Effects of Acyclovir, Foscarnet, and Ribonucleotides on HerpesSimplex Virus‑1 DNA Polymerase: Mechanistic Insights and a NovelMechanism for Preventing Stable Incorporation of Ribonucleotidesinto DNAAshwani Kumar Vashishtha and Robert D. Kuchta*

Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309-0215, United States

*S Supporting Information

ABSTRACT: We examined the impact of two clinically approved anti-herpes drugs, acyclovir and Forscarnet (phosphonoformate), on theexonuclease activity of the herpes simplex virus-1 DNA polymerase,UL30. Acyclovir triphosphate and Foscarnet, along with the closely relatedphosphonoacetic acid, did not affect exonuclease activity on single-strandedDNA. Furthermore, blocking the polymerase active site due to eitherbinding of Foscarnet or phosphonoacetic acid to the E−DNA complex orpolymerization of acyclovir onto the DNA also had a minimal effect onexonuclease activity. The inability of the exonuclease to excise acyclovir fromthe primer 3′-terminus results from the altered sugar structure directlyimpeding phosphodiester bond hydrolysis as opposed to inhibiting binding,unwinding of the DNA by the exonuclease, or transfer of the DNA from thepolymerase to the exonuclease. Removing the 3′-hydroxyl or the 2′-carbonfrom the nucleotide at the 3′-terminus of the primer strongly inhibitedexonuclease activity, although addition of a 2′-hydroxyl did not affect exonuclease activity. The biological consequences of theseresults are twofold. First, the ability of acyclovir and Foscarnet to block dNTP polymerization without impacting exonucleaseactivity raises the possibility that their effects on herpes replication may involve both direct inhibition of dNTP polymerizationand exonuclease-mediated destruction of herpes DNA. Second, the ability of the exonuclease to rapidly remove a ribonucleotideat the primer 3′-terminus in combination with the polymerase not efficiently adding dNTPs onto this primer provides a novelmechanism by which the herpes replication machinery can prevent incorporation of ribonucleotides into newly synthesizedDNA.

Herpes viruses are complex DNA viruses that areresponsible for a variety of indications, including oral

and genital herpes sores, chickenpox, viral encephalitis, etc.7

Herpes simplex virus 1 (HSV) encodes seven proteins essentialfor viral DNA replication: (a) the heterodimeric DNApolymerase−processivity factor complex (UL30/UL42), (b)the heterotrimeric helicase-primase (UL5-UL8-UL52), (c) anorigin binding protein (UL9), and (d) a single-stranded DNAbinding protein (UL29/ICP8).8−10 In addition to polymeraseactivity, UL30 also possesses 3′−5′ exonuclease activity thatproofreads the just-incorporated nucleotide.11,12

Acyclovir, gancicyclovir, and phosphonoformic acid areclinically useful anti-herpes drugs.13,14 Acyclovir is a remarkablypowerful treatment for α-herpes virus infections because it hasminimal side effects and problems with resistance.15−18 Onceconverted to the triphosphate by cellular and viral kinases,acyclovir triphosphate (ACVTP) acts as a chain terminator ofHSV polymerase.13,14,19−23 The formation of acyclovir-terminated DNA followed by binding of the next requireddNTP results in the formation of an extremely stable E−DNA−dNTP dead-end complex in the polymerase active site.24

Similarly, ganciclovir (as the biologically active GCVTP)primarily functions as a chain terminator during cytomegalo-virus DNA replication.25 On the other hand, phosphonoformicacid (Foscarnet) is a pyrophosphate analogue that functions bydirectly binding to the pyrophosphate binding site in thepolymerase active site.26−28

Derse et al. observed that the exonuclease activity of herpespolymerase (UL30/UL42) did not efficiently excise acyclovirmonophosphate that the polymerase activity had incorpo-rated.20 Similarly, Hanes et al. used transient kinetic methods todirectly show that the exonuclease does not efficientlyhydrolyze acyclovir-terminated DNA as compared to DNAcontaining deoxyguanine at the primer terminus (kexo of 12 s−1

for deoxyguanine-terminated DNA vs 5 × 10−3 s−1 foracyclovir-terminated DNA).29 These authors also proposedthat the presence of acyclovir at the 3′-terminus of the primerinterferes with movement of the DNA from the polymerase tothe exonuclease active sites.

Received: January 26, 2016Published: February 2, 2016

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© 2016 American Chemical Society 1168 DOI: 10.1021/acs.biochem.6b00065Biochemistry 2016, 55, 1168−1177

We recently showed that blocking the polymerase active sitevia formation of an UL30−DNA−aphidicolin dead-endcomplex has little or no effect on the exonuclease activity,indicating that the polymerase and exonuclease active sites haveindependent DNA binding domains.30 This result also raisesthe possibility that other inhibitors may block polymeraseactivity but leave the exonuclease unbothered.To improve our understanding of how acyclovir and

Foscarnet can impact herpes replication, we employed syntheticoligonucleotides of defined sequence to examine how formingE−DNAACV−dNTP or E−DNA−PFA complexes in thepolymerase active site affects exonuclease activity. In bothcases, forming these complexes did not affect exonucleaseactivity. The poor ability of the exonuclease to hydrolyzeacyclovir-terminated DNA results from the modified sugardirectly interfering with the hydrolysis reaction. Lastly, weobserved that while the presence of a 2′-hydroxyl at the primerterminus does not affect exonuclease activity, it potentlyinhibits polymerase activity. The biochemical and medicalsignificance of these results is discussed.

■ MATERIALS AND METHODS

Chemicals. All chemicals were of the highest grade availableand were used as purchased. T4 polynucleotide kinase was fromNew England Biolabs. dNTPs and ddNTPs were fromInvitrogen. [γ-32P]ATP was from Perkin-Elmer. T4 DNApolymerase, T7 DNA polymerase, and Klenow Fragment wereobtained from New England Biolabs. Phosphonoformic acid

and phosphonoacetic acid were from Sigma. Acyclovirtriphosphate and gancicyclovir triphosphate were obtainedfrom Wayne Miller (Burroughs-Welcome Corp., ResearchTriangle Park, NC).

Enzymes. His-tagged UL30 and UL30/42 were purifiedfrom SF9 insect cells infected with recombinant baculovirusesthat harbor the genes encoding these proteins as describedpreviously.31

Oligonucleotides. Oligonucleotides were obtained fromIntegrated DNA Technologies (Coralville, IA). Table 1 lists thesequences of all primers and templates used in this study. AllDNAs were gel purified using 20% denaturing polyacrylamidegel electrophoresis. Primers were radiolabeled at the 5′-endusing [γ-32P]ATP and T4 polynucleotide kinase by standardprocedures.32 DNA duplexes were formed by heating theprimer−templates in a molar ratio of 1:1.4 to 95 °C followed byslow cooling to room temperature.

Preparation of ACV, GCV, Dideoxyguanosine-Termi-nated Duplex and Single-Stranded DNAs. ACV-termi-nated primer−template was prepared by incubating 40 μMACVTP with 5 μM 5′-[32P]DNA15C in 50 mM Hepes (pH 7.6),5% glycerol, 0.1 mg/mL BSA, 1 mM DTT, and 10 mM MgCl2at 37 °C. The reaction was initiated with 500 nM KlenowFragment, allowed to proceed for 2 h, and then terminatedwhen the mixture was heated to 90 °C for 15 min. ExcessACVTP was removed from the reaction mixture by beingpassed through a G25 spin column. Denaturing polyacrylamidegel electrophoresis of the reaction products showed that >95%

Table 1. DNA Substrates Useda

aTemplating bases are underlined, and A*, G*, D*, and rG* indicate acyclovir-, ganciclovir-, dideoxy-, and ribose-terminated DNAs, respectively.

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of the starting DNA15C had been converted to DNA16ACV. Toprepare ACV-terminated single-stranded DNA, the radio-labeled acyclovir-terminated primer−template (DNA16ACV)was heated to 95 °C for 5 min and the ACV-terminatedprimer strand purified using 20% denaturing acrylamide gelelectrophoresis. GCV and dideoxyguanine-terminated single-stranded and duplex DNAs were prepared analogously.Simultaneous Polymerase/Exonuclease Assays. All

experiments were performed under steady-state conditions at37 °C. Assays typically contained 1 μM 5′-[32P]primer−template, 50 mM Hepes (pH 7.6), 5% glycerol, 0.1 mg/mLBSA, 1 mM DTT, 10 mM MgCl2, 10 μM dNTPs, and varyingconcentrations of inhibitor (ACVTP, PFA, or PAA). Reactionswere initiated by adding enzyme (typically 50 nM) andquenched at various times by adding 5 volumes of 90%formamide, 10 mM EDTA, 1× Tris/Borate/EDTA buffer, and0.1% bromophenol blue. Samples were heated for 2 min at 90°C and products separated by denaturing gel electrophoresis(20% acrylamide and 8 M urea) and analyzed by phosphor-imagery (Molecular Dynamics).Exonuclease Assays. All exonuclease assays were

performed under conditions of excess substrate as describedabove except that dNTPs were omitted from the assays. 5′-[32P]DNA (1 μM) was incubated with reaction buffer in thepresence of varying concentrations of ACVTP (0−200 μM).Reactions were initiated by adding enzyme (typically 5 nM forsingle-stranded DNA and 50 nM for duplex DNA) and aliquotsquenched at designated time intervals.Measurement of IC50 Values for Various DNAs. Assays

contained a fixed concentration of either 5′-[32P]DNA35ss or 5′-[32P]DNA35C (1 μM) and varying concentrations of anunlabeled DNA containing G, ddG, ACV, or GCV at theprimer 3′ terminus. The reciprocal of the fraction of DNA35ss orDNA35C hydrolyzed was plotted against DNA concentration toobtain the IC50 of the unlabeled DNA to inhibit exonucleaseactivity on the 32P-labeled DNA.

■ RESULTSWe previously showed that formation of an E−DNA−aphidicolin ternary complex where the DNA was bound inthe polymerase active site had no effect on exonucleaseactivity,30 raising the possibility that other inhibitors of herpespolymerase might likewise generate complexes with inhibitedpolymerase activity but active exonuclease. To test thispossibility, we examined the effects of two clinically usefulanti-herpes drugs, acyclovir and phosphonoformic acid, onexonuclease activity using synthetic oligonucleotides of definedsequence.We initially examined the effects of ACVTP, PFA, and PAA

on exonuclease activity using a single-stranded DNA as asubstrate (DNA35ss). Figure 1 shows the time course forexonuclease activity in the presence of increasing concen-trations of ACVTP. Even at saturating concentrations, ACVTPdid not inhibit the exonuclease activity on DNA35ss (Figure 1B).Similar results were obtained with PFA and PAA (Figures S1and S2 of the Supporting Information). Using long, partiallydouble-stranded DNAs as the substrate [oligo(dG)·poly(dC),33

radiolabeled activated calf thymus DNA,34 and radiolabeledEscherichia coli DNA35], previous work reported that both PFAand PAA inhibited exonuclease activity. However, we foundthat PFA only mildly inhibited exonuclease activity on 5′-[32P]DNA15C (Figure S3A). To ensure that the lack ofinhibition was not a consequence of the short length of

DNA15C and/or its sequence, we tested a somewhat longerDNA (DNA30C). Again, adding PFA did not inhibitexonuclease activity (Figure S3B).We next determined how these compounds impact

exonuclease activity under conditions where the polymerasecan simultaneously elongate a primer−template. We previouslyshowed that under conditions of excess DNA over UL30 (orthe UL30/UL42 complex), the enzyme processes some of theDNA via dNTP polymerization and some via exonucleaseactivity.30 Thus, these conditions allow simultaneous monitor-ing of both polymerase and exonuclease activity. Assayscontained 1 μM 5′-[32P]DNA15C, all four dNTPs, andincreasing concentrations of ACVTP, PFA, or PAA [Figure 2(ACVTP), Figure 3 (PFA), and Figure S4 (PAA, SupportingInformation)]. The DNA concentration is much greater thanits KD (approximately 10−50 nM) such that all of thepolymerase active sites should contain bound DNA. In theabsence of any inhibitor, the polymerase elongates some of theDNA to the end of the template while the exonucleasehydrolyzes a fraction of the DNA. Adding increasingconcentrations of ACVTP, PFA, or PAA inhibited dNTPpolymerization but had at most weak effects on exonucleaseactivity. Polymerase inhibition without significantly impactingthe exonuclease is consistent with the idea that the two activesites have independent DNA binding domains. Similar resultswere obtained with the UL30/UL42 complex, indicating thatUL42 does not affect the independence of the polymerase andexonuclease DNA binding domains (Figure S5 of theSupporting Information).PFA inhibits herpes replication by forming a UL30−DNA−

PFA ternary complex, while acyclovir can inhibit polymeraseactivity by forming either a UL30−DNAACV binary or UL30−DNAACV−dNTP ternary complex. To explicitly measure theeffects of these complexes on exonuclease activity, we generatedthe UL30−DNA−(±dNTP) complexes using a 5′-32P-labeled

Figure 1. ACVTP does not affect exonuclease activity on DNA35ss.UL30 was incubated with DNA (1 μM), and aliquots were taken out atvarious times. (A) Phosphorimages of the products of DNA35ssdegradation using UL30 at varying concentrations of ACVTP,including 0, 1, 5, 20, and 200 μM at time intervals of 0, 0.5, 1, 5,10, and 20 min. (B) Plot of exonuclease products as a function ofACVTP concentration at various time intervals. Note that the gelsshown are representative of experiments that were performed multipletimes.

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primer−template while simultaneously measuring exonucleaseactivity on a separate 5′-[32P]ssDNA (Figure 4). The two5′-32P-labeled DNAs were of different lengths; hence, theexonuclease activity on each DNA can be independentlymonitored. First, the effect of just a primer−templatecontaining a normal nucleotide at the primer terminus wasmeasured to control for the ability of a primer−template todirectly bind in the exonuclease site. Figure 4A shows thatwhereas significant exonuclease activity occurred on the single-stranded DNA, virtually no degradation occurred on theprimer−template (<5%), as expected in light of theexonuclease’s preference for single-stranded DNA. Addingeither PFA or PAA to assays containing a primer−template and5′-[32P]ssDNA had no effect on the rate at which UL30hydrolyzed the ssDNA (Table 2 and Figure 4A−C), indicatingthat formation of either an E−DNA−PFA or an E−DNA−PAAternary complex in the polymerase active site did not affect theexonuclease. Likewise, formation of either an E−DNAACV or an

E−DNAACV−dNTP complex in the polymerase active site didnot inhibit exonuclease activity (Figure 4D,E). Thus, evenunder conditions where the polymerase active site is blockeddue to binding and/or polymerization of various inhibitors, theexonuclease remains completely active.

Why Is DNA Containing ACV at the Primer 3′-Terminus a Poor Substrate for the Exonuclease? Previouswork showed that double-stranded DNA containing ACV at theprimer 3′-terminus is a very poor exonuclease substrate for theUL30/UL42 complex.20,24,29 Likewise, we observed that DNAcontaining ACV as the terminal nucleotide was a very poorsubstrate for the UL30 exonuclease (Table 3). The presence ofACV at the primer terminus (DNA16ACV) decreased the rate ofthe exonuclease by 23-fold compared to that of the identicalDNA containing dG at the 3′-terminus. This much slower rateclearly indicates that the sugar of the excised nucleotide greatlyimpacts phosphodiester bond cleavage. We therefore endea-vored to determine what features of the sugar are needed forefficient exonuclease activity and the mechanistic consequencesof eliminating these features.Compared to the canonical 2′-deoxyribose, the sugar in ACV

lacks a 3′-hydroxyl, lacks the hydrophobic 2′-methylene, and isconformationally much less constrained because of its acyclicnature. To determine which of these features contributes to thedecreased exonuclease efficiency, we synthesized primer−templates that contained either a 2′,3′-dideoxynucleotide (totest the importance of a 3′-hydroxyl) or ganciclovir (to test the

Figure 2. Effect of ACVTP on polymerase and exonuclease activitiesunder processive conditions. UL30 was incubated with DNA15C (1μM) in the presence of 0 or 10 μM dNTPs and varying concentrationsof acyclovir triphosphate (0−80 μM). Aliquots of each reactionmixture were analyzed at various times after the reaction had beeninitiated. (A) Phosphorimages of the products of DNA15C fullextension and degradation using UL30. (B) Plot of exonucleaseproducts as a function of ACVTP concentration at 6 min.

Figure 3. Effect of Foscarnet on polymerase and exonuclease activitiesunder processive conditions. UL30 was incubated with DNA15C (1μM) in the presence of 0 or 10 μM dNTPs and 0−80 μM Foscarnet.Aliquots were analyzed at 6 min. (A) Phosphorimages of the productsof DNA15C full extension and degradation using UL30. (B) Plot ofexonuclease products as a function of PFA concentration.

Figure 4. Formation of UL30−DNA15C−PFA, UL30−DNA15C−PAA,UL30−DNA16ACV, and UL30−DNA16ACV−dTTP complexes does notaffect the exonuclease activity on a second DNA. Assays containedDNA35ss and the additional DNAs and compounds as noted. All DNAswere present at 1 μM: (A) DNA15C, (B) DNA15C and 50 μM PFA, (C)DNA15C and 50 μM PAA, (D) DNA16ACV, and (E) DNA16ACV and 50μM dTTP. The time points for panels A−C were 0, 0.25, 0.75, 1, 2, 3,5, and 7 min. The time points for panels D and E were 0, 0.25, 0.5,0.75, 1, 2, and 5 min. In panels B−E, DNA15C has been omitted for thesake of clarity.

Table 2. Effects of Forming E−DNA−PFA, E−DNA−PAA,E−DNAACV−dTTP, and E−DNAACV Complexes on the 3′−5′ Exonuclease Activity of UL30 on DNA15C

inhibitor rate (nM/min)

no inhibitor 162 ± 450 μM PFA 156 ± 450 μM PAA 162 ± 31 μM DNA16ACV and 50 μM dTTP 160 ± 151 μM DNA16ACV 130 ± 20

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importance of an intact furan (Figure 5)) at the 3′-terminus.Just as the presence of acyclovir at the primer 3′-terminus

greatly decreased exonuclease activity, the presence of eitherganciclovir or a dideoxynucleotide at the primer 3′-terminusalso greatly decreased exonuclease activity (Table 3). UL30hydrolyzed a primer−template containing ganciclovir or 2′,3′-dideoxyG at the primer 3′-terminus 20- or 2900-fold lessefficiently, respectively, than if it contained a normal nucleotide(Table 3). UL42 does not impact the effects of the alteredsugars as UL30 and the UL30/UL42 complexes gave similarresults. The rates of DNA degradation obtained withDNA16ACV, DNA16GCV, and DNA16dd using UL30/UL42 were90-, 90-, and 2200-fold lower than that of DNA15G, respectively.Thus, the exonuclease activity of UL30 appears to beexquisitely sensitive to modifications in the sugar of thenucleotide at the 3′-terminus of the DNA.To determine if modifying the sugar at the primer 3′-

terminus affects the exonuclease associated with other DNApolymerases, we tested these modified DNAs with several otherproofreading exonucleases. Table 3 shows that just as withUL30, modifying the structure of the 3′-terminal nucleotidegreatly impacts the exonuclease of T4 DNA polymerase. Similarresults were obtained with T7 DNA polymerase and KlenowFragment when ACV-terminated DNA was used. These datasuggest that the exonucleases associated with at least some Aand B family polymerases are quite sensitive to modification ofthe sugar moiety.Mechanistically, three potential explanations could account

for the decreased exonuclease rates of UL30 upon removal ofthe 3′-hydroxyl from the 3′-terminal nucleotide or its

conversion into an acyclic sugar: (1) It greatly weakens bindingto the exonuclease. (2) Because the enzyme needs to transferthe DNA from the polymerase active site to the exonucleaseactive site and unwind 2 bp of the double-stranded DNA forthe exonuclease to hydrolyze the substrate,30 the modified sugarmight inhibit unwinding and/or DNA transfer. (3) The alteredsugar structure interferes with hydrolysis of the phosphodiesterlinkage. We initially tested the hypothesis that the altered sugarstructure interfered with binding to the exonuclease site.Exonuclease activity was measured on a 5′-32P-labeled single-stranded DNA (DNA35ss), and the effects of adding increasingamounts of a primer−template containing either ddG, ACV,GCV, or a normal dG at the primer 3′-terminus were measured.If altering the sugar structure interfered with binding to theexonuclease site, then the DNAs with altered sugars shouldhave inhibited exonuclease activity on the 5′-32P-labeled single-stranded DNA much less potently than the DNA with a normalsugar. However, all four DNAs inhibited exonuclease activitywith only small differences in potency, indicating thatmodifying the sugar did not significantly interfere with bindingof a primer−template to the exonuclease active site (Table 4).

Similarly, all four DNAs also inhibited exonuclease activity of anexogenous 5′-32P-labeled primer−template (DNA35C) to thesame extent (Table 4). To test the possibility that the modifiedsugars interfered with the unwinding and/or active site transferreactions as well as explicitly determine if the modified sugarinterferes with the hydrolysis reaction, we synthesized 5′-32P-labeled single-stranded DNAs containing dG, ACV, or ddG atthe 3′-terminus and measured exonuclease activity. Unlike withdouble-stranded DNA, neither unwinding nor transfer betweenpolymerase and exonuclease sites is needed for hydrolysis ofsingle-stranded DNA. Whereas the exonuclease rapidly hydro-lyzed the dG-terminated single-stranded DNA, it did notdetectably hydrolyze either ACV- or ddG-terminated DNA(Figure 6). Moreover, these single-stranded DNAs containingdG, ACV, or ddG at the 3′-terminus had very similar bindingaffinities for the exonuclease active site as measured by theirability to inhibit exonuclease activity on a second 5′-[32P]ssDNA (Table 5). Thus, just as with the primer−templates, a modified nucleotide at the primer 3′-terminusdoes not affect the ability of single-stranded DNAs to bind tothe exonuclease site. Additionally, because a modifiednucleotide in single-stranded DNAs blocked activity eventhough hydrolysis requires neither unwinding of any duplexDNA nor transfer between the polymerase and exonuclease

Table 3. Summary of Rates of Exonucleolytic Removal of the3′-Terminal Nucleotide of DNA15G, DNA16ACV, DNA16GCV,and DNA16dd Using UL30, T4 DNA Polymerase, T7 DNAPolymerase, and Klenow Fragmenta

rate (nM/min)

enzyme DNA15G DNA16ACV DNA16GCV DNA16dd

UL30 183 ± 5 8 ± 1(23-fold)

9 ± 1(20-fold)

0.063 ± 0.002(2900-fold)

T4 253 ± 4 11 ± 1(23-fold)

13 ± 1(20-fold)

0.50 ± 0.02(500-fold)

T7 297 ± 15 9.0 ± 0.2(33-fold)

NDb NDb

KF 99 ± 13 2.0 ± 0.1(50-fold)

NDb NDb

aIn each case, the x-fold decrease in rate is also noted. bNot done.

Figure 5. Nucleoside structures.

Table 4. Ability of Double-Stranded DNAs Containing dG(DNA15G), Acyclovir (DNA16ACV), Gancicyclovir(DNA16GCV), and DideoxyG (DNA16dd) To InhibitExonuclease Activity on 1 μM 5′-32P-Labeled Single-Stranded DNA (DNA35ss) or 1 μM 5′-32P-Labeled Double-Stranded DNA (DNA35C)

inhibitor DNA substrate DNA IC50 (μM)

DNA16GCV DNA35C 1.0DNA16ACV DNA35C 1.7DNA16dd DNA35C 2.5DNA15G DNA35C 1.3DNA16GCV DNA35ss 0.6DNA16ACV DNA35ss 1.0DNA16dd DNA35ss 0.3DNA15G DNA35ss 0.3

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sites, these data indicate that the modified sugar inhibitsexonuclease activity by interfering with phosphodiester bondcleavage.Substitution of the 2′-H with an OH Group Inhibits

Polymerase but Not Exonuclease Activity. To determinethe effect of replacing a 2′-H with an OH group, we preparedDNA containing a single ribonucleotide at the 3′-terminus andmeasured both exonuclease and polymerase activity. Panels Aand B of Figure 7 show the time courses for degradation ofDNA16riboss (a single-stranded DNA with a NMP at the 3′-terminus) and DNA16ribo (duplex DNA with a NMP at theprimer 3′-terminus), respectively. The polymerase degradedDNA16riboss and DNA15ss at similar rates (kcat values of 49 ± 3

and 67 ± 2 min−1, respectively), while it degraded DNA16riboslightly faster than DNA15G (kcat values of 10 ± 2 and 4 ± 1min−1, respectively). Thus, unlike the other modifications, thepresence or absence of a 2′-hydroxyl does not significantlyimpact the exonuclease.In contrast to the minimal effect on exonuclease activity, the

presence of even a single ribonucleotide at the primer 3′-terminus strongly inhibited dNTP polymerization. Adding asingle NMP to the 3′-end of a DNA primer (DNA16ribo)decreased kcat/Km for polymerization of the next correct dNTPby 3200-fold as compared to that with DNA containing adNMP at the 3′-terminus (Figure 8). Similarly, we previously

observed that herpes polymerase does not efficiently elongateRNA primers.36 To further test these results, we incubatedUL30 (exonuclease proficient) with excess primer−templatethat contained a ribonucleotide at the primer 3′-terminus andthe next correct dNTP. This resulted in a vast majority of theDNA being processed in the exonuclease site and only smallamounts of elongated product [<5% (Figure 9)].

■ DISCUSSIONTwo clinically useful inhibitors of herpes polymerase, ACVTPand PFA, result in E−DNA complexes in which the DNA istightly bound in the polymerase active site. In the case ofACVTP, the DNA resulting from incorporation (E−DNAACV)binds with moderate affinity. However, the next correct dNTPbinds very tightly (KD = 76 nM) to generate a very stable E−DNAACV−dNTP ternary complex.24 Importantly, the presenceof this bound DNA in the polymerase active site had no effecton 3′−5′ exonuclease activity. In the case of acyclovir, thisoccurred for both the E−DNAACV binary complex generatedimmediately after ACVTP polymerization and the E−DNAACV−dNTP ternary complex generated by ACVTPpolymerization followed by binding of the next correct

Figure 6. UL30 exonuclease does not efficiently remove acyclovir ordideoxyguanosine from single-stranded DNA. UL30 was incubatedwith various DNAs, and aliquots were removed at the noted times. AllDNAs were present at 1 μM: (A) DNA15ss, (B) DNA16ACVss, and (C)DNA16ddss. (D) Amount of exonuclease products generated for eachDNA.

Table 5. Effects of Varying the 3′-Terminal Nucleotide ofSingle-Stranded DNA on Their Ability To InhibitExonuclease Activity on a Second, 5′-32P-Labeled Single-Stranded DNA

inhibitor DNA substrate DNA IC50 (nM)

DNA35ss DNA15ss 55DNA16ACVss DNA35ss 35DNA16ddss DNA35ss 70

Figure 7. Time course for degradation of DNA16riboss and DNA16ribo.UL30 was incubated with DNA (1 μM), and aliquots were removed atvarious times. (A) Phosphorimages of the products of DNA16ribossdegradation using UL30 (1 nM) after 0, 0.25, 0.5, 1, 2, 5, 7, 10, 15, 20,25, and 30 min. (B) Phosphorimages of the products of DNA16ribodegradation using UL30 (25 nM) after 0, 0.25, 0.5, 1, 2, 5, 7, 10, 15,20, 25, and 30 min.

Figure 8. HSV pol does not efficiently polymerize dNTPs onto DNAcontaining a ribonucleotide at the 3′-terminus. UL30/UL42 (exo−)(50 nM) was incubated with DNAribo (1 μM) in the presence of 100μM TTP, and aliquots were removed after 15 min. (A)Phosphorimages of the products. (B) Plot of products as a functionof TTP concentration. Data were fit to the Michaelis−Mentenequation and gave a Vmax of 4.7 ± 0.4 nM/min and a KmdTTP of 163 ±53 μM.

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dNTP. Mechanistically, these data provide further support forUL30 containing at least two independent and nonoverlappingDNA binding domains, one for the exonuclease and one for thepolymerase.The exonuclease likely remains active throughout the

polymerase catalytic cycle (i.e., when the polymerase is in theopen and closed complexes). We previously showed that theexonuclease retains activity upon formation of either an E−DNA binary complex or an E−DNA−aphidicolin ternarycomplex.30 On the basis of the structures of other B-familypolymerases with just DNA or DNA and aphidicolin bound, thepolymerase active site is likely in the open complex in bothcases.37 In contrast, formation of the herpes polymerase−DNAACV−dNTP complex likely generates the closed complex.While the actual structure of this complex is not known, twodata suggest it will resemble the closed complex. (1) The nextcorrect dNTP binds very tightly to the E−DNAACV complex.

24

(2) Other B-family polymerases form the closed complex upongeneration of the E−DNA−dNTP ternary complexes wherethe DNA cannot be elongated (e.g., it contains a 2′,3′-dideoxynucleotide at the 3′-terminus).38−44 Likewise, bindingof PFA to the E−DNA binary complex to generate the E−DNA−PFA ternary complex probably converts UL30 into theclosed conformational state, despite the fact that the complex ispresent after phosphodiester bond formation but prior torelease of pyrophosphate. While the structure of this ternarycomplex has not been determined for UL30, the structure of achimeric version of the B-family polymerase RB69−DNA−PFAternary complex closely resembles that of the closed E−DNA−dNTP ternary complex.45 Importantly, these observationssuggest that the exonuclease remains active regardless ofwhether the polymerase is actively polymerizing dNTPs.a

Depending upon the assay conditions, we found that PFAand PAA either did not inhibit exonuclease activity or gave verymild inhibition. This, however, contrasts with previous studiesthat reported significant exonuclease inhibition by PFA and/orPAA.33−35 The likely cause of these differences is the DNAused. Whereas previous work used long DNAs whose structureis not well-defined [oligo(dG)·poly(dC),33 activated calfthymus DNA,34 and E. coli DNA35], we used two shorterDNAs of defined sequence (DNA15C and DNA30C). The latterDNA was designed to be able to completely fill the DNAbinding domain of the UL30/UL42 complex as defined by thenuclease protection studies of Challberg.46 This begets thequestion, however, of which DNA more closely resembles theDNA found in vivo. On the one hand, the length of calf thymusDNA or poly(dC) is certainly more similar to that found in

vivo. However, the structure and sequence of these DNAs willbe very different from what is found in vivo. For example, theactivated calf thymus DNA will contain loops, nicks, variablelengths of single- and double-stranded DNA, etc., dependingupon how it is handled. The DNAs we used will closelyresemble a typical primer−template in terms of structure, butthey are certainly much shorter than in vivo DNA.The observation that both acyclovir- and Foscarnet-mediated

polymerase inhibition minimally impact exonuclease activitymay have significant implications for how these compoundsimpact herpes replication. The potent polymerase inhibitionexhibited by both compounds is certainly critical for theirbiological effects as evidenced by the mechanisms by whichherpes can become resistant to these drugs.13,14 With bothdrugs, mutations that affect ACVTP polymerization and PFAbinding can give high-level resistance.b In the case of drugsensitive virus, however, if these inhibitors simply stopped thepolymerase from synthesizing new DNA, herpes couldpotentially continue replicating its DNA once the drugconcentration dropped below the value needed for effectivepolymerase inhibition. Inhibiting the polymerase but leavingthe exonuclease active could reduce this possibility becauseupon encountering a 3′-end of a DNA molecule, theexonuclease could degrade this already synthesized DNA.This destruction of preexisting DNA would be expected tonegatively impact the potential for herpes to resume DNAreplication upon diminution of the intracellular drug concen-tration.Similar to previous work, we found that the exonuclease very

poorly excises acyclovir from the 3′-terminus of DNA.20,29 Thiswas true for single-stranded DNA and a double-strandedprimer−template and if the polymerase active site was occupiedby a separate piece of DNA. Additionally, the presence ofacyclovir in the DNA did not significantly impact binding of theDNA to the exonuclease active site. Together, these dataindicate that the acyclovir inhibits exonuclease activity bydirectly blocking the hydrolytic reaction. This contrasts withthe conclusions of Hanes et al., who inferred that acyclovir-terminated DNA is a poor substrate for the exonucleasebecause of inefficient transfer of the DNA from the polymeraseto the exonuclease based on transient kinetic methods.29 Threeobservations from our studies indicate that this conclusion isincorrect. First, the presence of acyclovir at the 3′-terminus ofboth single-stranded DNA and a primer−template does notaffect binding of the DNA to the exonuclease active site.Second, the exonuclease inefficiently hydrolyzes acyclovir-terminated single-stranded DNA, a DNA that never has to betransferred between the two active sites. Third, blocking thepolymerase active site with another DNA still results ininefficient excision of acyclovir even though the blockedpolymerase site eliminates the possibility of DNA transferbetween the polymerase and exonuclease. Thus, inefficienthydrolysis of acyclovir results from the altered sugar structuredirectly impacting the exonuclease, not from an effect ontransfer.The herpes exonuclease exhibits surprising specificity with

respect to the sugar but not the base. As would be expected fora proofreading exonuclease, the enzyme readily excises any ofthe four natural dNMPs. In contrast, removing the 3′-hydroxyland/or the 2′-methylene greatly inhibits exonuclease activity.While interactions of the exonuclease active site with the sugarof the 3′-terminal nucleotide are not important for binding ofthe nucleic acid, these interactions likely directly stabilize the

Figure 9. UL30 preferentially degrades DNA containing aribonucleotide at the primer 3′-terminus rather than adding the nextcorrect dNTP. UL30 (exo+) (70 nM) was incubated with DNAribo (1μM) in the presence of 100 μM dTTP, and aliquots were removed atvarious time intervals. Phosphorimages of the products of DNAextension and degradation using 100 μM dTTP, which is the nextcorrect incoming dNTP.

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transition state for phosphodiester bond cleavage and/orproperly align the phosphodiester bond for attack by H2O.Unlike the inhibitory effects of removing the 3′-hydroxyl or

the 2′-methylene from the terminal nucleotide, the presence ofa 2′-hydroxyl had no effect on exonuclease activity such that theenzyme removed NMPs and 2′-dNMPs at similar rates.Conversely, the presence of a single NMP at the 3′-end ofthe primer reduced the efficiency of addition of the next correctdNTP by 3200-fold compared to that of a primer containing adNMP at the 3′-terminus. Similarly, previous studies showedthat herpes polymerase elongates RNA primers much lessefficiently than DNA primers (700−26000-fold). The com-parable effects indicate that the 2′-hydroxyl at the primer 3′-terminus causes much, if not all, of the slow elongation of RNAprimers.36

The greatly reduced rate of dNTP polymerization onto aribonucleotide in combination with an active exonuclease alsosuggests a simple and novel “proofreading” mechanism bywhich the herpes replication machinery may minimize thestable incorporation of NMPs into DNA. Upon incorporationof a ribonucleotide, the greatly reduced rate of further dNTPpolymerization will provide time for the exonuclease to removethe NMP prior to the polymerase eventually incorporating thenext correct dNTP. This ribonucleotide proofreading isanalogous to how UL30 can proofread incorrectly polymerizeddNTPs, although it should be noted that the efficiency of NTPproofreading will likely be less efficient than that for incorrectdNTP proofreading. Whereas UL30 discriminated againstribonucleotide elongation by 3200-fold, UL30 discriminatesagainst mismatch elongation by 104−105-fold.47To the best of our knowledge, this ability of UL30 to

efficiently remove incorporated ribonucleotides via proof-reading distinguishes it from all other replicative DNApolymerases. DNA pol α, δ, and ε replicate eukaryotic nuclearDNA, and none of them efficiently proofreads incorporatedribonucleotides. Pol α efficiently elongates primers containingone (or more) ribonucleotides at the primer 3′-terminus andlacks a proofreading exonuclease.48 The 3′−5′ exonuclease ofpol δ does not proofread ribonucleotides incorporated by polδ’s polymerase activity, while the 3′−5′ exonuclease of pol εonly weakly proofreads ribonucleotides incorporated by pol ε’spolymerase activity.49,50 This inability of pol α, δ, and ε toefficiently remove just incorporated ribonucleotides from newlysynthesized DNAs results in eukaryotic cells employing RNaseH2 to perform this essential function.49 Likewise, bacterial polIII and Φ29 polymerase incorporate NTPs and then continuedNTP polymerization.51,52 In the case of herpes, UL30 can useits proofreading function to minimize incorporation of a stableribonucleotide into DNA and potentially eliminate the need forRNaseH2 or other postreplicative mechanisms for excisingribonucleotides.The apparently complete independence of the polymerase

and 3′−5′ exonuclease active sites of UL30 has two importantimplications. First, it suggests a novel mechanism by whichcurrent herpes treatments may fatally disrupt herpes replication,the destruction of already synthesized herpes DNA. Second, itraises the questions of where on UL30 the DNA bindingdomain for the exonuclease resides and how the exonucleaseand polymerase activities are coordinated at the replicationfork. Studies to answer these questions are in progress.

■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.bio-chem.6b00065.

Additional data showing the effects of Foscarnet onexonuclease activity using single-stranded DNA as asubstrate (Figure S1), effects of PAA on exonucleaseactivity using single-stranded DNA as a substrate (FigureS2), effects of Foscarnet on exonuclease activity usingdouble-stranded DNA as a substrate (Figure S3), effectsof PAA on simultaneous polymerase and exonucleaseactivity (Figure S4), and effects of Foscarnet onsimultaneous polymerase and exonuclease activity(Figure S5) (PDF)

■ AUTHOR INFORMATION

Corresponding Author*Department of Chemistry and Biochemistry, University ofColorado, Boulder, CO 80309-0215. E-mail: Kuchta@colorado.edu. Phone: (303) 492-7027. Fax: (303) 492-5894.

FundingThis work was supported by National Institutes of HealthGrant AI59764 to R.D.K.

NotesThe authors declare no competing financial interest.

■ ABBREVIATIONSACV, acyclovir; ACVTP, acyclovir triphosphate; BSA, bovineserum albumin; ddNTP, 2′,3′-dideoxyribonucleoside 5′-triphosphate; DNAACV, DNA containing acyclovir at the primer3′-terminus; DNAGCV, DNA containing ganciclovir at theprimer 3′-terminus; DTT, dithiothreitol; EDTA, ethylenedia-minetetraacetic acid; GCV, ganciclovir; GCVTP, gancicyclovirtriphosphate; Hepes, 4-(2-hydroxyethyl)-1-piperazineethane-sulfonic acid; HSV, herpes simplex virus; PAA, phosphono-acetic acid; PFA, phosphonoformic acid (Foscarnet).

■ ADDITIONAL NOTESaWhile unlikely, an alternative mechanism that we cannotabsolutely rule out is the presence of two populations ofpolymerase, one with an active polymerase and one with anactive exonuclease. To the best of our knowledge, this has neverbeen observed with a polymerase.bThe primary mechanism by which HSV-1 becomes resistantto acyclovir is via mutations in the viral thymidine kinase genesuch that HSV-infected cells no longer accumulate thebiologically active ACVTP upon being treated with acyclo-vir.1−4 Secondarily, resistance is also mediated by mutations inthe UL30 gene such that the polymerase does not readilyincorporate ACVTP.5,6

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