(Received for publication, August 7, 1995)
From the
The subunit structure of mitochondrial DNA polymerase from Drosophila embryos has been examined by a combination of
physical and immunological methods. A highly specific rabbit antiserum
directed against the native enzyme was developed and found to recognize
specifically its two subunits in immunoblot and immunoprecipitation
analyses. That and the potent inhibition by the rabbit antiserum of the
DNA polymerase and 3` 5` exonuclease activities of the nearly
homogeneous mitochondrial DNA polymerase provide strong evidence for
the physical association of the 3`
5` exonuclease with the two
subunit enzyme. An immunoprecipitation analysis of crude enzyme
fractions showed that the two subunits of Drosophila mitochondrial DNA polymerase are intact, and an in situ gel proteolysis analysis showed that they are structurally
distinct. Template-primer DNA binding studies demonstrated formation of
a stable and discrete enzyme-DNA complex in the absence of accessory
proteins. Photochemical cross-linking of the complexes by UV light
indicated that the
but not the
subunit of mitochondrial DNA
polymerase makes close contact with DNA, and limited digestion of the
native enzyme with trypsin showed that an
65-kDa proteolytic
fragment of the
subunit retains the DNA binding function.
Of the five eucaryotic DNA polymerases (,
,
,
,
), the mitochondrial DNA polymerase (pol
) (
)is the least abundant and perhaps the least well studied.
With the recent discovery of mtDNA diseases(1) , and the
realization that drugs used to combat cancer and viruses affect mtDNA
function(2) , there is renewed interest in it. Studies
describing the identification and relative abundance of pol
have
demonstrated that it is the only DNA polymerase found in animal
mitochondria(3) , and that it accounts for only about 1% of the
total cellular DNA polymerase activity(4) . Notwithstanding the
enzyme's low relative abundance, in surveying Drosophila at six developmental stages, we showed that the level of pol
activity varies 180-fold during development and is greatest in early
embryos (5) . This allowed its purification to
near-homogeneity(5) , and characterization of its subunit
structure and catalytic mechanism (6, 7, 8, 9, 10, 11, 12) .
It is now apparent that other animal mitochondrial DNA polymerases
including mammalian enzymes have similar catalytic and structural
features(13, 14, 15) .
We proposed that Drosophila pol is a heterodimer comprising a 125-kDa
polymerase catalytic subunit and a 35-kDa polypeptide of unknown
function(5) . Likewise, pol
from frog(13) ,
pig(14) , and human cells (15) has been shown to
contain a large catalytic subunit and several smaller polypeptides,
some of which appear to result from in vitro proteolysis. A
genomic clone of the polymerase catalytic subunit of yeast mtDNA
polymerase encodes a 140-kDa polypeptide(16) ; whether or not
the yeast enzyme contains a small subunit is unknown.
We and others
have shown that pol contains a potent and highly mispair-specific
3`
5` exonuclease, which proofreads errors during in vitro DNA
synthesis(6, 7, 14, 17, 18, 19) .
Ito and Braithwaite (20) have shown that the deduced amino acid
sequence of the yeast catalytic subunit (MIP1) can be aligned with the
family A DNA polymerases, of which Escherichia coli DNA
polymerase I (Eco pol I) and bacteriophage T7 DNA polymerase
are members, and that some amino acid residues that are critical in the
DNA polymerase and 3`
5` exonuclease domains of Eco pol
I are invariant in MIP1. In fact, Foury has shown by site-directed
mutagenesis, that substitutions in conserved exo-domain aspartates
result in a mutator phenotype upon production of the recombinant
protein in yeast(21) . Thus, although a subunit assignment for
the 3`
5` exonuclease has not been made in any of the animal
mitochondrial DNA polymerases, it is most likely that the 3`
5`
exonuclease function resides in the polymerase catalytic subunit.
Interestingly, however, while the 3`
5` exonuclease resides in
the polymerase catalytic subunit in Bacillus subtilis DNA
polymerase III, it exists as a separate subunit in E. coli DNA
polymerase III(22) .
Here we report further studies of the
subunit structure and enzymatic activities of Drosophila pol
using a combination of physical and immunological approaches. We
have also explored the role of the two subunits in template-primer DNA
binding.
Figure 1:
Reactivity of rabbit antiserum against
native Drosophila DNA polymerase . A, immunoblot
analysis of pol
Fraction IV. Polymerase
Fraction IV in the
amount of 12 units (lane 1, 1.9 µg total protein,
8%
pure) or 24 units (lane 2) was denatured, electrophoresed in a
10% SDS-polyacrylamide gel, and transferred to nitrocellulose. The blot
was incubated with rabbit antiserum (1:1000) for 2 h at 24 °C and
then probed with
I-protein A and autoradiographed as
described under ``Methods.'' B, immunoprecipitation
of pol
Fraction III. Polymerase
Fraction III (20 units, 3.4
µg total protein) was incubated with an equal volume of rabbit
preimmune serum (lane 1) or antiserum (lane 2) for 2
h at 0 °C. Immune complexes were precipitated and then
electrophoresed in a 10% SDS-polyacrylamide gel, and the proteins were
transferred to nitrocellulose and detected by immunoblotting as
described under ``Methods.''
Figure 3:
Immunoprecipitation of Drosophila pol from crude enzyme fractions. Freshly harvested embryos
were rapidly processed, and whole embryo and mitochondrial extracts
were precipitated with either preimmune (lanes 4 and 7) or immune serum (lanes 2, 3, 5,
and 6) as described under ``Methods.'' The
immunoprecipitates were then electrophoresed in a 10%
SDS-polyacrylamide gel, and the proteins were transferred to
nitrocellulose and detected by immunoblotting as described under
``Methods.'' Lane 1 represents a control of pol
Fraction VI (75 ng); lanes 2-4 represent embryonic
fractions (6.4 mg of protein derived from 100 mg of embryos); lanes
5-7 represent mitochondrial fractions (2 mg of protein
derived from 600 mg of embryos).
Template-primer DNA binding by pol was examined in a gel
electrophoretic mobility shift assay as follows. Polymerase
Fraction VI (28 fmol) was incubated with the
[
P]dAMP-labeled BrdUMP-substituted 40-mer (0.22
pmol) for 10 min at 30 °C in standard reaction buffer containing 50
mM Tris
HCl (pH 8.5), 4 mM MgCl
, 5
mM dithiothreitol and 30 mM KCl, followed by the
addition of bromphenol blue and glycerol to 0.01 and 5%, respectively,
and electrophoresed in a 4.5% native polyacrylamide gel (13x13x0.15 cm)
in 45 mM Tris borate (pH 8.3) and 1 mM EDTA. After
electrophoresis, the gel was dried under vacuum and exposed at -80
°C to Kodak X-Omat AR x-ray film using a DuPont NEN Quanta III
intensifying screen.
Photochemical cross-linking with UV light was
performed after incubation of pol Fraction VI with
template-primer DNA as described above. The reaction mixtures were
irradiated for 15 min at 0 °C with UV light (300 nm) from a
germicidal bulb (Fotodyne, 4
15 watts) at a distance of 8 cm.
After irradiation, the samples were made 1
in Laemmli sample
buffer(23) , denatured, and electrophoresed in a 7.5%
SDS-polyacrylamide gel (13
13
0.15 cm). The gel was
dried under vacuum and autoradiographed as described above.
In
experiments where pol was subjected to digestion with trypsin
prior to photochemical cross-linking, the DNA binding mixtures
contained higher levels of pol
(0.6 pmol), template-primer DNA
(1.5 pmol), and trypsin (200 or 400 ng) and were incubated for 15 min
at 20 °C. The digestion was terminated by addition of sodium
metabisulfite to 20 mM and leupeptin to 20 µg/ml. The
samples were then irradiated, processed and electrophoresed as
described above.
Figure 2:
Inhibition of Drosophila pol
by rabbit antiserum. Polymerase
Fraction VI (0.3 units) was
preincubated for 60 min at 0 °C with the indicated concentrations
of preimmune (closed circles) or immune (open
circles) serum. Aliquots were assayed under standard conditions (9) for DNA polymerase activity (A) or 3`
5`
exonuclease activity (B), on singly primed M13 DNA containing
paired or mispaired primers, respectively.
In order to make a subunit assignment for the 3` 5`
exonuclease function, we carried out extensive studies by gel
filtration and velocity sedimentation in the presence of denaturants,
to achieve dissociation and separation of the two subunits of Drosophila pol
with retention of catalytic activity.
However, we found that subunit dissociation in the presence of
guanidine
HCl, urea, or ethylene glycol occurs only upon partial
denaturation and substantial loss of enzyme activity; enzyme assay and
immunoblot analyses indicate that
95% of both DNA polymerase and 3`
5` exonuclease activity is lost before subunit dissociation
occurs (data not shown).
To demonstrate that the and
subunits of Drosophila pol
are structurally
distinct, we performed an in situ gel proteolysis analysis
with N-chlorosuccinimide (Fig. 4). The
and
subunits of Drosophila pol
were purified by
SDS-polyacrylamide gel electrophoresis and then cleaved with N-chlorosuccinimide and re-electrophoresed. They yield
completely distinct polypeptide patterns; partial cleavage of the
subunit yields 11 polypeptides ranging from 9 to 33 kDa (lane
3), none of which correspond to the four products derived from the
subunit (lane 5).
Figure 4:
Cleavage of Drosophila pol
subunits with N-chlorosuccinimide. The
and
subunits of pol
(Fraction VI, 3 µg) were purified by
SDS-polyacrylamide gel electrophoresis and subjected to in situ proteolysis with N-chlorosuccinimide(24) , and
then re-electrophoresed in a 5-15% linear gradient
SDS-polyacrylamide slab gel and stained with silver(25) . Lane 1, pol
Fraction VI (50 ng); lane 2,
gel-purified
subunit (150 ng); lane 3, N-chlorosuccinimide-digested
subunit; lane 4,
gel-purified
subunit (150 ng); lane 5, N-chlorosuccinimide-digested
subunit; lane B,
sample buffer only.
Figure 5:
Binding and photochemical cross-linking of Drosophila pol to template-primer DNA. A,
template-primer DNA binding by pol
. Polymerase
Fraction VI
(0.36 units, 28 fmol) was incubated with radiolabeled
bromodeoxyuridylate-substituted template-primer DNA (shown in C, 0.22 pmol) for 10 min at 30 °C as described under
``Methods,'' and the reaction products were electrophoresed
in a 4.5% native polyacrylamide gel and the gel autoradiographed. Lanes 1 and 2 represent no protein and no DNA
controls, respectively. Lanes 3-5 represent samples
containing both pol
and radiolabeled DNA substrate in the absence (lane 3) or presence of unlabeled competitor DNA (lane
4, 2.2 pmol; lane 5, 4.4 pmol). B, photochemical
cross-linking of pol
to template-primer DNA. Polymerase
was
incubated with template-primer DNA as in A. The samples were
then irradiated with UV light (300 nm) for 15 min at 0 °C,
processed, and electrophoresed in a 7.5% SDS-polyacrylamide gel, and
the gel was autoradiographed as described under ``Methods.'' Lanes 1 and 2 represent no protein and no irradiation
controls, respectively. Lanes 3-5 represent irradiated
samples containing both pol
and radiolabeled DNA substrate in the
absence (lane 3) and presence of unlabeled competitor DNA (lane 4, 2.2 pmol; lane 5, 4.4 pmol). C,
template-primer DNA. A partially double-stranded deoxyoligomer (40 nt)
containing [
P]-dAMP (*) and bromodeoxyuridylate (B in sequence) at its 3`-terminus was prepared as described
under ``Methods.''
To probe the involvement of the two
subunits of Drosophila pol in template-primer DNA
binding, we subjected the enzyme-DNA complexes to photochemical
cross-linking in the presence of UV light and then analyzed denatured
cross-linked complexes by SDS-polyacrylamide gel electrophoresis and
autoradiography (Fig. 6). We found that the
but not the
subunit in native pol
can be cross-linked by UV light to
the radiolabeled BrdUMP-substituted template-primer DNA, and that
complex formation can be competed completely by a
30-fold excess of
unlabeled DNA substrate. This result indicates either that the
subunit does not bind to DNA directly or that it does not make close
contact with template-primer DNA in the native enzyme.
Figure 6:
Photochemical cross-linking of
trypsin-digested Drosophila pol to template-primer DNA.
Polymerase
Fraction VI (7 units, 0.6 pmol) was incubated for 15
min at 20 °C with radiolabeled BrdUMP-substituted template-primer
DNA (1.5 pmol), in the absence (lanes 1 and 2) or
presence of 200 ng (lane 3) or 400 ng (lanes
4-6) of trypsin, and the digestion was terminated by
addition of a large excess of protease inhibitors as described under
``Methods.'' Following incubation, the samples were
irradiated with UV light for 15 min at 0 °C, processed, and
electrophoresed in a 7.5% SDS-polyacrylamide gel, and the gel was
autoradiographed. Lane 1, a UV-irradiated, no protein control; lane 2, pol
irradiated without prior trypsin digestion; lanes 3 and 4, pol
digested with 200 or 400 ng
of trypsin, respectively, prior to UV irradiation; lane 5, a
UV-irradiated, trypsin only control; lane 6, as in lane 2 except that bovine serum albumin was substituted for pol
.
We showed
previously in an in situ gel assay that the subunit of Drosophila pol
contains the DNA polymerase
function(5) . To begin to dissect functional domains in pol
, we subjected the native enzyme to limited tryptic digestion in
the presence of template-primer DNA, followed by UV cross-linking and
then SDS-polyacrylamide gel electrophoresis and autoradiography. We
found that limited tryptic digestion of native pol
produces a
form of the enzyme that retains DNA binding activity, at a level that
produces a cross-linked product comparable in intensity to the intact
enzyme (Fig. 6, lanes 3 and 4 versus lane 2).
In the proteolyzed form, the polymerase catalytic subunit is trimmed
from a 125- to an
65-kDa DNA-binding polypeptide. Staining of the
SDS-polyacrylamide gel with silver indicates two predominant digestion
products of the
subunit of
65 and 55 kDa, and an intact
subunit (data not shown). Notably, the same result is obtained
when template-primer DNA is added before or after digestion with
trypsin. However, the binding of template-primer DNA appears to protect
the enzyme from further degradation, because while nearly quantitative
conversion of the
subunit from a 125- to an
65-kDa
DNA-binding polypeptide is observed in the presence of template-primer
DNA (Fig. 5, lanes 3 and 4), it is cleaved
further to yield smaller polypeptides in the absence of template primer
DNA, at a point where
50% of the
subunit remains intact
(data not shown). These data suggest that the 65-kDa polypeptide
represents both a structural and a functional domain of the
subunit with respect to DNA binding. Whether or not this form of the
enzyme exhibits either DNA polymerase or 3`
5` exonuclease
activity, or if the apparently intact
subunit remains associated,
remains to be determined.
The subunit structure of mitochondrial DNA polymerase is an
unresolved issue. Based on recent studies of the Drosophila(5) , Xenopus(13) , pig(14) , and
human (15) enzymes, we can propose a consensus subunit
structure for animal mitochondrial DNA polymerase, in which a large
polypeptide of 125-140 kDa containing the DNA polymerase
function, is associated quantitatively with a smaller subunit of
35-50 kDa. However, because the frog (13) and pig (14) preparations contain polypeptides of intermediate size,
some of which retain DNA polymerase activity, it is possible that all
of the smaller polypeptides result from proteolysis of the polymerase
catalytic subunit. To address this issue, we examined the intactness of Drosophila pol in a comparison by immunoprecipitation of
crude versus nearly homogeneous enzyme fractions. We found
that both the size and the apparent stoichiometry of the
and
subunits are the same in extracts as compared to that observed in
the isolated enzyme. Furthermore, we found by limited in vitro proteolysis of the two subunits that they are structurally
distinct. We conclude that the 125- and 35-kDa polypeptides that
copurify with the DNA polymerase activity are bona fide subunits of Drosophila pol
. However, the
possibility remains that the two subunits are derived from a transient
precursor polypeptide that is not detected in whole embryo extracts.
This issue can only be resolved when the nuclear genes encoding the two
subunits are isolated.
We explored structural and functional
relationships in native Drosophila pol , in enzyme
inhibition studies, and by limited in vitro proteolysis and
photochemical cross-linking. We developed a potent and highly specific
rabbit antiserum against native pol
and found it to inhibit its
5`
3` DNA polymerase and 3`
5` exonuclease to similar
extents, providing strong evidence of the physical association of the
latter with the two subunit enzyme. In numerous experiments we could
not demonstrate subunit separation with retention of exonuclease
activity, so we are still unable to make a subunit assignment for the
3`
5` exonuclease function. However, even though the
subunit may not contain either active site, our data show that it is
likely critical for maintenance of the catalytic efficiency of both the
DNA polymerase and 3`
5` exonuclease, suggesting a role in the
maintenance of the structural integrity of native pol
. In a
two-dimensional gel-electrophoretic analysis under nondenaturing
conditions, Longley and Mosbaugh (14) identified three forms of
pol
from porcine liver; one contained both DNA polymerase and 3`
5` exonuclease, and the other two contained either the former or
the latter, suggesting that the two activities reside in separate
subunits. In contrast, the MIP1 gene has been shown to encode
both the DNA polymerase and 3`
5` exonuclease activities of the
yeast enzyme(16) .
We examined template-primer DNA binding
by native Drosophila pol in gel electrophoretic mobility
shift and photochemical cross-linking analyses. We found that pol
forms a stable and discrete complex with a 40-nt template-primer DNA,
using a molar ratio as low as 0.1 pol
molecule/template-primer
DNA. Interestingly, several DNA polymerases known to associate with
accessory proteins for catalytic function, including bacteriophage T4
DNA polymerase(27) , E. coli DNA polymerase
III(28) , and calf thymus DNA polymerase
(26) do
not associate stably with template-primer DNA in the absence of the
auxiliary proteins. That Drosophila pol
does so, and
catalyzes relatively efficient DNA synthesis on a variety of
template-primer DNAs in the absence of accessory proteins (5, 6) , might suggest that such factors are not
required for mitochondrial DNA replication. In that regard, however, we
have shown recently that single-stranded DNA-binding protein increases
20-fold the rate of primer recognition and binding by pol
(11, 12) , raising the possibility that
polymerase accessory proteins may enhance its function in
vivo, but not be required under the in vitro conditions
examined.
Photochemical cross-linking of the pol
template-primer DNA complexes revealed that the
but not the
subunit makes close contact with the DNA. Furthermore, limited
proteolysis of the complexes with trypsin identified an
65-kDa
proteolytic intermediate of the
subunit, which retains DNA
binding activity and is stabilized by the presence of DNA during
protease digestion. We are currently evaluating the possibility that
this form of pol
retains enzymatic activity. Notably, the Klenow
fragment of Eco pol I (68 kDa) retains both DNA polymerase and
3`
5` exonuclease activity, while a 46-kDa C-terminal fragment
of the Klenow enzyme retains only DNA polymerase activity(29) .
Given the structural similarity identified among DNA polymerases for
which the three-dimensional structures have been
determined(30) , and the fact that both Eco pol I and
pol
belong to the family A DNA polymerase group(31) , it
seems reasonable to predict conservation of structure-function
relationships between the two enzymes. That considered, it will be
important to discern the structural features in mitochondrial DNA
polymerase that impart its high fidelity and processivity in DNA
synthesis, which distinguish it catalytically from Eco pol I.