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J. Biol. Chem., Vol. 276, Issue 26, 23832-23837, June 29, 2001

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Insights into the Molecular Mechanism of Mitochondrial Toxicity by AIDS Drugs^*

Joy Y. Feng^§¶, Allison A. Johnson^¶, Kenneth A. Johnson, and Karen S. Anderson^**

From the  Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut 06510 and  Institute for Cellular and Molecular Biology, University of Texas, Austin, Texas 78712

Received for publication, February 6, 2001, and in revised form, April 25, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several of the nucleoside analogs used in the treatment of AIDS exhibit a delayed clinical toxicity limiting their usefulness. The toxicity of nucleoside analogs may be related to their effects on the human mitochondrial DNA polymerase (Pol ), the polymerase responsible for mitochondrial DNA replication. Among the AIDS drugs approved by the FDA for clinical use, two are modified cytosine analogs, Zalcitabine (2',3'-dideoxycytidine (ddC)) and Lamivudine (-D-(+)-2',3'-dideoxy-3'-thiacytidine (()3TC])). ()3TC is the only analog containing an unnatural L() nucleoside configuration and is well tolerated by patients even after long term administration. In cell culture ()3TC is less toxic than its D(+) isomer, (+)3TC, containing the natural nucleoside configuration, and both are considerably less toxic than ddC. We have investigated the mechanistic basis for the differential toxicity of these three cytosine analogs by comparing the effects of dideoxy-CTP), (+)3TC-triphosphate (TP), and ()3TC-TP on the polymerase and exonuclease activities of recombinant human Pol . This analysis reveals that Pol  incorporates ()3TC-triphosphate 16-fold less efficiently than the corresponding (+)isomer and 1140-fold less efficiently than dideoxy-CTP, showing a good correlation between incorporation rate and toxicity. The rates of excision of the incorporated analogs from the chain-terminated 3'-end of the DNA primer by the 3'-5'-exonuclease activity of Pol  were similar (0.01 s¹) for both 3TC analogs. In marked contrast, the rate of exonuclease removal of a ddC chain-terminated DNA occurs at least 2 orders of magnitude slower, suggesting that the failure of the exonuclease to remove ddC may play a major role in its greater toxicity. This study demonstrates that direct analysis of the mitochondrial DNA polymerase structure/function relationships may provide valuable insights leading to the design of less toxic inhibitors.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The in vitro and in vivo mechanisms of cytotoxicity of the nucleoside analogs used as anti-HIV¹ drugs are not well understood. Various forms of toxicity, including peripheral neuropathy, myopathy, and pancreatitis, are observed after long term use of nucleoside analogs 3'-azido-3'-deoxythymidine (AZT) (1), 2',3'-dideoxyinosine (ddI) (2), 2',3'-dideoxycytidine (ddC) (3, 4), and 2',3'-didehydro-3'-deoxythymidine (5). The profile for toxicity varies with each analog. For instance, whereas all of the three, AZT, ddI, and ddC, exert cytotoxic effects on human muscle cells and induce functional alternation of mitochondria, only AZT, but not ddI and ddC, can induce a myopathy in HIV-1-infected patients (6). The fact that toxicity of different drugs is cell-, tissue-, and organ-specific (6) indicates that the differences in subcellular bioavailability and pharmacokinetic profile involving the activation and deactivation of the drugs can all contribute to the clinical toxicity. Many mechanisms have been proposed for the origins of nucleoside analog toxicity, but the most obvious sites of action are the enzymes responsible for host cell DNA replication and repair: DNA polymerases , , and , which are responsible for chromosomal DNA replication; DNA polymerase , which is involved in DNA repair; and DNA polymerase  (Pol ), which carries out mitochondrial DNA synthesis (7-9). The toxicity toward proliferating tissues, such as the well documented bone marrow toxicity of AZT, is believed to be due to the inhibition of one or more of the chromosomal DNA polymerases (10). The toxicity manifested in many nonproliferating tissues is thought to result more specifically from the effects of inhibition of DNA Pol  by nucleoside analogs (10-14). In support of this postulate, the recognizable clinical syndromes associated with nucleoside analog toxicity are reminiscent of mitochondrial dysfunctions associated with mitochondrial genetic disorders (12, 15-18); moreover, prolonged exposure of the cell to nucleoside analogs results in decreased mitochondrial DNA synthesis, decreased cell viability, and changes in the mitochondrial ultrastructure (19). Some in vitro studies have shown that for many of the nucleoside analogs tested, Pol  is more sensitive than any of the other DNA polymerases (20), but there are conflicting reports (19, 21). Earlier studies to assess the interaction of nucleoside analogs have used partially purified preparations of native enzyme. However, recently, highly purified homogenous preparations of recombinant human Pol  have become available through cloning and overexpression in a baculovirus expression system (22, 23-26). Human DNA Pol  is composed of a catalytic subunit of 140 kDa and an accessory subunit of ~54 kDa (22). The large catalytic subunit provides both polymerase and 3'-5'-exonuclease activities (23, 24), whereas the smaller accessory subunit (25) facilitates substrate binding and dNTP incorporation, thereby improving processivity (26). The cloning, overexpression, purification, and reconstitution of the human Pol  has now afforded a highly purified enzyme preparation, allowing a more rigorous analysis of the reactions governing nucleotide selectivity during incorporation and removal by the proofreading exonuclease (27).

Among the anti-HIV nucleoside analogs used clinically, the -L-()-2',3'-dideoxy-3'-thiacytidine (()3TC, lamivudine) is the only one with the unnatural L configuration, and most interestingly, it has been shown to be more potent and less toxic than the D isomer, (+)3TC (Fig. 1). Effective doses of ()3TC are well tolerated during therapy (28-30), and there is little or no evidence of mitochondrial injury (13, 30). In contrast, ddC is one of the more toxic of the nucleoside analogs approved for AIDS therapy by the Federal Drug Administration. The mechanistic basis for observed stereochemical differential toxicity for the two 3TC isomers and enhanced toxicity of ddC are not understood, and a number of factors may come into play. The differences in toxicity may be a combination of factors including cellular uptake (31, 32), transport (10), metabolic activation (31-33), incorporation (31, 33, 34), and removal or degradation from the system (10, 27, 33, 35, 36). A quantitative analysis of each factor is clearly required.

The mechanism of inhibition of DNA Pol  by the nucleoside analogs involves in vivo phosphorylation to form the triphosphate and subsequent incorporation into the DNA primer strand, resulting in chain termination. Thus in vitro studies have focused on evaluating the triphosphate form of analogues. Earlier steady-state kinetic studies have examined the effect of the (+) and ()3TC-TP incorporation into DNA by Pol  (21, 31) and suggest a 5-fold difference in K_i values. However, these steady-state kinetic studies were conducted under conditions that employed a low concentration of Pol , and according the to the K_d value of 35 nM for formation of the heterodimer (26), the polymerase would not have existed as the holoenzyme complex. Indeed, the significance of earlier studies using native purified enzyme is unclear due to the variable activity resulting from the loss of the small subunit during purification.

Similar to many of the DNA polymerases, Pol  has 3'-5'-exonuclease activity, which is believed to perform a proofreading function (22, 37). It is possible that the failure of 3'-5'-exonuclease of Pol  to efficiently remove chain terminators may play a role in mitochondrial toxicity of nucleoside analogs. However, discrepancies exist in the literature concerning the efficiency of the exonuclease in removal of these analogs and correlation to drug toxicity. It has been proposed that efficient removal of 3'-terminal ()3TC-monophosphates (MPs) by the 3'-5'-exonuclease activity of Pol  could be responsible for the low toxicity of ()3TC (33); however, similar rates of exonuclease removal were reported for the excision of dCMP, ddC-MP, and ()3TC-MP from the 3'-terminus of a DNA primer, even though there are significant differences in mitochondrial toxicity (33). This assessment was made with a partially purified preparation of Pol  that may not represent the holoenzyme complex due to the loss of the accessory subunit. Moreover, a novel cytosolic exonuclease was isolated from H9 cells that can remove chain-terminated dNMP and may be a contaminating activity in partially purified Pol  (27). Finally, in cell culture studies, ddC showed higher inhibition of mitochondrial DNA synthesis than both the (+) and ()3TC (31, 39). Thus, a detailed study using pure human Pol  is needed.

Previously, we have studied the stereochemical selectivity between the (+) and ()3TC triphosphate for inhibition of HIV-1 reverse transcriptase (RT) (40). We have also determined the detailed kinetic properties of human mitochondrial DNA polymerase for both the catalytic subunit and the holoenzyme (23, 26). We now describe our transient kinetic analysis of incorporation and excision of dideoxycytidine triphosphate (ddCTP), (+)3TC, and ()3TC, with the goal of understanding how these clinically important modified cytosine analogs interact with the Pol  holoenzyme complex in comparison to dCTP. An in-depth understanding of the mechanism of inhibition of the DNA  polymerase is important in overcoming problems associated with cytotoxicity with nucleoside analogs. Knowledge of the mechanistic similarities and differences may identify key features important in selectivity for the interaction with the viral DNA polymerase (HIV-1 reverse transcriptase) over the DNA  polymerase and thereby assist in the design of better AIDS drugs.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The large and small subunits of Pol  were purified as described (23, 26). The protein concentration was determined spectrophotometrically at 280 nm, with the extinction coefficients 234,420 and 71,894 M¹ cm¹ for the large and small subunits, respectively. A synthetic DNA duplex, 23·45 bp (40), was used in which the next correct base for incorporation was dCTP. Concentrations of the oligonucleotides were estimated spectrophotometrically at 260 nm using the calculated extinction coefficients 226,750 M¹ cm¹ and 491,960 M¹ cm¹ for the 23- and 45-mer, respectively. Before annealing, the primer was 5'-labeled with T4 polynucleotide kinase and [-³²P]ATP. The dCTP and ddCTP were purchased from Sigma, and the (+) and ()3TC-TP isomers were the kind gift of Dr. Raymond Schinazi at Emory University.

Experiments to measure polymerization kinetics were performed as described (40). Rapid quench experiments were done using KinTek Instruments Model RQF-3 rapid quench-flow apparatus (41). Unless mentioned otherwise, all concentrations refer to concentrations during reactions after mixing on the quench apparatus. Typically, the experiments were done by first incubating the duplex DNA (250 nM) with pre-mixed large (67 nM) and small subunits (440 nM) in reaction buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.8). A sample of this solution (15 µl) was rapidly mixed with an equal volume of a solution of dCTP analogs (at variable concentrations) and MgCl₂ (2.5 mM) in reaction buffer. The reactions were quenched by ~67 µl of 0.5 M EDTA at pH 8.0. All reactions were performed at 37 °C. Single nucleotide incorporation was monitored by extension of 5'-³²P-labeled 23-24-bp oligonucleotides. All reaction mixtures were analyzed on 20% denaturing polyacrylamide sequencing gels (8 M urea), imaged on a Bio-Rad GS-525 Molecular Imager System, and quantified using Molecular Analysis (Bio-Rad). Data were fitted by nonlinear regression using the program KaleidaGraph (Synergy Software). The pre-steady-state burst experiments (Fig. 3) were fitted to a burst equation: [product] = A[1  exp(-k_obsdt) + k_sst], where A represents the amplitude of the burst that correlates with the concentration of enzyme in active form, k_obsd is the observed first-order rate constant for dNTP incorporation, and k_ss is the observed steady-state rate constant.

The dissociation constant, K_d, for dNTP binding to the complex of Pol  and 23·45-bp DNA duplex is calculated by fitting the data into the following hyperbolic equation: k_obsd = (k_pol × [dNTP])/(K_d + [dNTP]), where k_pol is the maximum rate of dNTP incorporation, [dNTP] is the corresponding concentration of dNTP, and K_d is the equilibrium dissociation constant for the interaction of dNTP with the enzyme-DNA complex.

The 24-bp primers were prepared by incorporating the corresponding dCTP analogs into the 23-bp DNA. A mixture of 23·45 DNA duplex (1000 pmol), HIV-1 RT (300 pmol), dCTP analogs (5-50 µM), and 10 mM MgCl₂ in reaction buffer (50 mM Tris-HCl, 50 mM NaCl, pH 7.8) was incubated at 37 °C for 10-30 min. The 24-mer DNA products were purified by gel electrophoresis.

The exonuclease activity was studied by measuring the rate of formation of the cleavage products in the absence of dCTP or dCTP analogs. Products formed from the early time points were plotted as a function of time. The slope of the line was divided by the active enzyme concentration in the reaction, and a k_exo for exonuclease activity was obtained. The reaction was initiated by adding MgCl₂ (2.5 mM) into a solution of catalytic subunit (40 nM), accessory subunit (270 nM), and 1500 nM 24·45-bp DNA duplex in reaction buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.8) and quenched with 0.3 M EDTA. For exonuclease activity of the catalytic subunit, 16 nM catalytic subunit and 1000 nM 24·45-bp DNA duplex were used.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The underlying mechanisms of toxicity were investigated by examining the kinetics of the two different reactions catalyzed by human mitochondrial DNA Pol : DNA-dependent DNA synthesis and 3'-5'-exonuclease cleavage. This analysis was carried out by examining the single nucleotide incorporation using synthetically prepared dCTP, ddCTP, and two modified deoxycytidine analogs, (+)3TC-TP and ()3TC-TP, opposite a template deoxyguanosine for DNA-dependent DNA polymerization by the recombinant Pol  holoenzyme complex. The exonuclease cleavage was investigated by evaluating the excision of dCMP, ddCMP, (+)3TC-MP, ()3TC-MP, and dTMP from the 3'-terminus of the corresponding DNA 23- or 24-mer primer by the catalytic subunit alone and the holoenzyme complex of Pol . All of the incorporation studies were carried out at protein concentrations high enough to maintain the holoenzyme Pol  complex and on a time scale short enough to follow a single turnover of enzyme.

Using a transient kinetic approach, we determined the rate of polymerization (k_pol), the dissociation constant for the ground state nucleotide binding (K_d), and the incorporation efficiency or specificity constant (k_pol/K_d) for the natural substrate, dCTP, as well as for ddCTP, (+)3TC-TP, and ()3TC-TP (shown in Fig. 1). Enzyme specificity is defined by the ratio k_cat/K_m, representing the apparent second-order rate constant for substrate binding. In the case of DNA polymerases, our previous work (42) has shown that the ground state binding of nucleoside triphosphates is in a rapid equilibrium, and accordingly, the K_d measured under single turnover conditions would equal the K_m, and the k_pol would equal k_cat. We employed a synthetic 23/45-mer DNA/DNA primer-template (Fig. 2) to perform a series of pre-steady-state burst and single turnover experiments.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structures of deoxycytidine analogs.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Sequence of the oligonucleotide substrates.

In each case a pre-steady-state burst of product formation was observed, indicating that at saturating concentrations of dNTP, a step after the chemical step is rate-limiting (Fig. 3, A and B). The rate-limiting step after incorporation of a single nucleotide is the release of the DNA from the enzyme (26).² In each panel, the solid line represents the best fit of the data to a burst equation, from which the burst amplitude, A, the observed rate of incorporation, k_obsd, and the observed steady-state rate, k_ss, were obtained.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Pre-steady-state burst experiments for incorporation of dCTP analogs into DNA/DNA 23/45 primer-template by human mitochondrial DNA polymerase. A, the pre-steady state burst curve for incorporation of the natural substrate, dCTP (). For this experiment, 67 nM catalytic subunit and 440 nM accessory subunit was preincubated with 200 nM 23/45-mer DNA duplex. The polymerization was initiated by adding an equal volume of solution containing dCTP (10 µM) and MgCl2 (2.5 mM). The reactions were quenched with 0.3 M EDTA at each indicated time. The formation of elongated product DNA 24-mer was plotted as a function of time. The solid line represents the fit of data to the burst equation as described, and the curve shown represents fit with burst amplitude A = 34.5 ± 0.7 nM. The observed burst rate constant kobsd = 39 ± 3 s1, and the observed steady-state rate constant kss = 0.062 ± 0.007 s1. B, the pre-steady-state burst curves for incorporation of (+)3TC-TP (15 µM) () and ()3TC-TP (20 µM) () by human mitochondrial DNA polymerase. The reaction conditions are identical to those in A. For (+)3TC-CTP, the curve-fitting generated A = 34 ± 4 nM, kobsd = 0.31 ± 0.05 s1, and kss = 0.029 ± 0.01 s1. For incorporation of ()3TC-TP, the curve-fitting provided A = 30 ± 7 nM, kobsd = 0.12 ± 0.04 s1, and kss = 0.009 ± 0.008 s1. Note the difference in time scale between panels A and B.

A series of experiments were conducted using varying concentrations of dCTP, ddCTP, and (+)- and ()3TC-TP to examine the dNTP concentration dependence of the observed burst rate for each dNTP substrate. At low concentrations of (+)- and ()3TC-TP, a rather shallow burst of product formation was observed because the burst rate was comparable with the steady-state rate. Therefore, to measure the rate of incorporation, the reactions were studied under single turnover conditions with enzyme in excess of DNA as described in Johnson et al. (26) and Feng and Anderson (40).

The interaction of dCTP with the enzyme-DNA complex was assessed by fitting the concentration dependence of the burst rates to the hyperbolic equation k_obsd = (k_pol × [dNTP])/(K_d + [dNTP]), where k_pol is the maximum rate of incorporation, [dNTP] is the concentration of the nucleoside triphosphate, and K_d is the equilibrium dissociation constant for the ground state binding of dNTP with the enzyme-DNA complex. The concentration dependence of the observed polymerization rate for the incorporation of dCTP, ddCTP, (+)3TC-TP, and ()3TC-TP into a DNA/DNA 23/45-mer primer-template by Pol  holoenzyme is shown in Fig. 4, A-C.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Dependence of the observed first-order rate constant kobsd for dNTP incorporation by human mitochondrial DNA polymerase on the concentration of dCTP , (+)- or ()3TC-TP, and ddCTP. A, the first-order rates versus the dCTP concentration. The data () were fit to a hyperbola (solid line) to provide a Kd value of 1.1 ± 0.1 µM for dCTP dissociation constant and a maximum rate of incorporation (kpol) of 44 ± 2 s1. B, the first-order rate constant versus the concentration of ddCTP. The observed rate constants were fit to a hyperbola (solid line), and the curve shown represents a fit of Kd = 0.041 ± 0.007 µM and kpol = 0.66 ± 0.02 s1. C, the first-order rate constant versus the concentration (µM) of (+)3TC-TP () and ()3TC-TP (). The observed rate constants were fit to a hyperbola (solid line), and the curves shown represent fit with Kd = 1.5 ± 0.1 µM and kpol = 0.35 ± 0.01 s1 for (+)3TC-TP () and Kd = 9.2 ± 0.9 µM and kpol = 0.125 ± 0.005 s1 for ()3TC-TP (). Note the difference in scale for observed rates and concentration (µM) between panels A, B, and C.

A complete summary of k_pol, K_d, and k_pol/K_d values for each dCTP analog is shown in Table I. The k_pol and the K_d value for dCTP are all in good agreement with previous studies (26).

A comparison of the analogs reveals that the natural substrate, dCTP, serves as the best substrate for human mitochondrial DNA polymerase, as illustrated by the observed tight binding and fast rate of polymerization. In contrast, ddCTP is incorporated 70-fold more slowly than dCTP, but binds 27-fold more tightly, so the overall incorporation efficiency is only 2.6-fold lower. The ()3TC-TP and (+)3TC-TP analogs are incorporated 125- to 350-fold more slowly than dCTP, respectively. The dCTP and (+)3TC-TP share a similar high affinity for the enzyme-DNA complex, indicating chemistry or the conformational change has become rate-limiting for (+)3TC-TP incorporation. In contrast, the affinity of ()3TC-TP for the enzyme-DNA complex is 6-8-fold weaker than that of the analog that of (+)3TC-TP, containing the natural nucleoside configuration. Accordingly, Pol  incorporates the () isomer at least 16-fold less efficiently than the (+) isomer, as defined by the specificity constants summarized in Table I. This difference in incorporation efficiency is in good agreement with cell culture studies with the nucleoside form of the compounds where the (+)3TC isomer has been reported to be 25- and 12-fold more toxic than the ()3TC in terms of inhibition of cell growth and mitochondrial DNA synthesis, respectively (36).

We conducted an in-depth analysis of Pol  3'-5'-exonuclease removal of ddC and (+) and ()3TC MPs from duplex DNA with highly purified Pol  catalytic subunit and reassociated holoenzyme complex. Our analysis was carried out using DNA substrates that were 3'-terminated with ddCMP (D24-ddC), (+)3TC-MP (D24-(+)3TC), or ()3TC-MP (D24-(+)3TC) along with the corresponding unmodified DNA 23- and 24-mers containing 3'dTMP (D23-dT), and dCMP (D24-dC). We enzymatically synthesized the three DNA 24-mer primers that were 3'-terminated with dideoxy nucleoside analogs, ddCMP, (+)3TC-MP, and ()3TC-MP, respectively. To eliminate the sample variability that may be introduced during preparation, the DNA 23- and 24-mers containing dTMP and dCMP, respectively, were prepared in parallel to those containing modified nucleosides.

A gel illustrating the time course for product formation during the exonuclease reaction is shown in Fig. 5. For clarity, only representative time points have been selected for dCMP (D24-ddC), ddCMP (D24-ddC), (+)3TC-MP (D24-(+)3TC), or ()3TC-MP (D24-()3TC). A summary of the rates of exonuclease activity (k_exo) for either the catalytic subunit or the holoenzyme complex of Pol  using different D24-mer substrates is shown in Table II.

View larger version (37K): [in this window] [in a new window]   Fig. 5.   Gel analysis comparing the nature and kinetics the exonuclease activity of human mitochondrial DNA Pol  using different DNA substrates. Four different kinds of 24/45-mer DNA duplex were tested. At the 3'-end of each of the DNA 24-mer, the dNMP was either a dCMP, a ddCMP, a (+)3TC-MP, or a ()3TC-MP. The representative time courses are 0, 0.05, 0.5, 3, and 12 h. The reaction was initiated by adding MgCl2 (2.5 mM)-containing buffer into a solution of catalytic subunit (40 nM), accessory subunit (270 nM), and 1500 nM D24/D45. The reaction was quenched at various times with 0.3 M EDTA, and products were quantified as described in Feng and Anderson (40).

View this table: [in this window] [in a new window]   Table II Kinetic parameters for the exonuclease activity of human mitochondrial DNA polymerase  toward different DNA substrates

The unmodified correctly base-paired primers, D23-dT and D24-dC, exhibited the fastest excision rates; however, these rates are still small and consistent with the "cost of proofreading" due to the removal of ~1% of the correctly paired bases at the 3'-end of the primers (42). Excision of the (+) and () isomers of 3TC was 2-fold slower than removal of dCMP, and the presence of the accessory protein had minimal effect on this excision rate. The presence of the small accessory subunit of Pol  decreased the exonuclease activity 5-6-fold for the DNA substrates that contained natural nucleotides (DNA 23-dT or 24-dC) at their 3'-ends. These observations are in contrast to a recent study indicating that the accessory subunit may stimulate the exonuclease activity (25). Despite the slow excision rate, the exonuclease should still effectively remove the chain terminators with a half-life of ~1 min. Although this will slow the rate of mitochondrial DNA replication, the low rate of 3TC-TP incorporation will minimize the toxic effects.

In contrast, little or no DNA cleavage was observed for D24-ddC after either 4 h with the catalytic subunit or 12 h with the holoenzyme. The insusceptibility of the 3'-terminal ddCMP to base excision was also reported for a 3'-5'-exonuclease with porcine liver DNA polymerase  (43). Similar results have been reported in studies on yeast mitochondrial DNA polymerases, where the terminally incorporated ddTMP, AZTMP, and ddCMP were not removed by the 3'-5'-exonuclease activity of Pol  (44). This striking observation may explain why inhibition of mitochondrial DNA synthesis by ddC is so profound.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our studies have shown that ()3TC-TP is less efficiently incorporated than (+)3TC-TP into DNA by Pol , and both are incorporated less efficiently than ddCTP. Moreover, the efficiency of incorporation by Pol  is directly correlated with toxicity of each nucleoside analog. These observations point to incorporation by Pol  into mitochondrial DNA as a major site of toxicity of nucleoside analogs. The availability of reliable preparations of Pol  now affords a rapid method to screen drugs for toxicity and opens the path toward further structure/function studies to aid in the design of less toxic analogs.

3TC is well tolerated in the clinic (28, 29), and no subclinical signs of mitochondrial toxicity resulting from ()3TC therapy for 6 months have been observed (30). Apparently to date ()3TC has shown little evidence of mitochondrial injury (13). The unnatural L-() configuration of ()3TC seems to play an important role in preserving the anti-viral activity while decreasing the inhibitory effect on the host Pol . This example suggests other unnatural nucleoside analogs may show similar effectiveness combined with low toxicity.

In contrast, the effects of inhibition on mitochondrial DNA synthesis in vivo and in vitro by ddC are profound (38, 45). The IC₅₀ value for ddC relative to the (+) and () isomers of 3TC indicated that ddC is 180- and 2300-fold more toxic than the (+) and () isomer, respectively (36). A number of factors may clearly come into play including uptake, transport, metabolic activation, degradation, and removal from the system, but our studies have shown a lack of exonucleolytic removal of a 3'-ddC that may significantly contribute to drug toxicity. Conversely, analogs that activate the exonuclease removal by Pol  may exhibit even lower toxicity, and this may provide a new avenue for exploration of less toxic drugs.

The accessory subunit appears to enhance discrimination by the proofreading exonuclease since slower rates of excision by the holoenzyme were observed for correctly base-paired DNA 23-dT- and 24dC-mers. When the 3'-terminus is occupied by a correctly base-paired natural dNMP with a 3'-OH, the accessory subunit may slow down the excision of this dNMP by facilitating the retention of the 3'-end at the polymerase site. Clearly, additional studies are required to identify the structural features of nucleoside analogs that may be utilized to obtain selectivity for HIV-1 RT over DNA Pol .

In summary, our studies have demonstrated that the (+)3TC-TP is a more potent inhibitor for human mitochondrial DNA Pol  than the () isomer through tighter binding and faster incorporation rate into a DNA duplex, leading to greater toxicity. Using highly purified Pol , there is no significant difference in the excision of the incorporated (+)- and ()3TC-MP by the 3'-5'-exonuclease activity of Pol  from the 3'-terminus of a DNA primer, indicating this step likely does not contribute to the low toxicity of ()3TC. Additionally, our studies have shown that a terminally incorporated ddCMP is not efficiently removed by the 3'-5'-exonuclease activity of Pol , suggesting this may play a role in the high level of inhibition of ddC on mitochondrial DNA synthesis observed in vitro and in vivo. The overall toxicity of a nucleoside analog toward replication will be a function of the frequency of incorporation of the chain terminator and the kinetics of excision by the proofreading exonuclease relative to the rate of DNA replication required to sustain growth (Table III). We can now quantify these parameters and begin to assess the structural/functional determinants that govern nucleotide selectivity for Pol  in each step.

An in-depth understanding of the mechanism of inhibition of DNA polymerase  is important in defining the cytotoxicity of anti-HIV drugs toward mitochondrial replication. A knowledge of the mechanistic and structural similarities and differences between Pol  and HIV-1 RT may identify key features important for selective drug incorporation by the viral HIV-1 RT but not by Pol  and, therefore, facilitate the design of less toxic, viral-specific AIDS drugs.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grants GM44613 (to K. A. J.) and GM49551 (to K. S. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Current address: Triangle Pharmaceuticals, 4 University Place, 4611 University Dr., Durham, NC 27707.

^¶  These authors contributed equally to this work.

^** To whom correspondence should be addressed: Dept. of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-4526; Fax: 203-785-7670; E-mail: karen. anderson{at}yale.edu.

Published, JBC Papers in Press, April 27, 2001, DOI 10.1074/jbc.M101156200

² A. A. Johnson and K. A. Johnson, unpublished results.

	ABBREVIATIONS

The abbreviations used are: HIV, human immunodeficiency virus; AIDS, acquired immunodeficiency syndrome; AZT, 3'-azido-3'-deoxythymidine; ddC, 2',3'-dideoxycytidine; ddI, 2',3'-dideoxyinosine; d-, deoxy; dd, dideoxy; Pol , DNA polymerase ; MP, monophosphate; TP, triphosphate; RT, reverse transcriptase; (+)3TC-TP, -D-(+)-2',3'-dideoxy-3'-thiacytidine-5'-TP; ()3TC-TP, -L-()-3TC-TP; 3TC, 2',3'-dideoxy-3'-thiacytidine; bp, base pair(s).

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				J. W. Hanes and K. A. Johnson Exonuclease Removal of Dideoxycytidine (Zalcitabine) by the Human Mitochondrial DNA Polymerase Antimicrob. Agents Chemother., January 1, 2008; 52(1): 253 - 258. [Abstract] [Full Text] [PDF]

				A. Basavapathruni and K. S. Anderson Reverse transcription of the HIV-1 pandemic FASEB J, December 1, 2007; 21(14): 3795 - 3808. [Abstract] [Full Text] [PDF]

				L. Durand-Gasselin, D. Da Silva, H. Benech, A. Pruvost, and J. Grassi Evidence and Possible Consequences of the Phosphorylation of Nucleoside Reverse Transcriptase Inhibitors in Human Red Blood Cells Antimicrob. Agents Chemother., June 1, 2007; 51(6): 2105 - 2111. [Abstract] [Full Text] [PDF]

				E. C. Spikings, J. Alderson, and J. C. St. John Regulated Mitochondrial DNA Replication During Oocyte Maturation Is Essential for Successful Porcine Embryonic Development Biol Reprod, February 1, 2007; 76(2): 327 - 335. [Abstract] [Full Text] [PDF]

				E.-W. Lee, Y. Lai, H. Zhang, and J. D. Unadkat Identification of the Mitochondrial Targeting Signal of the Human Equilibrative Nucleoside Transporter 1 (hENT1): IMPLICATIONS FOR INTERSPECIES DIFFERENCES IN MITOCHONDRIAL TOXICITY OF FIALURIDINE J. Biol. Chem., June 16, 2006; 281(24): 16700 - 16706. [Abstract] [Full Text] [PDF]

				M. S. Janes, B. J. Hanson, D. M. Hill, G. M. Buller, J. Y. Agnew, S. W. Sherwood, W. G. Cox, K. Yamagata, and R. A. Capaldi Rapid Analysis of Mitochondrial DNA Depletion by Fluorescence In Situ Hybridization and Immunocytochemistry: Potential Strategies for HIV Therapeutic Monitoring J. Histochem. Cytochem., August 1, 2004; 52(8): 1011 - 1018. [Abstract] [Full Text] [PDF]

				G. E. Dutschman, S. P. Grill, E. A. Gullen, K. Haraguchi, S. Takeda, H. Tanaka, M. Baba, and Y.-C. Cheng Novel 4'-Substituted Stavudine Analog with Improved Anti-Human Immunodeficiency Virus Activity and Decreased Cytotoxicity Antimicrob. Agents Chemother., May 1, 2004; 48(5): 1640 - 1646. [Abstract] [Full Text] [PDF]

				J. Y. Feng, E. Murakami, S. M. Zorca, A. A. Johnson, K. A. Johnson, R. F. Schinazi, P. A. Furman, and K. S. Anderson Relationship between Antiviral Activity and Host Toxicity: Comparison of the Incorporation Efficiencies of 2',3'-Dideoxy-5-Fluoro-3'-Thiacytidine-Triphosphate Analogs by Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Human Mitochondrial DNA Polymerase Antimicrob. Agents Chemother., April 1, 2004; 48(4): 1300 - 1306. [Abstract] [Full Text] [PDF]

				Y. Lai, C.-M. Tse, and J. D. Unadkat Mitochondrial Expression of the Human Equilibrative Nucleoside Transporter 1 (hENT1) Results in Enhanced Mitochondrial Toxicity of Antiviral Drugs J. Biol. Chem., February 6, 2004; 279(6): 4490 - 4497. [Abstract] [Full Text] [PDF]

				E. Murakami, J. Y. Feng, H. Lee, J. Hanes, K. A. Johnson, and K. S. Anderson Characterization of Novel Reverse Transcriptase and Other RNA-associated Catalytic Activities by Human DNA Polymerase {gamma}: IMPORTANCE IN MITOCHONDRIAL DNA REPLICATION J. Biol. Chem., September 19, 2003; 278(38): 36403 - 36409. [Abstract] [Full Text] [PDF]

				L. J. Stuyver, S. Lostia, M. Adams, J. S. Mathew, B. S. Pai, J. Grier, P. M. Tharnish, Y. Choi, Y. Chong, H. Choo, et al. Antiviral Activities and Cellular Toxicities of Modified 2',3'-Dideoxy-2',3'-Didehydrocytidine Analogues Antimicrob. Agents Chemother., December 1, 2002; 46(12): 3854 - 3860. [Abstract] [Full Text] [PDF]

				A. S. Ray, A. Basavapathruni, and K. S. Anderson Mechanistic Studies to Understand the Progressive Development of Resistance in Human Immunodeficiency Virus Type 1 Reverse Transcriptase to Abacavir J. Biol. Chem., October 18, 2002; 277(43): 40479 - 40490. [Abstract] [Full Text] [PDF]