©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Structure of Mitochondrial DNA Polymerase from Drosophila Embryos
PHYSICAL AND IMMUNOLOGICAL STUDIES (*)

(Received for publication, August 7, 1995)

Matthew W. Olson Yuxun Wang Rhoderick H. Elder (§) Laurie S. Kaguni (¶)

From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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 alpha but not the beta 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 alpha subunit retains the DNA binding function.


INTRODUCTION

Of the five eucaryotic DNA polymerases (alpha, beta,,,), the mitochondrial DNA polymerase (pol ) (^1)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.


EXPERIMENTAL PROCEDURES

Materials

Nucleotides and Nucleic Acids

Unlabeled deoxyribonucleoside triphosphates were purchased from P-L Biochemicals. [^3H]dTTP was purchased from ICN Biochemicals; [P]dATP was purchased from DuPont NEN. A recombinant M13 viral DNA (10,650 nt) was prepared by standard laboratory methods for use in DNA polymerase and 3` 5` exonuclease assays. Synthetic oligodeoxynucleotides (15 nt) complementary to M13 viral DNA and containing a 3`-terminal base pair (dAMPbulletdTMP) or a 3`-terminal mispair (dAMPbulletdAMP), and the 36-mer 5`-TCTCCATAATTTGAGGGCCTGACTCACCAGGCCGTC3` were synthesized in an Applied Biosystems model 477 oligonucleotide synthesizer.

Enzymes and Proteins

Drosophila pol was prepared as described by Wernette and Kaguni(5) . T4 polynucleotide kinase and E. coli DNA polymerase I Klenow fragment were purchased from New England Biolabs. Bovine serum albumin and prestained and unstained SDS molecular weight protein markers were purchased from Sigma. Protein A-alkaline phosphatase conjugate and protein A-agarose were purchased from Sigma and Boehringer Mannheim, respectively. I-Protein A was purchased from ICN.

Chemicals

Nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate were purchased from Sigma, prepared as 50 mg/ml stocks and stored at 4 °C. N-Chlorosuccinimide and trypsin (type XIII) were purchased from Sigma. Sodium metabisulfite and leupeptin were purchased from the J.T. Baker Chemical Co. and the Peptide Institute, Minoh-Shi, Japan, respectively.

Methods

DNA Polymerase Assay

Polymerase Fraction VI was assayed for DNA polymerase and 3` 5` exonuclease activity on singly primed M13 DNA containing paired or mispaired primers, respectively, as described by Olson and Kaguni(9) . In antibody inhibition studies, preincubation with rabbit antiserum was performed for 60 min at 0 °C under standard assay conditions (9) in the absence of template-primer DNA. Following preincubation, singly primed M13 DNA was added, and DNA polymerase and 3` 5` exonuclease activities were determined after incubation for 20 min at 30 °C.

Preparation of Antiserum

Antiserum directed against Drosophila pol was prepared with the nearly homogeneous Fraction VI enzyme(5) . A virgin female New Zealand White rabbit was immunized with 2.5 µg of pol in Freund's complete adjuvant by injection at or near the popliteal lymph node. Seven booster immunizations, each containing 2 µg of Fraction VI enzyme were administered in Freund's incomplete adjuvant at 2-4-week intervals. Bleedings were performed 8-10 days after each boost.

Preparation of Drosophila Extracts

All operations were performed at 0-4 °C. Drosophila melanogaster (Oregon R) embryos of average age (9 h) were collected immediately before use and suspended at a ratio of 4 ml/gm in 25 mM Hepes (pH 8.0), 10% glycerol, 0.3 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10 mM sodium metabisulfite, 2 µg/ml leupeptin, and 2% sodium cholate and homogenized by 8 strokes in a 7-ml glass homogenizer. The homogenate (embryo extract) was frozen immediately in liquid nitrogen and stored at -80 °C. Mitochondrial extracts were prepared from freshly harvested embryos as described by Wernette and Kaguni(5) .

Protein Gel Electrophoresis, Transfer, and Immunoblotting

SDS-polyacrylamide gel electrophoresis was performed according to Laemmli(23) . Proteins were transferred to nitrocellulose membranes (0.45 µg, Schleicher & Schuell) using a Hoefer Transphor electrophoresis unit model TE22 for 6 h at 70 V in 190 mM glycine, 25 mM Tris base, and 20% (v/v) methanol. The membranes were washed in 20 mM TrisbulletHCl (pH 7.5), 150 mM NaCl and 0.05% (v/v) Tween 20 (TBST) for 10 min and blocked for 2 h in 10 mM TrisbulletHCl (pH 7.5), 150 mM NaCl, and 5% (w/v) nonfat milk. The membranes were probed with anti-DNA polymerase serum (1:1000 in 20 mM TrisbulletHCl (pH 7.5), 150 mM NaCl (TBS) and 0.5% nonfat milk) for 6-10 h and then washed three times for 10 min each in TBST and once for 5 min in TBS. After the washes were complete, incubation (2.5 h) was performed with protein A-alkaline phosphatase conjugate (1:2000 (v/v) or I-protein A (2 µCi/20 ml) in TBS (pH 8.0). The protein A-alkaline phosphatase-treated membranes were washed three times for 10 min each in TBST, once for 5 min in TBS and once for 5 min in 100 mM TrisbulletHCl (pH 9.5), 100 mM NaCl, and 5 mM MgCl(2) (alkaline phosphatase buffer). The membranes were developed by incubation in alkaline phosphatase buffer containing nitro blue tetrazolium (330 µg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (165 µg/ml). The I-protein A-treated membranes were washed three times for 10 min each in TBST and exposed to Kodak XAR-5 film using a DuPont NEN Quanta III intensifying screen.

Immunoprecipitation of Drosophila DNA Polymerase

Polymerase (Fraction I, III or embryo extract, as indicated in the legends to Fig. 1and Fig. 3) was diluted 1:1 with 10 mM NaPO(4) (pH 7.0) and 154 mM NaCl (PBS) and incubated with preimmune serum or polyclonal antiserum overnight on ice. Immune complexes were precipitated by incubation with preswollen protein A-agarose (60 µl of a 50% slurry) for 2 h with gentle rotation. The precipitates were collected by centrifugation and washed once with PBS containing 0.05% (v/v) Tween and twice with PBS, suspended in Laemmli sample buffer (23) , heated for 10 min at 85 °C, and re-centrifuged. The supernatant fractions were then subjected to immunoblot analysis as described above.


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).



Chemical Cleavage of Drosophila DNA Polymerase

Polymerase Fraction VI (3 µg) was subjected to SDS-polyacrylamide gel electrophoresis, and gel slices containing the 125- and 35-kDa polypeptides were excised and subjected to proteolysis with N-chlorosuccinimide as described by Lischwe and Ochs (24) . The proteolytic products were electrophoresed in a 5-15% linear gradient SDS-polyacrylamide gel and stained with silver as described by Wray et al.(25) .

Binding and Photochemical Cross-linking of Drosophila DNA Polymerase to Template-Primer DNA

A radiolabeled deoxyoligomer (40 nt) containing a 3`-terminal bromodeoxyuridylate residue was prepared by incubating the 36-mer (22 pmol) described under ``Materials'' with E. coli DNA polymerase I Klenow fragment (0.4 units) for 10 min at 37 °C in the presence of BrdUTP (16 nmol) and [alpha-P]dATP (26 pmol), in a reaction mixture (0.2 ml) containing 50 mM TrisbulletHCl (pH 8.5), 10 mM MgCl(2), 4 mM dithiothreitol, and 200 µg/ml bovine serum albumin. The mixture was incubated further for 30 min at 37 °C after addition of unlabeled dATP (5 nmol). The reaction was terminated by addition of EDTA to 10 mM, and the mixture was then heated for 15 min at 65 °C. The resulting radiolabeled 40-mer was precipitated with ethanol, resuspended in 10 mM TrisbulletHCl (pH 8.0), 1 mM EDTA, and its length and purity were analyzed by polyacrylamide gel electrophoresis in the presence of 7 M urea.

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 TrisbulletHCl (pH 8.5), 4 mM MgCl(2), 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 times 15 watts) at a distance of 8 cm. After irradiation, the samples were made 1times in Laemmli sample buffer(23) , denatured, and electrophoresed in a 7.5% SDS-polyacrylamide gel (13 times 13 times 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.


RESULTS

Specificity of Antiserum Developed against Native Drosophila DNA Polymerase

We have described Drosophila mitochondrial DNA polymerase as a heterodimer of 125- and 35-kDa subunits, containing both 5` 3` DNA polymerase and 3` 5` exonuclease activities(5, 8) . To begin to elucidate structure-function relationships in pol , we developed a highly specific rabbit antiserum against the nearly homogeneous enzyme (see ``Methods''). In an immunoblot analysis, the rabbit antiserum detects only the 125- and 35-kDa subunits of pol in the Fraction IV enzyme, which is only 8% pure (Fig. 1A, and (5) ). Immunoprecipitation of pol Fraction III yields the same two polypeptides upon subsequent immunoblotting (Fig. 1B). The data indicate that neither the alpha nor the beta subunits of Drosophila pol have been proteolyzed during the course of purification from the Fraction III to Fraction IV stage, and that the antiserum is highly specific and recognizes the native enzyme. Further, notwithstanding the non-quantitative nature of the immunological procedures employed, both the immunoblot and immunoprecipitation analyses of the crude enzyme fractions are consistent with the 1:1 subunit stoichiometry that we determined for the nearly homogeneous Drosophila pol by SDS-polyacrylamide gel electrophoretic analysis, and in hydrodynamic studies that yield a calculated native molecular mass of 160 kDa (5) .

Inhibition of DNA Polymerase and 3` 5` Exonuclease in Drosophila pol by Rabbit Antiserum

The DNA polymerase and 3` 5` exonuclease in Drosophila pol copurify quantitatively(8) . To provide further evidence that the two activities reside in the two subunit enzyme, we performed enzyme inhibition studies of nearly homogeneous pol using the rabbit antiserum. The rabbit antiserum is a potent inhibitor of both DNA polymerase and exonuclease activity (Fig. 2). In contrast, no inhibition of the DNA polymerase or 3` 5` exonuclease activities of E. coli DNA polymerase I is observed (data not shown). Because the antiserum is highly specific for the two subunit enzyme (Fig. 1) and inhibits its DNA polymerase and exonuclease activities to similar extents at equal antiserum concentrations, we conclude that both activities reside in native Drosophila pol .


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 guanidinebulletHCl, urea, or ethylene glycol occurs only upon partial denaturation and substantial loss of enzyme activity; enzyme assay and immunoblot analyses indicate that geq95% of both DNA polymerase and 3` 5` exonuclease activity is lost before subunit dissociation occurs (data not shown).

The Two Subunits of Drosophila pol Are Intact and Distinct

To confirm the proposed subunit structure of native Drosophila pol , immunoprecipitation and in situ gel proteolysis studies were pursued. Immunoprecipitation of native pol from crude enzyme fractions shows that its alpha and beta subunits have most likely not been proteolyzed during the course of purification (Fig. 3). In this analysis, freshly harvested embryos were processed rapidly into whole embryo (lanes 2-4) and mitochondrial (lanes 5-7) extracts which were precipitated with either rabbit antiserum (lanes 2, 3, 5, and 6) or preimmune serum (lanes 4 and 7). It is evident that only the alpha and beta polypeptides in the nearly homogeneous enzyme are identified in the crude protein fractions upon subsequent immunoblotting. Notably, no polypeptides larger than the 125-kDa alpha subunit are observed, and the alpha/beta polypeptide ratio is similar in the crude versus nearly homogeneous fractions.

To demonstrate that the alpha and beta subunits of Drosophila pol are structurally distinct, we performed an in situ gel proteolysis analysis with N-chlorosuccinimide (Fig. 4). The alpha and beta 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 alpha subunit yields 11 polypeptides ranging from 9 to 33 kDa (lane 3), none of which correspond to the four products derived from the beta subunit (lane 5).


Figure 4: Cleavage of Drosophila pol subunits with N-chlorosuccinimide. The alpha and beta 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 alpha subunit (150 ng); lane 3, N-chlorosuccinimide-digested alpha subunit; lane 4, gel-purified beta subunit (150 ng); lane 5, N-chlorosuccinimide-digested beta subunit; lane B, sample buffer only.



Drosophila pol Forms a Discrete Complex with Template-Primer DNA That Involves Close Contact with the alpha Subunit

Drosophila pol catalyzes efficient DNA synthesis on a variety of template-primer DNAs(5, 6) . To begin to elucidate the roles of its two subunits in enzyme function, we examined template-primer DNA binding by native pol . To do so, we used a radiolabeled, partially double-stranded oligomer of 40 nt that contains a 3`-terminal bromodeoxyuridylate residue (see ``Methods'' and Fig. 5C). We observed a single discrete enzyme-DNA complex with Drosophila pol upon analysis of DNA binding by a gel electrophoretic mobility shift assay (Fig. 5A). In contrast, stable complexes involving DNA polymerases alpha and were observed by gel shift assay only in the presence of accessory proteins or cofactors and after glutaraldehyde fixation(26) . Furthermore, the single discrete enzyme-DNA complex with Drosophila pol was observed over an 10-fold range of pol to template-primer DNA molecules (data not shown), and stable complex formation could be competed nearly completely by a 20-fold excess of unlabeled DNA substrate (Fig. 5A).


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 alpha but not the beta 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 leq30-fold excess of unlabeled DNA substrate. This result indicates either that the beta 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 alpha 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 alpha subunit of 65 and 55 kDa, and an intact beta 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 alpha 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 alpha subunit remains intact (data not shown). These data suggest that the 65-kDa polypeptide represents both a structural and a functional domain of the alpha 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 beta subunit remains associated, remains to be determined.


DISCUSSION

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 alpha and beta 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 beta 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 bullet template-primer DNA complexes revealed that the alpha but not the beta subunit makes close contact with the DNA. Furthermore, limited proteolysis of the complexes with trypsin identified an 65-kDa proteolytic intermediate of the alpha 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM45295. 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.

§
Present address: CRC Dept. of Carcinogenesis, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, Manchester, M20 9BX, United Kingdom.

To whom correspondence should be addressed. Tel.: 517-353-6703; Fax: 517-353-9334; lskaguni@pilot.msu.edu.

(^1)
The abbreviations used are: pol, DNA polymerase; nt, nucleotide(s); TBS, Tris-buffered saline; TBST, TBS-Tween; PBS, phosphate-buffered saline.


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