©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Efficient Incorporation of Anti-HIV Deoxynucleotides by Recombinant Yeast Mitochondrial DNA Polymerase (*)

(Received for publication, December 19, 1994; and in revised form, March 17, 1995)

Staffan Eriksson (§) Baoji Xu (¶) David A. Clayton

From theDepartment of Developmental Biology, Beckman Center for Molecular and Genetic Medicine, Stanford University School of Medicine, Stanford, California 94305-5427

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Saccharomyces cerevisiae mtDNA polymerase, isolated as a single 135-kDa recombinant polypeptide, showed high processivity and a capacity to use poly(dA)bulletoligo(dT), poly(rA)bulletoligo(dT), or primed bacteriophage M13 DNA as a template. In a primer extension assay, the enzyme exhibited an intrinsic 3`-5`-exonuclease activity. By optimizing the polymerization reaction conditions, apparent K and V(max) values could be determined for the incorporation of dTTP, 2`-3`-dideoxy-TTP (ddTTP), 3`-azido-TTP (AZTTP), 3`-fluoro-TTP, dCTP, 2`-3`-dideoxy-CTP, and didehydro(d4)CTP. The yeast mtDNA polymerase used ddTTP, 3`-fluoro-TTP, and ddCTP almost as efficiently as natural deoxynucleoside triphosphates. Both 3`-AZTTP and d4CTP were each significantly less efficient as substrates. Overall, the kinetic data with mtDNA polymerase were very similar to those of the recombinant human immunodeficiency virus reverse transcriptase control. Terminally incorporated AZTTP or ddTTP was not removed by the 3`-5` exonuclease activity of mtDNA polymerase. This may explain the inhibition of mtDNA replication observed in anti-human immunodeficiency virus treatment with dideoxynucleoside analogs and suggests that screening of antiviral nucleosides for their effects on mtDNA polymerase could be of value in future rational drug design.


INTRODUCTION

Current therapies against AIDS rely on the use of dideoxynucleoside analogs such as zidovudine (3`-azido-3`-deoxythymidine, AZT^1), dideoxycytidine (ddC), and dideoxyinosine as inhibitors of HIV replication with reverse transcriptase as the target(1, 2, 3) . Although these drugs result in clinical improvement in patients with AIDS, there are several severe side effects. Bone marrow toxicity is observed early when high doses of AZT are used, and on prolonged treatment a substantial number of patients show myopathies of the ``ragged-red fiber'' type, indicative of mitochondrial dysfunction(4, 5) . In case of ddC treatment, the dose-limiting side effect is a peripheral neuropathy(2, 6) . Cell culture studies as well as experiments with isolated mitochondria have shown that AZT, ddC, 3`-fluoro-3`-dideoxythymidine and, to a lesser degree, several other dideoxynucleosides can impair overall mtDNA synthesis, which should cause subsequent defects in mitochondrial function leading to delayed cell toxicity(7, 8, 9, 10, 11) .

The underlying mechanism for the mitochondrial side effects of dideoxynucleosides has been attributed to the capacity of mtDNA polymerase to incorporate dideoxynucleotide analogs (8, 9, 12, 13, 14) and thereby to be inhibited selectively. In accordance with this hypothesis, several studies with purified mammalian polymerase (13, 15, 16, 17, 18, 19) have shown that AZTTP and other dideoxynucleoside triphosphates are very efficient competitive (and noncompetitive) inhibitors of the enzyme. However, there have been no studies with a purified and structurally defined mtDNA polymerase to address this issue directly.

Saccharomyces cerevisiae mtDNA polymerase, the product of the nuclear MIP1 gene(20) , is the first and thus far only member of this polymerase family that has been cloned and sequenced. The gene encodes a protein of 144 kDa that contains sequence similarities to eukaryotic polymerases, as well as reverse transcriptases. In the N-terminal region, there is both a presumed mitochondrial targeting presequence and several motifs characteristic of 3`-5` exonuclease domains of other DNA polymerases. By site-directed mutagenesis of key residues in these motifs, it was shown that they were required for exonuclease activity and that the mutant cells exhibited a mitochondrial mutator phenotype(21) . These studies, which did not involve enzyme purification, and earlier studies of partially purified yeast mtDNA polymerase (22) revealed overall biochemical similarities between the yeast enzyme and other mtDNA polymerases isolated from higher eukaryotes (19, 23, 24, 25, 26, 27, 28) .

We describe here the purification of recombinant yeast mtDNA polymerase and characterization of its kinetic properties with regard to several pharmacologically important dideoxynucleotides. For the latter, we have adopted an approach recently used by Copeland et al.(29) for studying incorporation of AZTTP and ddCTP into defined oligonucleotide templates catalyzed by purified human DNA polymerases alpha and beta. The assay system tests nucleotide incorporation during two distinct phases of DNA synthesis; i.e. during the initial extension of the primer and then subsequent elongation by processive synthesis(30, 31) . We first tested as substrates ddTTP and 3`-FTTP in addition to AZTTP. The former is an analog with as high or higher anti-HIV activity in cell culture and animal model studies as the latter(32) . In addition, ddCTP and d4CTP, both efficient anti-HIV replication inhibitors(33) , were used as substrates, and in most cases parallel reactions with recombinant HIV-RT and homogeneous human DNA polymerase alpha catalytic subunit were performed. Overall, the results show significant enzyme kinetic similarities between mtDNA polymerase and HIV-RT in the capacity to incorporate dideoxynucleotides.


EXPERIMENTAL PROCEDURES

Materials

Single-subunit human DNA polymerase alpha was a generous gift from W. C. Copeland and T. S.-F. Wang (Stanford University School of Medicine, Stanford, CA) and was purified as described previously(29) . Pure recombinant HIV-RT was a gift from T. Unge (Department of Molecular Biology, The Biomedical Center, Uppsala, Sweden) and was isolated from Escherichia coli as described previously(34) . Both 3`-FTTP and AZTTP were provided by B. Öberg, (Medivir AB, Huddinge, Sweden) and A. Karlsson (Department of Biochemistry I, The Karolinska Institute, Stockholm, Sweden). The d4CTP was generously provided by J. Balzarini (The Rega Institute, Leuven, Belgium). The 2-deoxynucleotides, dideoxynucleotides, DEAE-Sephacel, heparin-Sepharose, butyl-Sepharose, poly(dA)bulletoligo(dT) and poly(rA)bulletoligo(dT) were from Pharmacia Biotech, Inc. Protease inhibitors were purchased from Sigma or Boehringer Mannheim. Activated calf thymus DNA was from Sigma. The oligonucleotide primer-templates were as described previously(29) : for thymidine-containing nucleotides and standing start synthesis (Template 1), 18-mer 5`-TGACCATGTAACAGAGAG-3` with 36-mer template 3`-ACTGGTACATTGTCTCTCATTCTCTCTCTCTTCTCT-5` and for the running start primer, 16-mer 5`-CGCCCACGCGGCAGA-3` with 36-mer template 3`-GCGGGTGCGCCGTCTCTTACCTCTTCTCTCTTCTCT-5`. For cytidine-containing substrates, standing start synthesis was performed with 18-mer 5`-TGACCATGTAACAGAGAG-3` with 36-mer template 3`-ACTGGTACATTGTCTCTCGTTCTCTCTCTCTTCTCT-5`.

Overexpression of Yeast mtDNA Polymerase

The entire MIP1 coding region was amplified by polymerase chain reaction on the MIP1-containing plasmid YEpT7-3 ((20) , kindly provided by Dr. Françoise Foury, Universite Catholique de Louvain, Belgium) using a primer with a SacI site and a primer with a ClaI site. The polymerase chain reaction product was purified by electrophoresis onto DEAE-cellulose membrane NA-45 (Schleicher and Schuell). The purified DNA fragment was digested with SacI and ClaI and ligated into pBluescript II KS- digested with the same enzymes to generate the plasmid pMIP1.1. The SphI-XbaI region of the MIP1 gene in the pMIP1.1 was replaced with the same region of the genomic clone to remove possible polymerase chain reaction mutations in this region; this produced plasmid pMIP1.2. The remaining nucleotide sequence upstream and downstream of the SphI-XbaI region was verified, and the entire MIP1 coding region in pMIP1.2 was subcloned into the yeast expression vector pYES2.0 to construct the yeast mtDNA polymerase overexpression plasmid pMIP1.3. The expression of MIP1 in pMIP1.3 is under the control of the Gal1 promoter. Yeast mtDNA polymerase was overexpressed in yeast strain DBY2670 (alpha, pep4::His3, prblDelta 1.6R, his3-Delta 200, ura3-52, can1) as described for yeast mtRNA polymerase(35) . After induction by galactose, yeast mtDNA polymerase expression was increased more than 50-fold.

Purification of Yeast mtDNA Polymerase

Yeast cells harboring the MIP1 overpression plasmid pMIP1.3 were grown to early stationary phase in 500 ml of the synthetic complete medium without uracil(35) . This culture was diluted 50-fold into 20 liters of SGal medium (0.67% yeast nitrogen base without amino acids, 2% galactose) to induce the expression of yeast mtDNA polymerase. When the culture reached A = 2, the cells were harvested by centrifugation for 5 min at 2,700 g. The cells were washed once with cold water and resuspended in 120 ml of buffer D (25 mM Tris-Cl, pH 7.9, 20% glycerol, 1 mM DTT, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 2 mM benzamidine, 1 µg/ml of E64, and 2 µg/ml each of pepstatin A, leupeptin, and antipain) containing 0.1% Triton X-100 and 0.25 M KCl. The cell suspension was placed in a 250-ml bead beater chamber with 80 ml of glass beads (425-600 microns in size) and beaten for five 1-min pulses alternating with 1 min of cooling. The mixture was centrifuged at 100,000 g for 60 min. The cleared extract was diluted with buffer D to lower the KCl concentration to 0.1 M and loaded onto a 50-ml of DEAE-Sephacel column. The column was washed extensively with buffer D containing 0.1 M KCl until the absorbance at 280 nm reached a base line. Yeast mtDNA polymerase was eluted with buffer D containing 0.3 M KCl. The fractions containing proteins were pooled and loaded onto a 12-ml phosphocellulose 11 (Whatman) column (1.5 cm 6.8 cm) equilibrated with buffer D plus 0.3 M KCl. After a wash with 30 ml of buffer D containing 0.3 M KCl, the column was eluted with a 100-ml linear gradient of 0.3-0.8 M KCl in buffer D. Fractions containing DNA polymerase activity (eluting at 0.6 M KCl) were pooled and dialyzed against buffer E (the same as buffer D except that 25 mM Tris-Cl, pH 7.9, was replaced with 25 mM HEPES-KOH, pH 7.4, and the EDTA concentration was lowered to 1 mM) plus 0.25 M KCl. The dialyzed sample was adjusted to 0.3 M KCl and loaded onto a 3-ml heparin-Sepharose column (1 cm 3.8 cm). After a wash with 10 ml of buffer E containing 0.3 M KCl, the column was eluted with a 30-ml linear gradient from 0.3 to 0.8 M KCl in buffer E. Yeast mtDNA polymerase eluted at 0.5 M KCl. Active fractions were pooled, and 3 M ammonium sulfate was added slowly to a final concentration of 0.7 M. This mtDNA polymerase pool was applied to a 1.5-ml butyl-Sepharose (Pharmacia) column. The column was washed with buffer E containing 0.8 M ammonium sulfate and eluted with an 18-ml linear reverse gradient from 0.8 M ammonium sulfate in buffer E to buffer E alone. Fractions containing mtDNA polymerase were pooled, dialyzed against enzyme storage buffer (10 mM Tris-Cl, pH 7.9, 50% glycerol, 1 mM dithiothreitol, 1 mM EDTA, 0.1 M KCl, 2 mM benzamide, 2 µg/ml each of pepstatin A, leupeptin, and antipain) and stored at -80 °C. Before dialysis, mtDNA polymerase exhibits low activity due to the high salt concentration.

DNA Polymerase Assays

Standard reaction mixtures contained 10 mM Tris-Cl, pH 7.9, 10 mM MgCl(2), 40 mM KCl, 0.1 mg/ml BSA, 50 µg/ml aphidicolin, 1 mM DTT, 6 µM [P]dTTP (10,000 cpm/pmol), and 20 µg/ml poly(dA)bulletoligo(dT) in a total volume of 25 µl. Reactions were performed at 30 °C for 30 min and terminated by dilution into 2 ml of ice cold 5% trichloroacetic acid and 50 mM sodium pyrophosphate. Incorporation of radioactive dTMP into acid-insoluble products was assayed as described previously(35) . One unit of enzyme activity was defined as the amount of enzyme that incorporated 1 pmol of dTMP in 30 min at 30 °C.

The processivity of mtDNA polymerase was determined using primed M13mp19 single-strand DNA as the template. The M13-20 sequence primer (5`-GTAAAACGACGGCCAGT-3`) was end-labeled with T4 polynucleotide kinase and [-P]ATP. A 25-µl reaction contains 10 mM Tris-Cl, pH 7.9, 10 mM MgCl(2), 1 mM DTT, 0.1 mg/ml BSA, 40 mM KCl, 0.017 pmol of primed M13mp19, 100 µM each of dATP, dCTP, dGTP, and dTTP, and 0.071 pmol of mtDNA polymerase or 0.5 units of Klenow fragment of E. coli DNA polymerase I. The reaction was incubated at 30 °C for 30 min and terminated by the addition of an equal volume of proteinase K mixture (0.5 mg/ml proteinase K, 1% SDS, and 20 mM EDTA). The mixture was incubated at 37 °C for 30 min and was extracted once with phenol/chloroform (1:1). The products were precipitated by ethanol in the presence of 10 µg of E. coli tRNA, dissolved in 12 µl of alkaline loading buffer (50 mM NaOH, 1 mM EDTA, 3% Ficoll), and separated on a 1% alkaline agarose gel. After electrophoresis, the gel was fixed in 10% methanol, 10% acetic acid, dried, and exposed to x-ray film.

To prepare the primer templates for the running and standing start reactions, primer oligonucleotides were 5`-end labeled with T4 polynucleotide kinase and [-P]ATP, gel purified, and annealed to the respective templates in a 1:2 ratio as described previously(29) . The reactions were performed in a volume of 10 µl for 10 min at 37 °C with 20 mM Tris-Cl, pH 7.9, 10 mM MgCl(2), 0.2 mg/ml BSA, 1 mM DTT, 100 mM KCl, 0.25 pmol of labeled primer-template, 1-3 ng of mtDNA polymerase, and different concentrations of deoxynucleoside triphosphates. When HIV-RT or DNA polymerase alpha was used, KCl was omitted. The reactions were terminated by the addition of 2 volumes of 90% formamide, 0.25 M EDTA, and the products were analyzed on 16 or 19% polyacrylamide-7 M urea gels that were exposed to x-ray film (Hyperfilm MP, Amersham Corp.) without an intensifier screen at -70 °C. The films were scanned with a Molecular Dynamics model 300A densitometer (Sunnyvale, CA), and the percentage of radioactivity incorporated into elongated products was determined relative to total loaded radioactivity. Care was taken to remain within the linear range of the film. The kinetic constants were calculated using a reiterative curve fitting program for the Michaelis-Menten equation.

The 3`-5`-exonuclease reactions were performed with the standing start template 1 as a control. For the synthesis of a primer terminated with 3`-AZTTP or ddTTP, HIV-RT was used in 10-fold higher amounts and with 50 µM AZTTP or 10 µM ddTTP in a standard 60-min reaction with template 1. This converted more than 90% of 18-mer primer into 19-mer product. The template solutions were extracted with phenol-chloroform, diluted 10-fold with 50 mM Tris-Cl, pH 8.0, 10 mM EDTA buffer and concentrated in a Centricon (Amicon Apparatus) filter apparatus. In addition to double-stranded templates, exonuclease reactions contained 30 ng of mtDNA polymerase, 20 mM Tris-Cl, pH 7.9, 10 mM MgCl(2), 150 mM KCl, 0.1 mg/ml BSA, 1 mM DTT in a volume of 30 µl. Eight-µl samples were taken at different time points and analyzed on 19% polyacrylamide urea gels.

Other Analytical Methods

Protein was determined by the Pierce BCA protein assay reagent with BSA as the protein standard. SDS-polyacrylamide gels and silver staining for detection of polypeptides were performed as described previously(36, 37) .


RESULTS

Overexpression and Purification of Yeast mtDNA Polymerase

To facilitate purification of the enzyme, the coding region of the MIP1 gene was cloned into the high copy expression vector pYES2.0, which placed the gene under the control of the inducible Gal1 promoter. Using this system, yeast mtDNA polymerase was purified to homogeneity from whole cell extracts by a simple four-step procedure that included chromatographics over columns of DEAE-Sephacel, phosphocellulose 11, heparin-Sepharose, and butyl-Sepharose (Table1, Fig. 1). Approximately 140 µg of mtDNA polymerase could be obtained from 20 liters of yeast cell culture with an overall purification of 300-fold and a yield of 6%. It was important to include a mixture of protease inhibitors in the isolation buffers in order to obtain full-length mtDNA polymerase in reasonable yield. The MIP1 gene encodes a 144-kDa polypeptide including the mitochondrial targeting sequence, while its mature form migrates as a 135-kDa species under our electrophoresis conditions. The 135-kDa species is present in the whole cell extract (Fig.1) but not in the extract from yeast cells that do not overexpress the MIP1 gene (data not shown), suggesting that purified mtDNA polymerase is in intact, mature form. The estimated size difference of the MIP1 gene product with the reported 140-kDa estimate (21) is likely due to the use of different markers used in the two studies.




Figure 1: SDS-polyacrylamide gel analysis of fractions from the purification of yeast mtDNA polymerase. Samples from each purification step were analyzed on a denaturing 7% polyacrylamide gel. Proteins were visualized by silver staining. The apparent molecular weights of standards are shown on the left. WAC, whole cell extract of active component; DEAE, DEAE-Sephacel; P11, phosphocellulose 11; Heparin, heparin-Sepharose; Butyl, butyl-Sepharose.



Properties of mtDNA Polymerase

As expected from earlier studies, the enzyme required Mg (or Mn) for activity. The optimum Mg or Mn concentration was 6-16 or 2-8 mM, respectively, when poly(dA)bulletoligo(dT) was used as the substrate (data not shown). Addition of 40-150 mM KCl resulted in a 2-fold stimulation of the polymerase activity, while higher salt concentrations were inhibitory (data not shown).

Poly(dA)bulletoligo(dT) was chosen as the substrate throughout the purification since this DNA template gave the most reproducible activity in crude extracts. Enzyme assays using nicked calf thymus DNA as a template gave erroneously high measurements of activity at earlier stages of the purification, presumably due to nuclease contamination. More highly purified mtDNA polymerase showed a capacity to use poly(dA)bulletoligo(dT), poly(rA)bulletoligo(dT), activated calf thymus DNA, or primed M13 DNA as templates.

The processivity of the single-subunit mtDNA polymerase was investigated with a primed M13 DNA template assay using 5`-end-labeled primers and cold dNTPs. In this assay, the radioactivity contained in the reaction products represents the number of molecules synthesized. If a DNA polymerase has low processivity, DNA strand elongation will continue for short periods before the polymerase disengages the template and reinitiates on a new template. Multiple rounds of this process would result in apparent simultaneous elongation of all the DNA strands. Compared with Klenow fragment, yeast mtDNA polymerase is highly processive (Fig.2). Complete template-size products were visible after 2 min, while the majority of primer remained unelongated (Fig.2, lane1). In contrast, no template-sized product was observed even after a 20-min incubation with Klenow fragment (Fig.2, lane6). In this case, all products appeared to be elongated simultaneously (Fig.2, lanes4-6), as predicted for an enzyme having low processivity. In contrast to its high processivity, yeast mtDNA polymerase exhibits very low catalytic efficiency on primed M13 DNA templates. Only a small portion of the template was elongated despite the >3-fold ratio of mtDNA polymerase over primer termini on a molar basis.


Figure 2: Processivity of mtDNA polymerase and Klenow fragment of E. coli polymerase I on primed M13 template. Mitochondrial DNA polymerase (0.071 pmol) (lanes1-3) or 0.5 units of Klenow fragment (lanes4-6) was incubated with the template (0.017 pmol) for 2 (lanes1 and 4), 6 (lanes2 and 5), or 20 min (lanes3 and 6).



Substrate Specificity of Yeast mtDNA Polymerase

The main aim of the present study was to determine the kinetic properties of mtDNA polymerase with pharmacologically important dideoxynucleotides. We used an assay system that tested the capacity of the polymerase to incorporate a nucleoside triphosphate either as the first (standing start template) or as the third nucleotide (running start template). The properties of human DNA polymerases alpha and beta were recently described by Copeland et al.(29) using the same assay system. A major complicating factor in this work was an intrinsic 3`-5`-exonuclease activity of mtDNA polymerase, which is not found in the two nuclear DNA polymerases. Although yeast mtDNA polymerases with loss of function mutations in exonuclease domains have been reported, these mutations also changed the polymerization properties of the enzyme; e.g. lower polymerization activity and lower processivity(21) . This precluded the use of these exonuclease-defective mtDNA polymerases in the present study. However, by using low amounts of enzyme, short incubation periods, and adding 100 mM KCl to the reactions, the degradation of the primer-template hybrids was <20% (Fig.3, compare lanes1 and 2).


Figure 3: Gel analysis of the capacity of mtDNA polymerase to incorporate deoxynucleotides into the standing start primer template DNA. Assays were performed as described under ``Experimental Procedures'' with a primer-template as shown in lane1 (before incubation) and after 10 min at 37 °C incubation with mtDNA polymerase alone (lane2). Addition of dTTP (concentrations indicated) led to the synthesis of a 19-mer product (lane3). When 10 µM of both dATP and dGTP were added, some complete synthesis of a 36-mer occurred (lane4). Triphosphate analogs were tested, and the results quantified as described under ``Experimental Procedures.''



Since a low concentration of primer-template (25 nM) was used in nucleotide incorporation assays, the measured apparent K and V(max) may differ significantly from their true values. To overcome this problem of DNA concentration effects, we used relative efficiency (R) to compare the capacities of DNA polymerases to incorporate deoxynucleotide analogs. R is the ratio between V(max)/K values for an analog and the natural dNTP.

The elongation of the primer by mtDNA polymerase in the standing start assay was saturated at nM concentrations of dTTP, and the addition of the other deoxynucleotides led to synthesis of some complete 36-mer (Fig.3). However, smaller products are present that could represent failures to extend or hydrolysis products. When 3`-AZTTP was used as substrate, there was minimal incorporation, even at micromolar concentrations. The identity of the species at the position of a 20-mer is unknown (Fig.3, lane8). Conversely, 3`-FTTP (Fig.3; Table 2) and ddTTP (Table 2) served as relatively efficient substrates.



A second standing start primer-template, specific for cytidine nucleotide incorporation ((29) ; and see ``Experimental Procedures''), was used to determine the efficiency of dCTP, ddCTP, and d4CTP as substrates. Experiments were performed in the same manner as for thymidine-containing nucleotides, and the products were quantified by densitometry. The calculated apparent K, V(max) and R values for these substrates are shown in Table3.



A similar set of reactions was carried out with pure recombinant HIV-RT. In these assays, there was no degradation of the primer since HIV-RT lacks an exonuclease activity. The kinetic constants observed with mtDNA polymerase and HIV-RT were very similar overall. Both polymerases were able to use ddCTP, ddTTP, and 3`-FTTP as substrates. But the incorporation efficiencies (R) for these analogs were 2-17-fold lower than that for dTTP ( Table2and Table 3). The two polymerases were distinguished by the observation that 3`-AZTTP was a poor substrate for mtDNA polymerase, while it was almost as efficient as dTTP in the case of HIV-RT. With both polymerases, the unsaturated sugar analog d(4)-CTP was incorporated at significant levels but with roughly 100-fold lower efficiency as compared to dCTP. The d4CTP-containing product was chemically unstable, and reheating of the primer template resulted in >50% degradation of the d4CTP-containing primer.

With running start synthesis assays, both 3`-FTTP and ddTTP could be incorporated almost as well as dTTP by yeast mtDNA polymerase (Fig.4). In contrast to the standing start synthesis assays, the analog AZTTP was also a good substrate in the running start synthesis assays, although it required 30-fold higher concentration of AZTTP to obtain the same amount of incorporation as dTTP (Fig.4). Due to the exonuclease activity of yeast mtDNA polymerase, it was difficult to determine apparent K and V(max) values for the running start synthesis assays.


Figure 4: Gel analysis of the capacity of mtDNA polymerase to incorporate deoxynucleotides into the running start primer-template DNA. The 16-mer primer template is shown alone (lane17) and after incubation with enzyme and 5 µM dATP for 10 min at 37 °C leading to formation of 17- and 18-nucleotide products (lane1). Addition of dTTP (or analogs thereof) resulted in synthesis of a 19-nucleotide product, and when 10 µM dGTP was also included, there was synthesis of some complete 36-mer (lane3). The amount of 19-nucleotide product relative to total radioactivity in each lane was determined as described under ``Experimental Procedures.''



With running start synthesis assays HIV-RT was again found to be similar to mtDNA polymerase in the capacity to incorporate ddTTP and 3`-FTTP, but with HIV-RT, the K for AZTTP was 30-fold lower (data not shown). Copeland et al.(29) showed with the same assay that pure human DNA polymerase alpha was capable of incorporating AZTTP. We repeated their experiment and included assays with ddTTP and 3`-FTTP. Similar to the reported results (29) , the human nuclear DNA polymerase used AZTTP as a substrate, but only at very high concentrations (>200 µM). As a consequence, a direct estimate of the K could not be achieved. Both 3`-FTTP and ddTTP could be used more efficiently than AZTTP. However, saturation of DNA polymerase alpha required analog concentrations of >100 µM (data not shown). Thus, the nuclear enzyme discriminates very efficiently against the incorporation of dideoxynucleotides.

3`-5`-Exonuclease Activity of mtDNA Polymerase

A role for the proofreading activity of mtDNA polymerase 3`-5`-exonuclease domains has been clarified in vivo(21) . Therefore, it was relevant to determine if dideoxynucleotides incorporated as chain terminators during the polymerase reaction could be removed by the intrinsic exonuclease activity. We used HIV-RT for the synthesis of AZTTP or ddTTP 3` termini by priming with the standing start 18-mer. Yeast mtDNA polymerase (at higher enzyme and KCl concentrations compared with the elongation assays) degraded the primer strand of the matched standing start primer-template almost completely in 20 min at 37 °C (Fig.5). However, when the primer strand contained 3`-terminal ddTTP or AZTTP, there was no detectable degradation observed even after 2 h of incubation (Fig.5).


Figure 5: 3`-5`-exonuclease activity of mtDNA polymerase. Yeast mtDNA polymerase was incubated with a primer-template in which the primer was terminated with dTTP (lanes1-4), AZTTP (lanes5-8), or ddTTP (lanes9-12) at 37 °C for 1 min (lanes1, 5, and 9), 20 min (lanes2, 6, and 10), 40 min (lanes3, 7, and 11), and 60 min (lanes4, 8, and 12). The products were analyzed on a 19% polyacrylamide, 7 M urea gel.




DISCUSSION

Properties of Yeast mtDNA Polymerase

Earlier studies with yeast mtDNA polymerase have established that a single 144-kDa polypeptide contains both the polymerase and 3`-5` exonuclease activities(20, 21) . We demonstrate here that no other protein components are required for these functions, since they are found with the apparently homogeneous polypeptide. Furthermore, this single polypeptide enzyme exhibits high processivity and thus may not require any processivity factor(s); however, the physiological form of yeast mtDNA polymerase has not yet been detailed. These features differ from those reported for highly purified animal mtDNA polymerases(19, 23, 24, 25, 26, 27, 28) . For example, isolates of the human enzyme contain at least two major proteins (140 and 54 kDa), and additional factors are required for DNA synthesis with primed M13 templates(19) . At present, it is not known if the animal mtDNA polymerase subunits are derived from separate genes or by proteolysis of a single gene product.

Although there could be differences between the in vivo behavior of yeast mtDNA polymerase and the corresponding animal cell enzymes, the known overall basic functional characteristics are very similar. These include primer-template preferences, the presence of a 3`-5` exonuclease activity, and the absence of a 5`-3` exonuclease capacity(19, 20, 21, 22, 23, 24, 25, 26, 27, 28) .

Conservation between polymerases has been revealed by sequence similarities observed among a large number of DNA and RNA polymerases (38) . In most cases, the various types of polymerases (e.g. Family B of DNA-dependent DNA polymerases, including yeast polymerase I and human polymerase alpha) show stronger similarities within a group than between different polymerases of the same species. In the case of mtDNA polymerase, the most closely related enzyme currently known is that of bacteriophage T7. A corresponding sequence similarity was observed between S. cerevisiae mtRNA polymerase and T7 RNA polymerase(39) .

Therefore, we reasoned that an analysis of the kinetic properties of yeast mtDNA polymerase with pharmacologically important dideoxynucleotide analogs was warranted and that the results should have general significance for mtDNA replication. The advantage of using an assay system where the polymerase is either incorporating the substrate as the first or the third nucleotide in a primer extension reaction, followed by denaturing polyacrylamide gel electrophoresis, has been elegantly demonstrated by Goodman and co-workers(30, 31) . Since we used the same primer-templates as those used by Copeland et al. (29) to study incorporation of AZTTP and ddCTP by pure human DNA polymerases alpha and beta, we obtained the additional advantage of being able to compare directly our results with theirs. Previously, the assay has been used only with DNA polymerases that lack a 3`-5` exonuclease activity. The fact that mtDNA polymerase does degrade the primer during the reaction is an inherent complication. Future experiments with a mutated mtDNA polymerase (which conceivably could be altered to eliminate the exonuclease activity and not affect polymerization properties) may be warranted.

Consequences for Anti-HIV Chemotherapy

The use of anti-HIV chemotherapy is compromised primarily due to the occurrence of severe toxic side effects with all of the currently used nucleoside analogs. Certain late side effects, primarily with AZT and ddC treatment, are caused by impaired mitochondrial function, with the underlying mechanism attributed to the incorporation of dideoxynucleotides by mtDNA polymerase. It has been shown previously that several 3`-dTTP analogs, including ddTTP, 3`-FTTP, and AZTTP, inhibited mtDNA polymerase, but the same analogs appeared to be at least 10-fold more inhibitory to HIV-RT(13, 15) . In contrast, in our study, we find close similarity in the kinetic properties of mtDNA polymerase and recombinant HIV-RT. Izuta et al. (16) have analyzed the capacity of purified bovine mtDNA polymerase to use AZTTP as a substrate; the authors concluded that it was a good inhibitor but that it could not be incorporated into synthetic oligonucleotide primer-templates, even at 200 µM. This is at variance with the results presented here with yeast mtDNA polymerase, but with respect to ddTTP incorporation, the two enzyme preparations show similar properties. Daluge et al. (17) determined the K values of AZTTP and ddCTP with purified preparations of human mtDNA polymerase to be 8.7 µM and 15 nM, respectively, and the corresponding values with HIV-RT were 14 and 22 nM, respectively. Compared with earlier published work, we found lower K values for HIV-RT and human nuclear polymerase alpha with both natural and analog triphosphates(4, 13, 29) . This may be due to the assay conditions, since we used lower primer-template concentrations than in the other studies.

The results presented here support the hypothesis that the capacity of mtDNA polymerase to incorporate dideoxynucleotides is the underlying mechanism for the mitochondrial defects observed during anti-HIV treatment with those analogs. We also found that once incorporated, 3`-dideoxynucleoside monophosphates cannot be removed by the proofreading exonuclease activity of mtDNA polymerase, in agreement with results of Longley and Mosbaugh(40) . To serve as substrates for mtDNA polymerase, sufficient dideoxynucleoside triphosphates presumably must accumulate inside mitochondria. The synthesis of deoxynucleotides for mtDNA replication likely occurs (at least in mammalian cells) both via deoxyribonucleotide translocation across the mitochondrial membranes, as well as by salvage of deoxyribonucleosides(41) . The details of this metabolism are still unknown, and in yeast cells there are no known mitochondrial deoxyribonucleoside kinases. However, these salvage enzymes are most likely responsible for supplying mtDNA precursors in resting animal cells with very low cellular deoxyribonucleotide pools. It is in these type of tissues (i.e. muscles and nerve cells) that interference of mtDNA replication should lead to the mitochondrial functional defects seen following dideoxynucleoside treatments. Therefore, the properties of the salvage enzymes are of importance. Recently, human mitochondrial thymidine kinase and bovine mitochondrial deoxyguanosine kinase were purified, and their substrate specificities were determined with regard to dideoxynucleoside analogs(42, 43, 44) .

In order to make predictions about the capacity of a certain analog to block mtDNA replication, it is necessary to know the extent to which the compound can be phosphorylated and transported into the mitochondria in the cells of interest, as well as if it is capable of inhibiting mtDNA polymerase. Further studies on the enzymology and basic mechanism of mtDNA replication and mtDNA precursor synthesis may enable design of antiviral drugs that will not, in turn, interfere with mtDNA metabolism.


FOOTNOTES

*
This work was supported in part by Grant GM33088-23 from the National Institute of General Medical Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by grants from the Swedish Medical Research Council, the Swedish Society of Medicine, the Medical Faculty of the Karolinska Institute, and the Swedish University of Agricultural Sciences. Present address: Dept. of Veterinary Medical Chemistry, Swedish University of Agricultural Science, The Biomedical Center, S-751 23 Uppsala, Sweden.

A trainee in the graduate program of the Department of Biological Sciences, Stanford University, and supported by a predoctoral fellowship from The Rockefeller Foundation.

^1
The abbreviations used are: AZT, 3`-azido-3`-deoxythymidine; ddC, dideoxycytidine; HIV, human immunodeficiency virus; AZTTP, 3`-azido-TTP; FTTP, 3`-fluoro-TTP; RT, reverse transcriptase; BSA, bovine serum albumin; DTT, dithiothreitol; d4CTP, didehydro(d4)CTP.


ACKNOWLEDGEMENTS

We thank W. C. Copeland and T. S.-F. Wang for advice and reagents, G. S. Shadel for comments on the manuscript, and Betsy Lewis for help in preparation of the manuscript.


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