(Received for publication, December 19, 1994; and in revised form, March 17, 1995)
From the
Saccharomyces cerevisiae mtDNA polymerase, isolated as
a single 135-kDa recombinant polypeptide, showed high processivity and
a capacity to use poly(dA)oligo(dT), poly(rA)
oligo(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
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.
Current therapies against AIDS rely on the use of
dideoxynucleoside analogs such as zidovudine
(3`-azido-3`-deoxythymidine, AZT), 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 and
. 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
catalytic subunit were performed. Overall, the results show significant
enzyme kinetic similarities between mtDNA polymerase and HIV-RT in the
capacity to incorporate dideoxynucleotides.
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
, 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
, 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
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, 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.
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.
Poly(dA)oligo(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)
oligo(dT), poly(rA)
oligo(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).
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
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
/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
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
-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
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
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
required analog
concentrations of >100 µM (data not shown). Thus, the
nuclear enzyme discriminates very efficiently against the incorporation
of dideoxynucleotides.
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.
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 ) 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 and
, 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.
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.