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Insights into the Molecular Mechanism of Polymerization and Nucleoside Reverse Transcriptase Inhibitor Incorporation by Human PrimPol Andrea C. Mislak, Karen S. Anderson Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut, USA

Human PrimPol is a newly identified DNA and RNA primase-polymerase of the archaeo-eukaryotic primase (AEP) superfamily and only the second known polymerase in the mitochondria. Mechanistic studies have shown that interactions of the primary mitochondrial DNA polymerase ␥ (mtDNA Pol ␥) with nucleoside reverse transcriptase inhibitors (NRTIs), key components in treating HIV infection, are a major source of NRTI-associated toxicity. Understanding the interactions of host polymerases with antiviral and anticancer nucleoside analog therapies is critical for preventing life-threatening adverse events, particularly in AIDS patients who undergo lifelong treatment. Since PrimPol has only recently been discovered, the molecular mechanism of polymerization and incorporation of natural nucleotide and NRTI substrates, crucial for assessing the potential for PrimPolmediated NRTI-associated toxicity, has not been explored. We report for the first time a transient-kinetic analysis of polymerization for each nucleotide and NRTI substrate as catalyzed by PrimPol. These studies reveal that nucleotide selectivity limits chemical catalysis while the release of the elongated DNA product is the overall rate-limiting step. Remarkably, PrimPol incorporates four of the eight FDA-approved antiviral NRTIs with a kinetic profile distinct from that of mtDNA Pol ␥ that may manifest in toxicity.

N

ucleoside reverse transcriptase inhibitors (NRTIs) are an important class of antivirals that target the human immunodeficiency virus (HIV) polymerase, reverse transcriptase (RT). All FDA-approved NRTIs are nucleoside analogs that lack a 3=-hydroxyl moiety to terminate DNA chain extension upon incorporation by RT into the growing proviral DNA. While significant health advances have been achieved with the use of NRTIs, the necessity for lifelong treatment to control HIV infection is limited by NRTI-associated toxicities that arise from virus-versus-host polymerase selectivity wherein NRTIs also serve as substrates for host polymerases (1, 2). The most severe NRTI-associated toxicities predominantly manifest in mitochondrial dysfunction (1–6) and are attributed primarily to incorporation by the human mitochondrial DNA polymerase ␥ (mtDNA Pol ␥) (7–9). However, there are observed discrepancies between toxicity and the potential to inhibit mtDNA Pol ␥, suggesting alternative mechanisms and evaluation of additional host cell polymerases as potential perpetrators of antiviral toxicity (10, 11). Understanding the propensity for host cell polymerases to incorporate nucleoside analogs is critical for assessing safety in the design and development of antiviral, -parasitic, -bacterial, and -cancer nucleoside analog therapies. For instance, development of the antiviral ribonucleoside triphosphate analog BMS-986094 for the treatment of hepatitis C virus was halted in phase II after nine patients were hospitalized and one died (12). Investigational in vitro studies had identified that BMS986094 was incorporated by the human mitochondrial RNA polymerase 30-fold more efficiently than ribavirin, the then-current standard of care, with “tolerable” off-target effects (13), highlighting the importance of understanding nucleoside analog incorporation by host polymerases to prevent life-threatening adverse events. Toward this end, the NRTI incorporation efficiencies have been determined for human B-, X-, and Y-family DNA polymerases (1) while the recent discovery of PrimPol operating in the

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nucleus and the mitochondria suggests an unexplored mechanism of NRTI-associated toxicity (14–17). PrimPol has emerged as a DNA and RNA primase and DNAdependent translesion synthesis (TLS) polymerase (14–18). A 560-amino-acid protein belonging to the archaeo-eukaryotic primase (AEP) superfamily, PrimPol possesses a conserved N-terminal AEP polymerase domain with three highly conserved catalytic motifs and a C-terminal zinc finger domain similar to the viral UL52 primase domain (17, 19). Notably, it lacks 3=-to-5= exonuclease activity and preferentially binds the cofactor manganese (15, 20). PrimPol functions in replication restart downstream of stalled replication forks (16) and can perform translesion synthesis, bypassing oxidative and UV-induced lesions, including 8-oxoguanine (8oxoG) (15, 20, 21), abasic sites (15), (6-4) pyrimidinepyrimidone photoproducts [(6-4)pp] (16, 21), and cyclobutane pyrimidine dimers (CPDs) (16). PrimPol helps to maintain mtDNA copy numbers and replication rates and can utilize both deoxynucleoside triphosphates (dNTPs) and NTPs (15). Since PrimPol has only recently been discovered, little is known about the molecular mechanism of polymerization and nucleotide incorporation, critical parameters for evaluating NRTI

Received 16 September 2015 Returned for modification 8 October 2015 Accepted 5 November 2015 Accepted manuscript posted online 9 November 2015 Citation Mislak AC, Anderson KS. 2016. Insights into the molecular mechanism of polymerization and nucleoside reverse transcriptase inhibitor incorporation by human PrimPol. Antimicrob Agents Chemother 60:561–569. doi:10.1128/AAC.02270-15. Address correspondence to Karen S. Anderson, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02270-15. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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incorporation and assessing the potential for PrimPol-mediated NRTI-associated toxicity. In this study, we present the first transient-state kinetic analysis of the molecular mechanism of polymerization and incorporation of the four natural nucleotide substrates by PrimPol. Our study reveals that the chemical catalysis by PrimPol is governed by nucleotide selectivity and defines product release of the elongated primer-template DNA as the overall ratelimiting step in polymerization. Subsequently, we evaluate incorporation of all eight FDA-approved NRTIs by PrimPol and demonstrate that PrimPol efficiently incorporates four of them with a kinetic profile distinct from that of mtDNA Pol ␥, which may suggest an additional mechanism of toxicity. MATERIALS AND METHODS Expression and purification of recombinant human PrimPol. A fulllength cDNA of human PrimPol was obtained from the ATCC (GenBank accession number BC064600.1) and subcloned into pET28a vector to fuse a 6⫻His tag to the N terminus of the protein. Wild-type PrimPol was expressed in Escherichia coli BL21(DE3)/pRIL cells upon induction with 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) at an optical density at 600 nm (OD600) of ⬃0.6 to 0.8 for 2.5 h at 30°C. Cell pellets were lysed with 1⫻ BugBuster (EMD-Millipore) in lysis buffer of 50 mM Tris (pH 8.0), 1 M NaCl, 400 mM AcNH4, 10 mM imidazole, 10% glycerol, 2 mM ␤-mercaptoethanol, and complete EDTA-free protease inhibitor (1 tablet per 50 ml of buffer; Roche), and proteins were isolated by centrifugation. PrimPol was bound in batch by Ni2⫹-nitrilotriacetic acid (NTA) agarose (Qiagen) equilibrated with lysis buffer for 1 h at 4°C. The resin was packed into a column, washed with 10 column volumes of buffer A (50 mM Tris [pH 8.0], 1 M NaCl, 400 mM AcNH4, 20 mM imidazole, 10% glycerol, 2 mM ␤-mercaptoethanol) and then washed with 10 column volumes of buffer B (50 mM Tris [pH 8.0], 50 mM NaCl, 10% glycerol, 2 mM ␤-mercaptoethanol). Protein was eluted with buffer C (50 mM Tris [pH 8.0], 50 mM NaCl, 200 mM imidazole, 10% glycerol, 2 mM ␤-mercaptoethanol), and the PrimPol-containing fractions were loaded onto a heparin agarose column (MP Biomedicals), washed with 10 column volumes of buffer B, and eluted with a step gradient using buffer D (50 mM Tris [pH 8.0], 1 M NaCl, 10% glycerol, 2 mM ␤-mercaptoethanol). The PrimPol-containing fractions were further purified by size exclusion chromatography on a Superdex 200 (GE Healthcare) gel filtration column equilibrated in buffer E (50 mM Tris [pH 7.5], 300 mM NaCl, 10% glycerol, 1 mM dithiothreitol [DTT]). Total protein concentration was determined by UV absorbance at 280 nm. PrimPol active-site concentration was measured by presteady-state burst experiments as described below (22), and subsequent transient-state biochemical experiments were performed using active-site concentrations. PrimPol protein samples were stored at ⫺80°C. Nucleotides and oligonucleotides. Natural 2=-deoxynucleotides were purchased from GE Healthcare Biosciences (Pittsburgh, PA). The active triphosphate forms of the NRTIs were procured from commercial sources or synthesized in-house using established protocols. DNA oligonucleotides were purchased from Integrated DNA Technologies (Coraville, IA) and further purified using 20% polyacrylamide denaturing gel electrophoresis. The sequences of DNA primers and templates used for all kinetic experiments are listed in Table S1 in the supplemental material. Primers were 5=-32P labeled and annealed to the D45 template as previously described (22, 23). DNA-enzyme binding affinity assays. The equilibrium dissociation constant (Kd) of all DNA primer-template substrates for PrimPol were determined using electrophoretic mobility shift assays (EMSAs) as previously described (21, 24) but with minor modifications. Various active-site concentrations of PrimPol (0 to 5 ␮M) in 10 mM bis-Tris propane-HCl (pH 7.0), 1 mM DTT, and 10 mM MnCl2 or MgCl2 were incubated with 3 nM DNA primer-template substrate in a final volume of 20 ␮l for 60 min at room temperature. Then, the reaction mixtures were supplemented with 2 ␮l of 25% (wt/vol) Ficoll and resolved by electrophoresis on a 5%

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0.5⫻ Tris-borate-EDTA (TBE) polyacrylamide gel in 0.5⫻ TBE buffer at 150 V for 33 min at 4°C. The 5% 0.5⫻ TBE polyacrylamide gels were prerun at 150 V for 45 min at 4°C prior to sample loading. Reactions were visualized by phosphorimaging (Molecular Imager FX; Bio-Rad), and the fraction bound was quantified with using Quantity One, version 4.6.9, software (Bio-Rad). Burst polymerization kinetics. The overall rate-limiting step and active-site concentrations of PrimPol were determined by pre-steady-state burst experiments evaluating single-nucleotide incorporation using a Kintek Instruments RQF-3 rapid chemical quench apparatus. All concentrations are final after mixing. A slight excess of D23-D45 primer-template substrate (30 ␮M) relative to the total concentration of PrimPol (10 ␮M), accounting for the low affinity of PrimPol for the primer-template substrate, was rapidly mixed with a saturating concentration of the next incoming nucleotide, dCTP (200 ␮M), in reaction buffer (10 mM Bis-Tris propane [pH 7.0], 1 mM DTT) containing 10 mM MnCl2 at 37°C. The reactions were quenched with 0.3 M EDTA, pH 8.0, over a time course. The reaction mixtures were resolved by electrophoresis on a 20% polyacrylamide denaturing gel (8 M urea), visualized by phosphorimaging (Molecular Imager FX; Bio-Rad), and the extension of a 5=-32P-labeled D23-mer to D24-mer was quantified with Quantity One, version 4.6.9, software (Bio-Rad). The data were plotted using KaleidaGraph (Synergy Software) and fit to a burst equation, as follows: 关product兴 ⫽ A ⫻ 共1 ⫺ e⫺kobst兲 ⫹ A ⫻ kss ⫻ t, where A is the burst phase amplitude, kobs is the observed single exponential rate, kss is the steady-state rate, and t is the time. The percentage of active-site PrimPol averaged 40%. Subsequent transient-state biochemical experiments were performed using the activesite concentration. Single-nucleotide incorporation assays. Single-nucleotide incorporation experiments catalyzed by PrimPol were performed under singleturnover conditions to determine the equilibrium dissociation constant, Kd, and the maximum rate of incorporation, kpol, of each of the four nucleotides and all FDA-approved NRTIs. A preincubated solution of PrimPol (10 ␮M) and 5=-32P-labeled primer-template (300 nM) substrate in reaction buffer (10 mM Bis-Tris propane [pH 7.0], 1 mM DTT) was rapidly mixed with 10 mM MnCl2 and various concentrations of the next correct incoming nucleotide or NRTI at 37°C using an RQF-3 rapid chemical quench apparatus for the dNTPs or mixed manually in the case of the slower NRTI incorporation. The reaction mixtures were quenched and resolved by electrophoresis as described in the paragraph “Burst polymerization kinetics.” Plots of the concentration of product formed versus time obtained from single-turnover single-nucleotide incorporation experiments were fit to a single-exponential equation as follows: 关product兴 ⫽ A ⫻ 共1 ⫺ e⫺kobst兲, where A is amplitude, kobs is the observed first-order rate constant for the incoming dNTP or NRTI, and t is the time. When two products were observed, as was the case for dATP incorporation, the sum of the first dATP incorporation (n ⫹ 1) and the second dATP misincorporation (n ⫹ 2) was used to determine the total amount of product formed and fit to a double-exponential equation to determine the rate of correct incorporation and misincorporation, respectively, as follows: 关product兴 ⫽ A1 ⫻ 共1 ⫺ e⫺kobs1t兲 ⫹ A2 ⫻ 共1 ⫺ e⫺kobs2t兲. For all nucleotides and NRTIs except zidovudine triphosphate (AZTTP), to generate the dissociation constant (Kd) of the natural nucleotide/ NRTI for the PrimPol-DNA complex and the maximum rate of nucleotide/NRTI incorporation (kpol), a plot of kobs versus nucleotide/NRTI concentration was fit to a quadratic equation (2):

关dNTP兴 ⫽ 0.5共Kd ⫹ 关dNTP兴 ⫹ 关kpol兴兲 ⫺ 0.5兵共Kd ⫹ 关dNTP兴 ⫹ 关kpol兴兲2 ⫺ 4关dNTP兴关kpol兴其1⁄2 Concerning AZT-TP, there was no dependence of the rate on NRTI concentration, but, rather, the amplitude of the AZT-TP incorporation was concentration dependent. Thus, a plot of amplitude versus AZT-TP concentration was fit to a quadratic equation to generate the dissociation constant (Kd) and the maximum rate of incorporation (kpol). The concen-

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FIG 2 Transient-state kinetic analysis of polymerization by PrimPol. Singlenucleotide incorporation of dCTP into the DNA primer-template D23-D45 under pre-steady-state burst conditions as catalyzed by PrimPol demonstrates biphasic kinetics: a burst of product formation at a rate of 0.36 ⫾ 0.04 s⫺1 followed by a slower, linear phase with a rate of 0.046 ⫾ 0.006 s⫺1.

FIG 1 The binding affinity of PrimPol for the DNA primer-template substrates as determined by EMSA. Affinities of PrimPol for the DNA primertemplate substrates (where D20, D22, D23 and D30 are primers and D45 is the template) (see Table S1 in the supplemental material for sequences) used in the study were determined by gel mobility shift assay. DNA primer-template (3 nM) was mixed with 0 to 5 ␮M PrimPol protein and separated by electrophoresis. Quantitative analysis of autoradiograms was used to calculate the fraction of DNA primer-template bound to determine the Kd.

tration dependence of the amplitude involving AZT-TP incorporation has also been observed for mtDNA Pol ␥ (2). All data were fit to a quadratic function rather than a hyperbola because the concentration of PrimPol (10 ␮M) used in the assay necessary to overcome the weak affinity for DNA primer-template substrate was comparable to the nucleotide/NRTI Kd values.

RESULTS

PrimPol’s affinity for the DNA primer-template substrate. Recent studies indicate that PrimPol has a weaker affinity for DNA primer-templates than other polymerases and a preference for the cofactor manganese (20, 21, 24). It was important to provide a quantitative assessment of equilibrium dissociation constants for DNA binding as an essential component of defining appropriate burst and single-turnover conditions for incorporation studies. Therefore, we sought to determine PrimPol’s affinity for the radiolabeled DNA primer-templates used in this study (see Table S1 in the supplemental material). Electrophoretic mobility shift assays (EMSAs) indicate PrimPol-DNA binding affinities ranging from 340 to 720 nM (Fig. 1 and Table 1) in the presence of manganese, which is approximately 50-fold weaker than the DNA binding affinity of RT (22) and consistent with reported weak DNA affinity. In the presence of magnesium we obtained a high DNA binding affinity value of 8 ␮M, which is 11- to 24-fold weaker than in the presence of manganese. In light of these observations and with the understanding that PrimPol

TABLE 1 Equilibrium dissociation constants for PrimPol binding to DNA primer-templates Primer-templatea

Kd (nM)

D20-D45 D22-D45 D23-D45 D30-D45

720 ⫾ 20 380 ⫾ 10 410 ⫾ 10 340 ⫾ 10

a

See Table S1 in the supplemental material for sequences.

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and other TLS polymerases prefer manganese (25), we pursued all further kinetic analyses in the presence of manganese. Transient-state kinetic analysis to discern the mechanism of polymerization. In light of its recent discovery, a steady-state kinetic analysis was performed (20). While this approach provides an important initial characterization of the enzyme’s catalytic activity, it comprises a complex mix of rate constants that reflect the rate-limiting step. To gain mechanistic insight into the events occurring at the enzyme active site requires transient-state kinetic analysis (26). A transient-state kinetic approach reveals important parameters describing how substrates are recognized and utilized at the enzyme level. Thus, we conducted a transient-state kinetic analysis to investigate the molecular mechanism of PrimPol polymerization. We performed single-nucleotide incorporation under presteady-state burst conditions of 10 ␮M PrimPol protein and 30 ␮M DNA primer-template to achieve ⬎98% PrimPol bound to the DNA primer-template at the start of the reaction. Using a KinTek RQF-3 chemical quench, the DNA-PrimPol complex was rapidly mixed with saturating concentrations of incoming dNTP and manganese chloride in the reaction buffer. The elongated primer-template DNA product was quantitated and plotted as a function of time, and biphasic kinetics were observed (Fig. 2). The observation of a burst indicates that chemical catalysis is faster, with the slower step of product release being overall rate limiting. The data were fit to a burst equation to yield a burst rate of 0.36 ⫾ 0.04 s⫺1, which describes the rate of nucleotide incorporation during the first enzyme turnover, followed by a slower, linear phase with a rate of 0.046 ⫾ 0.006 s⫺1 that reflects the rate of subsequent enzyme turnovers and is limited by product dissociation. Product release is also the overall rate-limiting step for polymerization by HIV RT (22) and mtDNA Pol ␥ (27), among others. The burst amplitude defines the PrimPol active-site concentration and was used in designing the subsequent single-turnover studies to measure natural nucleotide and NRTI incorporation. Kinetic mechanism of natural nucleotide incorporation as catalyzed by PrimPol. To define the mechanistic basis of nucleotide selectivity, transient-state kinetic analysis is necessary to discern the individual rate constants, kpol, the maximum rate of incorporation, and Kd, the binding affinity, and the efficiency (kpol/Kd) that govern incorporation of the natural nucleotides by PrimPol. To more precisely define the rate of chemical catalysis for nucleotide incorporation, single-enzyme turnover experiments were performed in which enzyme is in excess of DNA prim-

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FIG 3 Transient-state kinetic analysis for correct nucleotide incorporation as catalyzed by PrimPol. The concentration dependence of correct nucleotide incorporation on rate was fit to a quadratic equation to generate rate constants kpol and Kd for dATP, dTTP, dCTP, and dGTP, as indicated.

er-template. The amount of elongated DNA product formed as a function of increasing nucleotide concentration defines these key kinetic parameters for nucleotide selectivity. We performed single-nucleotide incorporation studies under single-turnover conditions using a KinTek RQF-3 chemical quench to rapidly mix 10 ␮M PrimPol protein and 300 nM DNA primer-template with various concentrations of the next correct incoming nucleotide and manganese chloride in reaction buffer. The concentration dependence of correct nucleotide incorporation on rate is hyperbolic for all natural nucleotides (Fig. 3). Similar to other polymerases (HIV RT and T7 polymerase), this suggests that a rate-limiting conformational change following nucleotide binding precedes chemical catalysis. PrimPol incorporates the natural nucleotides at rates ranging from 0.307 to 4.2 s⫺1, with affinities ranging from 5 to 50 ␮M (Table 2). Overall, PrimPol incorporates dATP 10- to 20-fold more efficiently than the other

TABLE 2 Kinetic parameters and efficiency of correct nucleotide incorporation by PrimPol dNTP

kpol (s⫺1)

Kd (␮M)

Efficiency (kpol/Kd, ␮M⫺1 s⫺1)

dATP dTTP dCTP dGTP

4.2 ⫾ 0.4 3.2 ⫾ 0.1 1.43 ⫾ 0.04 0.307 ⫾ 0.005

5⫾2 50 ⫾ 6 18 ⫾ 2 7.0 ⫾ 0.5

0.840 0.064 0.079 0.044

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three nucleotides, followed by dCTP ⬎ dTTP ⬎ dGTP. The observed efficiencies are similar in range and magnitude to the natural nucleotide incorporation efficiencies observed for the Y-family polymerases Pol ␩, Pol ␬, Pol ␫, and Rev1 (1). Strikingly, in our dATP incorporation experiments we observed that PrimPol misincorporates a second dATP opposite a template dG in the n⫹2 position at a significant rate, even before complete turnover of the primer-template (Fig. 4). PrimPol misincorporates dATP opposite the n⫹2 template dG at a rate of 1.03 ⫾ 0.06 s⫺1 with an affinity of 7 ⫾ 1 ␮M, for an efficiency of 0.147 ␮M⫺1 s⫺1 (Table 3). A template dG also occupied the n⫹2 position when dTTP incorporation was evaluated; however, significant incorporation of dTTP opposite dG was not observed in the time frame examined at concentrations 6 times the Kd for dTTP. That PrimPol misincorporates dATP opposite a template dG to form a purine-purine mismatch and that it does so more efficiently than correctly inserting the other nucleotides are supported by observations of other investigators that PrimPol performs promiscuous DNA synthesis most efficiently when it utilizes dATP (21) and is most error prone when it is copying a template dG with cofactor manganese (20). In the context of its role as a TLS polymerase, PrimPol is reported to misincorporate dATP opposite the oxidative lesion 8oxoG 1.5-fold more efficiently than it performs error-free insertion of dCTP (15) although a later steady-state study reports that PrimPol accurately bypasses 8oxoG 4-fold more efficiently than it misinserts dATP (20).

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FIG 4 PrimPol readily misincorporates dATP at n ⫹ 2 (22 nt) opposite a template dG below and above the Kd for dATP. Correct dATP incorporation is observed

at n ⫹ 1 (21 nt) opposite a template dT while dATP misincorporation is observed at n ⫹ 2 (22 nt) opposite a template dG. PrimPol misincorporates at 1 ␮M (15 and 60 s), 5 ␮M (3, 10, and 30 s), and 25 ␮M dATP (5 s), even before all of substrate n (20 nt) is turned over.

NRTI incorporation as catalyzed by PrimPol. Previous work in our lab has evaluated the kinetic mechanism and NRTI-associated toxicity in the mitochondria using detailed mechanistic studies to examine incorporation of NRTIs by HIV RT and mtDNA Pol ␥ (9, 28–35). With the recent discovery of PrimPol in the mitochondria (15), an obvious question is whether additional mechanisms for NRTI-mediated toxicity may be plausible. The transient-kinetic studies defining the molecular mechanism of natural nucleotide incorporation described above laid the foundation for subsequent evaluation of all eight FDA-approved NRTIs. Using the incorporation efficiencies of the natural nucleotides as a reference, we sought to evaluate the enzyme’s propensity to utilize NRTIs. Thus, we examined the kinetics of incorporation as catalyzed by PrimPol for each FDA-approved NRTI: zidovudine triphosphate (AZT-TP), didanosine (ddI; the active metabolite is ddATP), zalcitabine TP (ddCTP), stavudine TP (d4T-TP), lamivudine TP [(⫺)-3TC-TP], abacavir (ABC; the active metabolite is carbovir-TP [CBV-TP]), tenofovir diphosphate (TFV-DP), and emtricitabine TP [(⫺)-FTC-TP]. Measurement of kpol and Kd to calculate the efficiency (kpol/Kd) for each nucleoside analog triphosphate and determine selectivity relative to the natural nucleotide substrate was performed under single-turnover conditions similar to those used for the natural nucleotide but over a longer time course. Of all eight FDA-approved NRTIs, PrimPol efficiently incorporates four of them: ddATP, AZT-TP, ddCTP, and CBV-TP (Table 4 and Fig. 5). The concentration dependence of NRTI incorporation on rate is hyperbolic for ddCTP and ddATP, and the concentration dependence of AZT incorporation on amplitude is hyperbolic, as has been observed previously for mtDNA Pol ␥ (2) (Fig. 6). For CBV-TP, we did not observe a hyperbolic dependence indicating that the rate-limiting step has changed. Overall, the NRTI affinities (Kd values) were in the low-micromolar range, similar to those of the natural nucleotides, and incorporation was substantially slower. While PrimPol preferred the natural nucleotides versus all NRTIs, it most efficiently incorporated CBV-TP, followed by ddCTP ⬎ ddATP ⬎ AZT-TP.

ddCTP and ddATP are the simplest nucleoside analogues that differ from their dNTP counterparts only in lacking the deoxyribose 3=-hydroxyl, but they are the two NRTIs with the highest clinical toxicities (36). PrimPol has similar affinities for ddCTP and ddATP as for dCTP and dATP, respectively, but incorporates the NRTIs 40- and 300-fold more slowly than the natural substrates. PrimPol most efficiently incorporated CBV-TP. Though the concentration dependence of the rate of CBV-TP incorporation was not hyperbolic, we observed that the maximum rate of CBV-TP incorporation was 0.013 ⫾ 0.002 s⫺1 at saturating concentrations of CBV-TP (300 ␮M). This rate was similar to that of ddATP incorporation. A quadratic fit of the rate data estimates PrimPol’s binding affinity for CBV-TP to be approximately 1 ␮M. This is an approximation because the high concentration of PrimPol necessary to saturate binding of DNA primer-template makes a precise measurement of the low value technically challenging. Single-turnover experiments were performed at lower concentrations of enzyme comparable to the observed Kd (2 ␮M); however, the reaction rate was limited by binding of the DNA primer-template substrate. AZT-TP, a dTTP analog with the 3=-hydroxyl replaced by a 3=-azido group, presents with side effects, including myopathy, cardiomyopathy, and anemia, that initially implicated the mitochondria in NRTI toxicity (37). PrimPol incorporates AZT-TP with an efficiency of 0.0001 ␮M⫺1 s⫺1, which is 9-, 20-, and 130-fold lower than that of ddATP, ddCTP, and CBV-TP, respectively.

TABLE 3 Kinetic parameters describing correct dATP incorporation opposite template dT at n ⫹ 1 and dATP misincorporation opposite template dG at n ⫹ 2

TABLE 4 Kinetic parameters for NRTI incorporation as catalyzed by PrimPol

Position of dATP incorporation n ⫹ 1 (21 nt) match template dT n ⫹ 2 (22 nt) mismatch template dG

kpol (s )

Kd (␮M)

Efficiency (kpol/Kd, ␮M⫺1 s⫺1)

4.2 ⫾ 0.4

5⫾2

0.840

1.03 ⫾ 0.06

7⫾1

0.147

⫺1

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DISCUSSION

In the present study, we performed the first transient-state kinetic analysis of polymerization as catalyzed by PrimPol and utilized this molecular mechanism of PrimPol catalysis to assess its potential as a perpetrator of NRTI-associated toxicity. The magnitude of PrimPol’s NRTI incorporation efficiencies has the potential to incite genomic damage and result in off-target toxicities, particularly in the mitochondria. The discontinued ribonucleoside BMS986094 was incorporated by human mitochondrial RNA polymerase with an efficiency of 0.00019 ␮M⫺1 s⫺1, and other

NRTI

kpol (s⫺1)

Kd (␮M)

Efficiency (kpol/Kd, ␮M⫺1 s⫺1)

ddATP AZT-TP ddCTP CBV-TP

0.0138 ⫾ 0.0008 0.0040 ⫾ 0.0008 0.033 ⫾ 0.002 0.013 ⫾ 0.002

15 ⫾ 3 38 ⫾ 6 18 ⫾ 4 ⬃1a

0.0009 0.0001 0.002 0.013a

a

See the discussion in the text.

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FIG 5 PrimPol incorporates four of the eight FDA-approved NRTIs. Depicted here are the structures of the metabolically active triphosphate forms of the four NRTIs: carbovir triphosphate (CBV-TP) which is the active metabolite of abacavir, didanosine triphosphate, which is the active metabolite of didanosine, zalcitabine TP, and zidovudine TP.

ribonucleoside antivirals that resulted in mitochondrial dysfunction were incorporated with efficiencies ranging from 10⫺1 to 10⫺6 ␮M⫺1 s⫺1 (13). The data suggest the potential for PrimPolmediated NRTI-associated toxicity, particularly considering that patients undergo lifelong antiviral treatment to control viral loads. To contextualize the potential for PrimPol-mediated NRTIassociated toxicity, an evaluation of PrimPol’s discrimination, a measure of the ability of a polymerase to select the natural nucleotide versus an NRTI, relative to that for mtDNA Pol ␥ and HIV RT is presented in Table 5. The ideal NRTI would have a lower level of discrimination for HIV RT and a much higher level for mtDNA Pol ␥ and PrimPol. The kinetic profile for antiviral NRTIs utilized by PrimPol is distinct from that of mtDNA Pol ␥. Similar to mtDNA Pol ␥, PrimPol efficiently incorporates the highly toxic nucleoside analog ddCTP, as well as ddATP, but it also efficiently incorporates AZT-TP and CBV-TP, two nucleoside analogs that are relatively highly discriminated against by mtDNA Pol ␥. AZT-TP is a poor substrate for and highly discriminated against by mtDNA Pol ␥ (2) but presents with adverse events indicative of mitochondrial dysfunction, such as myopathy, cardiomyopathy, and anemia (37). Here, we observe that PrimPol more readily incorporates AZT-TP than mtDNA Pol ␥ by a factor of 58, providing insight into possible mechanisms of AZT-TPinduced toxicity and the resulting mitochondrial dysfunction. PrimPol also discriminates against CBV-TP 270,000-fold less than mtDNA Pol ␥. Though we observed a change in the rate-limiting step concerning CBV-TP incorporation, the maximum rate of CBV-TP incorporation by PrimPol was 0.013 ⫾ 0.002 s⫺1, an order of magnitude faster than 0.0018 ⫾ 0.0001 s⫺1 (2), the rate of mtDNA Pol ␥ CBV-TP incorporation. Furthermore, mtDNA Pol ␥ (8) is highly discriminatory against CBV-TP incorporation, and there is limited information regarding the B-, X-, and Y-family polymerases (1) while PrimPol’s relatively low level of CBV-TP discrimination is novel and may provide insight into the lifethreatening hypersensitivity in a subset of patients upon treat-

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ment with CBV-TP (38). Of the four NRTIs incorporated by PrimPol, only CBV-TP is a better substrate for PrimPol than HIV RT by an order of magnitude. PrimPol is better able to discriminate against the highly toxic NRTIs ddATP and ddCTP than mtDNA Pol ␥ and HIV RT by 233- and 187-fold and by 14- and 3-fold, respectively, which indicates a low potential for PrimPol-mediated toxicity of these two analogues. Importantly, AZT-TP and CBV-TP, the NRTIs for which PrimPol has lower levels of discrimination than mtDNA Pol ␥, are both commonly administered in the treatment of HIV today. AZT-TP is a component of the fixed-dose combinations lamivudine-zidovudine (Combivir) and abacavir sulfate-lamivudine-zidovudine (Trizivir), and CBV-TP is also a component of Trizivir, abacavir-lamivudine (Epzicom), and a recently FDA-approved combination therapy with dolutegravir and lamivudine (Triumeq). Although it may be assumed that PrimPol performs fewer incorporation events than the primary replicative nuclear polymerases ␣, ε, and ␦ and mtDNA Pol ␥, NRTI incorporation by PrimPol at lesions, including 8oxoG (15, 20, 21), abasic sites (15), (6-4)pp (16, 21), and CPDs (16), can result in DNA breaks, genomic instability, and ultimately apoptosis (39). Our findings highlight one possible alternative mechanism of antiviral toxicity for consideration in the clinical use of these drugs and future nucleoside analog development. It was somewhat surprising that PrimPol did not incorporate TFV-DP, a dATP nucleoside analog, given that it most efficiently incorporated dATP of all the natural nucleotides (Table 2). Clinically administered in the treatment of HIV and hepatitis B virus, tenofovir has long been known to induce toxic side effects particularly in the mitochondria of patients’ kidneys (40, 41), but the underlying mechanism is not definitively known. Moreover, the relatively abundant expression of PrimPol mRNA in mouse kidneys (15) prompted us to investigate the role PrimPol might assume in contributing to the long-observed tenofovir toxicity. We

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Polymerization and NRTI Incorporation by Human PrimPol

FIG 6 Transient-state kinetic analysis for NRTI incorporation as catalyzed by PrimPol. The concentration dependence (amplitude for AZT) of NRTI incorporation on rate was fit to a quadratic equation to generate rate constants kpol and Kd for ddATP, AZT-TP, ddCTP, and CBV-TP, as indicated.

observed that PrimPol does not incorporate TFV-DP under the conditions examined at concentrations of up to 2 mM. In addition to discrimination, a key feature of NRTI antiviral activity is HIV RT’s lack of proofreading exonuclease activity to prevent removal of an incorporated NRTI. Importantly and in contrast to mtDNA Pol ␥, PrimPol also lacks a proofreading mechanism for excision of incorporated NRTIs. Understanding the consequences of PrimPol’s inability to remove incorporated NRTIs in the context of its role in restarting replication forks and bypassing lesions, particularly in the mitochondrial genome, is TABLE 5 PrimPol’s discrimination relative to that of mitochondrial polymerase ␥ and HIV RT, the primary off-target and target polymerases for NRTIs, respectively Discrimination (efficiencydNTP/efficiencyNRTI)a Analog

PrimPol

Pol ␥

HIV RT

ddATP AZT-TP ddCTP CBV-TP

933 640 39.5 3.4

4.0b 37,103b 2.9c 902,777c

5d 2.5e 15.7f 34g

a

Efficiency of the polymerase to select the natural nucleotide (dNTP) versus an NRTI. Reference 2. Reference 9. d Reference 45. e Reference 33. f Reference 34. g Reference 46. b c

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important for future investigations (15, 16, 20, 21). Furthermore, it remains to be understood what role auxiliary proteins (e.g., replication protein A [RPA], PCNA, and mitochondrial singlestranded binding protein [mtSSB]) (14, 42) or the C-terminal zinc finger domain, implicated in PrimPol’s fidelity (21), play in regulating polymerization and nucleotide selectivity. The mtDNA Pol ␥ holoenzyme contains a catalytic subunit, Pol ␥A, and a dimeric accessory subunit, Pol ␥B, that enhance activity of the catalytic subunit (27, 43). Obtaining ternary X-ray structures of PrimPol in complex with the natural nucleotides and NRTIs will provide structural insights into nucleotide selectivity and PrimPol’s incorporation of the relevant NRTIs relative to mtDNA Pol ␥ and HIV RT, facilitating the design of more selective and safe nucleoside analogs (44). Moreover, PrimPol’s ability to utilize NTPs indicates that it may also mediate ribonucleoside triphosphate analog toxicities (13) and warrants further investigation. The transient-state kinetic analyses presented here are the first to define the molecular mechanism of polymerization and nucleotide incorporation by PrimPol. We find that the chemical catalysis by PrimPol is most likely limited by a conformational change that governs nucleotide selectivity, and product release of the elongated primer-template DNA was identified as the overall ratelimiting step in polymerization. In light of PrimPol being only the second known polymerase in the mitochondria and given our involvement in elucidating the kinetic mechanisms of NRTIassociated toxicities, we subsequently evaluated PrimPol’s incorporation of all eight FDA-approved NRTIs. We discover that

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PrimPol can incorporate four NRTIs, zalcitabine, didanosine, zidovudine, and abacavir, a profile of NRTI incorporation that is distinct from that of mtDNA Pol ␥ and highlights one possible alternative mechanism of antiviral toxicity. The data suggest that NRTI incorporation into nuclear and mitochondrial DNA by PrimPol is possible in vivo. Our findings and the kinetic analysis presented here provide a foundation for future investigations of the mechanism of antiviral and anticancer nucleoside analog triphosphate off-target effects.

14.

15.

16.

ACKNOWLEDGMENTS We are grateful to the National Institutes of Health for research support (GM049551) and to the National Institute of Allergy and Infectious Diseases for fellowship support for A.C.M. (F31AI116322). The funding agencies had no role in study design, data collection or interpretation of the results, or the decision to submit the work for publication. We declare that we have no competing financial interests.

17.

18.

FUNDING INFORMATION HHS | National Institutes of Health (NIH) provided funding to Karen S. Anderson under grant number GM049551. HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID) provided funding to Andrea Christine Mislak under grant number F31AI116322.

19.

20.

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