42 or PCNA.     

Linear diffusion on DNA despite high-affinity binding by a DNA polymerase processivity factor.

The oligomeric "sliding clamp" processivity factors, such as PCNA, are thought to rely on a loose, topological association with DNA to slide freely along dsDNA. Unlike PCNA, the processivity subunit of the herpes simplex virus DNA polymerase, UL42, is a monomer and has an intrinsic affinity for dsDNA that is remarkably high for a sequence-independent DNA binding protein. Using a DNase footprinting assay, we demonstrate that UL42 translocates with the catalytic subunit of the polymerase during chain elongation. In addition, footprinting and electrophoretic mobility shift assays show that, despite its tight DNA binding, UL42 is capable of linear diffusion on DNA at a rate of between 17 and 47 bp/s. Our results thus suggest that, despite profound biochemical differences with the sliding clamps, UL42 can freely slide downstream with the catalytic subunit during DNA replication.     

Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity

The interactions of the herpes simplex virus processivity factor UL42 with the catalytic subunit of the viral polymerase (Pol) and DNA are critical for viral DNA replication. Previous studies, including one showing that substitution of glutamine residue 282 with arginine (Q282R) results in an increase of DNA binding in vitro, have indicated that the positively charged back surface of UL42 interacts with DNA. To investigate the biological consequences of increased DNA binding by UL42 mutations, we constructed two additional UL42 mutants, including one with a double substitution of alanine for aspartic acid residues (D270A/D271A) and a triple mutant with the D270A/D271A and Q282R substitutions. These UL42 mutants exhibited increased and prolonged DNA binding without an effect on binding to a peptide corresponding to the C terminus of Pol. Plasmids expressing any of the three UL42 mutants with an increased positive charge on the back surface of UL42 were qualitatively competent for complementation of growth and DNA replication of a UL42 null mutant on Vero cells. We then engineered viruses expressing these mutant proteins. The UL42 mutants were more resistant to detergent extraction than wild-type UL42, suggesting that they are more tightly associated with DNA in infected cells. All three UL42 mutants formed smaller plaques on Vero cells and replicated to reduced yields compared with results for a control virus expressing wild-type UL42. Moreover, mutants with double and triple mutations, which contain D270A/D271A mutations, exhibited increased mutation frequencies, and mutants containing the Q282R mutation exhibited elevated ratios of virion DNA copies per PFU. These results suggest that herpes simplex virus has evolved so that UL42 neither binds DNA too tightly nor too weakly to optimize virus production and replication fidelity.Processivity factors of DNA polymerases promote long-chain DNA synthesis by preventing dissociation of the DNA polymerase from the primer/template. Processivity factors also can influence DNA replication fidelity, as indicated by numerous in vivo and in vitro studies (1-3, 5, 6, 11, 12, 18, 28, 36). A major class of processivity factors known as “sliding clamps” includes proliferating cell nuclear antigen (PCNA) of eukaryotic cells (23) and gp45 of T4 bacteriophage (27). Sliding clamps are homodimers or homotrimers that encircle DNA and interact with the catalytic subunits (Pols) of their cognate DNA polymerases to promote processive DNA synthesis.A second class of processivity factors includes those encoded by herpesviruses and is exemplified by herpes simplex virus (HSV) UL42. UL42 forms a heterodimer with the HSV Pol. Both subunits are essential for production of infectious virus and for viral DNA replication (20, 26). UL42 can stimulate long-chain DNA synthesis by Pol, and template challenge experiments established that this stimulation is due to increased processivity (15). In addition to its interaction with Pol, which is mediated by the C terminus of Pol, UL42 also binds DNA directly with high affinity (14, 15, 30, 37). This mode of DNA binding differs from that of sliding clamps, which do not form high-affinity direct interactions with DNA (13) but must be loaded onto DNA with the aid of ATP-dependent clamp loaders for their normal functioning (16). Nevertheless, the structure of UL42 is very similar to a monomer of the sliding clamp PCNA (39). Like other processivity factors, UL42 also plays a role in maintaining DNA replication fidelity both in vivo and in vitro (5, 18).The “back face” (opposite face to the side that binds Pol) of a UL42 molecule contains several positively charged residues. By titrating the effects of cations on UL42 DNA binding, it was determined that charge-charge interactions are involved in the interaction (22). Substitutions of alanine for any of four arginine residues on the back face of UL42 resulted in substantial reductions in DNA binding without affecting the binding to peptide corresponding to the C terminus of Pol in vitro (31), while substitutions of lysine for arginine had little or no effect on DNA binding affinity (22). A UL42 mutant (Q282R) containing a substitution of arginine for a negatively charged glutamine residue on the back face of UL42 exhibited a fourfold increase in DNA binding without altering the interaction with the Pol C-terminal peptide in vitro (22). Therefore, the positively charged surface of UL42 is important for the interaction between UL42 and DNA. A question raised by these studies is whether UL42 could bind DNA so tightly as to affect HSV replication.Mutant viruses engineered to encode individual arginine-to-alanine substitution mutations in UL42 exhibit several phenotypes, including a delayed onset of viral DNA replication, reduced virus yields, and reduced fidelity of DNA replication (18). Recombinant viruses expressing UL42 with multiple substitutions of alanine for arginine residues exhibit even greater effects on viral DNA replication and virus yields (19). Thus, reducing DNA binding by UL42 deleteriously affects viral growth and DNA replication fidelity. However, these studies did not address whether increasing DNA binding by UL42 would have any effects on viral DNA replication, replication fidelity, or virus production.In this study we engineered two new UL42 mutant proteins (with the D270A/D271A or Q282R/D270A/D271A mutations) that contain less negative charge on the back face and examined the effects of these substitutions on DNA and Pol peptide binding. In addition, recombinant viruses were constructed to examine the effect of these multiple substitutions and the single Q282R substitution on virus production, DNA replication, and the fidelity of DNA replication.     

Interactions of gene 2.5 protein and DNA polymerase of bacteriophage T7.

Bacteriophage T7 gene 2.5 protein has been shown to interact with T7 DNA polymerase (the complex of T7 gene 5 protein and Escherichia coli thioredoxin) by affinity chromatography and fluorescence emission anisotropy. T7 DNA polymerase binds specifically to a resin coupled to gene 2.5 protein and elutes from the resin when the ionic strength of the buffer is raised to 250 mM NaCl. In contrast, T7 gene 5 protein alone binds more weakly to gene 2.5 protein, eluting when the ionic strength of the buffer is 50 mM NaCl. Thioredoxin does not bind to gene 2.5 protein. Steady-state fluorescence emission anisotropy gives a dissociation constant of 1.1 +/- 0.2 microM for the complex of gene 2.5 protein and T7 DNA polymerase, with a ratio of gene 2.5 protein to T7 DNA polymerase in the complex of 1:1. Nanosecond emission anisotropic analysis suggests that the complex contains one monomer each of gene 2.5 protein, gene 5 protein, and thioredoxin. The ability of T7 gene 2.5 protein to stimulate the activity and processivity of T7 DNA polymerase is compared with the ability of three other single-stranded DNA-binding proteins: E. coli single-stranded DNA-binding protein, T4 gene 32 protein, and E. coli recA protein. All except E. coli recA protein stimulate the activity and processivity of T7 DNA polymerase; E. coli recA protein inhibits these activities.     

Functional interaction between the herpes simplex virus type 1 polymerase processivity factor and origin-binding proteins: enhancement of UL9 helicase activity

The origin (ori)-binding protein of herpes simplex virus type 1 (HSV-1), encoded by the UL9 open reading frame, has been shown to physically interact with a number of cellular and viral proteins, including three HSV-1 proteins (ICP8, UL42, and UL8) essential for ori-dependent DNA replication. In this report, it is demonstrated for the first time that the DNA polymerase processivity factor, UL42 protein, provides accessory function to the UL9 protein by enhancing the 3'-to-5' helicase activity of UL9 on partially duplex nonspecific DNA substrates. UL42 fails to enhance the unwinding activity of a noncognate helicase, suggesting that enhancement of unwinding requires the physical interaction between UL42 and UL9. UL42 increases the steady-state rate for unwinding a 23/38-mer by UL9, but only at limiting UL9 concentrations, consistent with a role in increasing the affinity of UL9 for DNA. Optimum enhancement of unwinding was observed at UL42/UL9 molecular ratios of 4:1, although enhancement was reduced when high UL42/DNA ratios were present. Under the assay conditions employed, UL42 did not alter the rate constant for dissociation of UL9 from the DNA substrate. UL42 also did not significantly reduce the lag period which was observed following the addition of UL9 to DNA, regardless of whether UL42 was added to DNA prior to or at the same time as UL9. Moreover, addition of UL42 to ongoing unwinding reactions increased the steady-state rate for unwinding, but only after a 10- to 15-min lag period. Thus, the increased affinity of UL9 for DNA most likely is the result of an increase in the rate constant for binding of UL9 to DNA, and it explains why helicase enhancement is observed only at subsaturating concentrations of UL9 with respect to DNA. In contrast, ICP8 enhances unwinding at both saturating and subsaturating UL9 concentrations and reduces or eliminates the lag period. The different means by which ICP8 and UL42 enhance the ability of UL9 to unwind DNA suggest that these two members of the presumed functional replisome may act synergistically on UL9 to effect initiation of HSV-1 DNA replication in vivo.     

Characterization of human herpesvirus 8 ORF59 protein (PF-8) and mapping of the processivity and viral DNA polymerase-interacting domains

Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118-126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.     

Functional analysis of the herpes simplex virus UL42 protein.

The herpes simplex virus UL42 gene encodes a multifunctional polypeptide (UL42) that is essential for virus DNA replication. To further understand the relationship between the structure of UL42 and the role that it plays during virus replication, we analyzed an extensive set of mutant UL42 proteins for the ability to perform the three major biochemical functions ascribed to the protein:binding to DNA, stably associating with the virus DNA polymerase (Pol), and acting to increase the length of DNA chains synthesized by Pol. Selected mutants were also assayed for their ability to complement the replication of a UL42 null virus. The results indicated that the N-terminal 340 amino acids of UL42 were sufficient for all three biochemical activities and could also support virus replication. Progressive C-terminal truncation resulted in the loss of detectable DNA-binding activity before Pol binding, while several mutations near the N terminus of the polypeptide resulted in an altered interaction with DNA but had no apparent affect on Pol binding. More dramatically, an insertion mutation at residue 160 destroyed the ability to bind Pol but had no effect on DNA binding. This altered polypeptide also failed to increase the length of DNA product synthesized by Pol, and the mutant gene could not complement the growth of a UL42 null virus, indicating that the specific interaction between Pol and UL42 is necessary for full Pol function and for virus replication. This study confirms the validity of the Pol-UL42 interaction as a target for the design of novel therapeutic agents.     

The herpes simplex virus type 1 UL42 gene product: a subunit of DNA polymerase that functions to increase processivity.

Genetic experiments have shown that the products of the herpes simplex virus type 1 (HSV-1) DNA polymerase (UL30) and UL42 genes are both required for viral DNA replication, and a number of studies have suggested that these two proteins specifically interact. We have confirmed and extended these findings. The viral DNA polymerase from HSV-1-infected cells has been purified as a complex containing equimolar quantities of the UL30 (Pol, the catalytic subunit) and UL42 polypeptides. Sedimentation and gel filtration analyses of this complex are consistent with the idea that the complex consists of a heterodimer of Pol and UL42. A complex with identical physical and functional properties was also purified from insect cells coinfected with recombinant baculoviruses expressing the two polypeptides. Therefore, the formation of the Pol-UL42 complex does not require the participation of any other HSV-encoded protein. We have compared the catalytic properties of the Pol-UL42 complex with those of the isolated subunits of the enzyme purified from recombinant baculovirus-infected insect cells. The specific activity of the catalytic subunit alone was nearly identical to that of the complex when assayed on activated DNA. When assayed on a defined template such as singly primed M13 DNA, however, the combination of Pol and UL42 utilized fewer primers and formed larger products than Pol alone. Template challenge experiments demonstrated that the Pol-UL42 complex was more highly processive than Pol alone. Our data are consistent with the idea that the UL42 polypeptide is an accessory subunit of the DNA polymerase that acts to increase the processivity of polymerization.     

Cloning, sequencing, and functional characterization of the two subunits of the pseudorabies virus DNA polymerase holoenzyme: evidence for specificity of interaction.

The pseudorabies virus (PRV) genes encoding the two subunits of the DNA polymerase were located on the genome by hybridization to their herpes simplex virus type 1 (HSV-1) homologs, pol and UL42, and subsequently were sequenced. Like the HSV-1 homologs, in vitro translation products of the PRV gene encoding the catalytic subunit (pol) possessed activity in the absence of the Pol accessory protein (PAP). However, the PRV PAP stimulated the activity of Pol fourfold in the presence of 150 mM KCl, using an activated calf thymus DNA template. The stimulation of Pol activity by PAP under high-salt conditions and the inhibition of Pol activity by PAP when assayed in low salt (0 mM KCl) together were used to determine the specificity with which PAP interacted with Pol. Despite functional similarity, HSV-1 UL42 and PRV PAP could neither stimulate the noncognate Pols at high salt nor inhibit them at low salt. Furthermore, a PRV Pol mutant lacking the 30 C-terminal amino acids retained basal Pol activity but could be neither stimulated nor inhibited by the PRV PAP. Sequence comparisons of the Pol proteins of the alphaherpesviruses reveal a conserved domain in the C terminus which terminates immediately before the last 41 residues of both PRV and HSV-1 proteins. These results indicate that the ability and specificity for interaction of the PRV Pol with PAP most likely resides predominantly in the extreme Pol C terminus.     

ATP activation of DNA polymerase III holoenzyme from Escherichia coli. II. Initiation complex: stoichiometry and reactivity

DNA polymerase III holoenzyme (holenzyme) has an ATPase activity elicited only by a primed DNA template. Reaction of preformed ATP.holoenzyme complex with a primed template results in hydrolysis of the ATP bound to the holoenzyme, release of ADP and Pi, and formation of an initiation complex between holoenzyme and the primed template. Approximately two ATP molecules are hydrolyzed for each initiation complex formed, a value in keeping with the number bound in the ATP.holoenzyme complex. The possibility that the latter and the initiation complex contain two holoenzyme molecules is supported by the presence of two beta monomers in the initiation complex. Holoenzyme action in the absence of ATP resembles that of pol III (the holoenzyme core) or DNA polymerase III (holoenzyme lacking the beta subunit), with or without ATP, in sensitivity to salt and in processivity of elongation. The initiation complex formed by ATP-activated holoenzyme resists a level of KCl (150 mM) that completely inhibits nonactivated holoenzyme and the incomplete forms of the holoenzyme, and displays a processivity at least 20 times greater. Upon completing replication of available template, holoenzyme can dissociate and form an initiation complex with another primed template, provided ATP is available to reactivate the holoenzyme. By inference, no essential subunits are lost in the cycle of initiation, elongation and dissociation.     

Crystal Structure of Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

The DNA polymerase processivity factor of the Epstein-Barr virus, BMRF1, associates with the polymerase catalytic subunit, BALF5, to enhance the polymerase processivity and exonuclease activities of the holoenzyme. In this study, the crystal structure of C-terminally truncated BMRF1 (BMRF1-ΔC) was solved in an oligomeric state. The molecular structure of BMRF1-ΔC shares structural similarity with other processivity factors, such as herpes simplex virus UL42, cytomegalovirus UL44, and human proliferating cell nuclear antigen. However, the oligomerization architectures of these proteins range from a monomer to a trimer. PAGE and mutational analyses indicated that BMRF1-ΔC, like UL44, forms a C-shaped head-to-head dimer. DNA binding assays suggested that basic amino acid residues on the concave surface of the C-shaped dimer play an important role in interactions with DNA. The C95E mutant, which disrupts dimer formation, lacked DNA binding activity, indicating that dimer formation is required for DNA binding. These characteristics are similar to those of another dimeric viral processivity factor, UL44. Although the R87E and H141F mutants of BMRF1-ΔC exhibited dramatically reduced polymerase processivity, they were still able to bind DNA and to dimerize. These amino acid residues are located near the dimer interface, suggesting that BMRF1-ΔC associates with the catalytic subunit BALF5 around the dimer interface. Consequently, the monomeric form of BMRF1-ΔC probably binds to BALF5, because the steric consequences would prevent the maintenance of the dimeric form. A distinctive feature of BMRF1-ΔC is that the dimeric and monomeric forms might be utilized for the DNA binding and replication processes, respectively.     

A novel processive mechanism for DNA synthesis revealed by structure, modeling and mutagenesis of the accessory subunit of human mitochondrial DNA polymerase

Mitochondrial DNA polymerase (pol gamma) is the sole DNA polymerase responsible for replication and repair of animal mitochondrial DNA. Here, we address the molecular mechanism by which the human holoenzyme achieves high processivity in nucleotide polymerization. We have determined the crystal structure of human pol gamma-beta, the accessory subunit that binds with high affinity to the catalytic core, pol gamma-alpha, to stimulate its activity and enhance holoenzyme processivity. We find that human pol gamma-beta shares a high level of structural similarity to class IIa aminoacyl tRNA synthetases, and forms a dimer in the crystal. A human pol gamma/DNA complex model was developed using the structures of the pol gamma-beta dimer and the bacteriophage T7 DNA polymerase ternary complex, which suggests multiple regions of subunit interaction between pol gamma-beta and the human catalytic core that allow it to encircle the newly synthesized double-stranded DNA, and thereby enhance DNA binding affinity and holoenzyme processivity. Biochemical properties of a novel set of human pol gamma-beta mutants are explained by and test the model, and elucidate the role of the accessory subunit as a novel type of processivity factor in stimulating pol gamma activity and in enhancing processivity.