(Received for publication, February 26, 1997)
From the Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824-1319
A full-length cDNA of the accessory ()
subunit of mitochondrial DNA polymerase from Drosophila
embryos has been obtained, and its nucleotide sequence was determined.
The cDNA clone encodes a polypeptide with a deduced amino acid
sequence of 361 residues and a predicted molecular mass of 41 kDa. The
gene encoding the
subunit lies within 4 kilobase pairs of that for
the catalytic subunit in the Drosophila genome, on the left
arm of chromosome 2. The two genes have similar structural features and
share several common DNA sequence elements in their upstream regions,
suggesting the possibility of coordinate regulation. A human cDNA
homolog of the accessory subunit was identified, and its nucleotide
sequence was determined. The human sequence encodes a polypeptide with a predicted molecular mass of 43 kDa that shows a high degree of amino
acid sequence similarity to the Drosophila
subunit. Subunit-specific rabbit antisera, directed against the recombinant catalytic and accessory subunit polypeptides overexpressed and purified
from Escherichia coli, recognize specifically and
immunoprecipitate the native enzyme from Drosophila
embryos. Demonstration of the physical association of the two subunits
in the Drosophila enzyme and identification of a human
accessory subunit homolog provide evidence for a common heterodimeric
structure for animal mitochondrial DNA polymerases.
Animal mitochondria are essential energy-producing organelles that
contain multiple copies of their double-stranded circular DNA genome.
Accumulating evidence documents the involvement of specific
mitochondrial DNA (mtDNA)1 mutations in the
pathogenesis of genetic and aging-related degenerative diseases in
humans that involve the central nervous system, heart, muscle,
endocrine system, kidney, and liver (1). The mitochondrial genome
encodes proteins required for oxidative phosphorylation; it does not,
however, encode any of the proteins that are required for its faithful
duplication. A single nuclear encoded DNA polymerase (pol ) is
involved in the replication of animal mtDNA (2). Because pol
and
all of the other proteins required for mitochondrial DNA replication
are encoded by nuclear genes and imported into mitochondria from the
cytoplasm, mutations in nuclear genes can also affect the integrity of
mtDNA. In fact, human diseases resulting from multiple mtDNA deletions
have been shown to be caused by alterations in nuclear encoded genes
(3). At the same time, antiviral drugs (such as zidovudine)
administered in long term therapy have been shown to induce
mitochondrial dysfunction resembling that in mitochondrial genetic
disease as a result of their inhibitory effects on pol
(4).
We are studying Drosophila embryos as an animal model of
mitochondrial function and have shown that Drosophila pol
in its native form is a heterodimer of 125- and 35-kDa subunits (5, 6). The large (
) subunit contains both 5
3
DNA polymerase and
3
5
exonuclease activities (5, 7). Although we have been unable
to assign a specific biochemical function to the accessory (
)
subunit, we have shown that it is important in maintaining the
catalytic efficiency and/or the structural integrity of the holoenzyme
(6). We have recently cloned and characterized biochemically the
catalytic subunit of Drosophila pol
(7). The catalytic subunit has also been cloned from several yeasts, Xenopus,
man, and mouse (8-11). However, the subunit structure of native pol
from these sources remains an unresolved issue.
We report here the cloning and molecular analysis of the subunit of
Drosophila pol
and demonstrate its association in the
native enzyme. We also describe the identification of a human accessory
subunit homolog, providing evidence for a common heterodimeric structure in animal mitochondrial DNA polymerases.
Materials
Nucleotides and Nucleic AcidsUnlabeled deoxyribonucleoside
triphosphates were purchased from P-L Biochemicals.
[-32P]ATP was purchased from ICN. Plasmid pUC119,
pET-11a, and
gt11 DNAs were prepared by standard laboratory methods.
Synthetic oligodeoxynucleotides as indicated below were synthesized in
an Applied Biosystems Model 477 oligonucleotide synthesizer.
Drosophila pol Fraction
VI was prepared as described by Wernette and Kaguni (5). T4
polynucleotide kinase, T4 DNA ligase, and S1 nuclease were purchased
from Boehringer Mannheim. Taq DNA polymerase and exonuclease
III were from Life Technologies, Inc.
Escherichia
coli LE392 (hsdR514, hsdM, supE44, supF58, lac Y1 or
(lacIZY)6, galK2, galT22, metB1, trpR55) was used for
screening a
gt11 ovarian cDNA library from Drosophila
melanogaster (the generous gift of Dr. Chuen-Sheue Chiang,
Stanford University). E. coli XL1-Blue (recA1, endA1,
gyrA96, thi, hsdR17, supE44, relA1, lac(F
proAB,
lacIqZ
M15, Tn10(tetr))) was
used to subclone the
subunit cDNA for sequence analysis. E. coli BL21(
DE3) (ompT,
rB
MB
) was used for
the expression of pET-11a constructs.
Isopropyl--D-thiogalactopyranoside,
nitro blue tetrazolium, and 5-bromo-4-chloro-3-indolyl phosphate were
purchased from Sigma. Sodium metabisulfite and leupeptin were purchased
from J. T. Baker Inc. and the Peptide Institute (Minoh-Shi, Japan), respectively.
Methods
Sequence Analysis of D. melanogaster polD.
melanogaster pol was prepared as described by Wernette and
Kaguni (5). The enzyme (255 pmol), purified from 1120 g of embryos
with an average age of 9 h, was denatured for 3 min at 65 °C in
1 × Laemmli sample buffer (12) and was electrophoresed on a
5-15% linear gradient SDS-polyacrylamide gel (13 × 13 × 0.15 cm) as described by Laemmli (12). After electrophoresis, the gel
was soaked for 10 min in transfer buffer (10 mM CAPS, pH
11.0, 10% (v/v) methanol, and 0.005% (w/v) SDS), and the 35-kDa
subunit polypeptide was transferred to polyvinylidene difluoride
membrane (Westran, Schleicher & Schuell; presoaked for 10 min in
methanol and then for 15 min in transfer buffer) for 16 h at 150 mA using a Hoefer Transphor electrophoresis unit (Model TE22). The
polyvinylidene difluoride membrane was then stained for 90 s in
0.5% Ponceau S (Sigma) and 1% (v/v) acetic acid and destained for
90 s in 1% (v/v) acetic acid, and the protein-containing membrane
(80 mm2) was excised and rinsed with water. The membrane
was submitted for protein sequence analysis to Harvard MicroChem
(Harvard University), where the membrane-bound protein was digested
with trypsin, and the resulting polypeptides were fractionated by
microbore HPLC and sequenced by automated sequential Edman degradation
using an Applied Biosystems Model 477A pulse liquid peptide sequenator with an on-line Model 120A phenylthiohydantoin-derivative analyzer. The
amino-terminal sequences of five tryptic peptides are indicated in Fig.
1.
Cloning of the
We
used the peptide sequence IVPHDLAEDLNPNDYQAIDIR to generate two
degenerate oligonucleotide primers,
5-GAT(CT)T(CG)GC(ACGT)GA (AG)GA(CT)(CT)T-3
(forward) and
5
-AT(AG)TC(AGT)AT(ACGT)GC (CT)TG(AG)TA-3
(reverse), for use in PCR
synthesis on a
gt11 cDNA library derived from D. melanogaster ovarian mRNA. The PCR amplification was performed for 30 cycles of 94 °C for 90 s, 50 °C for 90 s, and
72 °C for 60 s in a reaction volume of 0.1 ml containing 20 mM Tris-HCl, pH 8.4, 50 mM KCl, 4 mM MgCl2, 1 mM dithiothreitol, 200 µM dATP, 200 µM dCTP, 200 µM
dGTP, 200 µM dTTP, 0.5 µg of
gt11 cDNA, 50 pmol
of forward oligonucleotide primer, 4 pmol of 5
-32P-labeled
reverse oligonucleotide primer (1.1 × 106 cpm/pmol),
and 1.25 units of Taq DNA polymerase. The expected PCR
product of 47 bp was obtained, purified by electrophoresis on a 15%
polyacrylamide gel, and sequenced by the method of Maxam and Gilbert
(13). Based on the nucleotide sequence obtained, a 23-nucleotide
deoxyoligomer, 5
-AGGACCTCAATCCCAACGACTAC-3
, was synthesized and used
to screen the
gt11 ovarian cDNA library by the method of Benton
and Davis (14) using E. coli LE392 as the bacterial
host.
Seven tertiary-screen positive clones were obtained, and one plaque-pure isolate with a 1.7-kilobase pair insert was subcloned and sequenced in its entirety on both strands. The cDNA insert was amplified by PCR and subcloned into plasmid pUC119 at its unique EcoRI site using E. coli XL1-Blue as the bacterial host. Fourteen nested deletion plasmids were generated using exonuclease III and S1 nuclease (15). A nucleotide sequence of 1569 bp was determined on both DNA strands by automated fluorescent DNA sequencing using the Applied Biosystems Catalyst 800 for Taq cycle sequencing and a Model 373 DNA Sequencer for the analysis of products. The complete cDNA sequence was assembled using the Sequencher Version 2.1.1 software package.
Bacterial Subcloning, Overexpression and Purification of theThe 1083-bp coding
sequence of the subunit was engineered by PCR amplification of the
gt11 cDNA to contain NdeI restriction endonuclease
sites at its ends. The PCR-amplified DNA fragment was purified by gel
electrophoresis, cleaved with NdeI, and cloned into the
bacteriophage T7 promoter-based expression vector pET-11a (Novagen) at
its unique NdeI site. The E. coli strain
BL21(
DE3) (Novagen) was used for transformation, and
ampicillin-resistant plasmid-containing cells were screened for insert
size and orientation of recombinant DNA by restriction analysis.
For overexpression of the subunit, the recombinant
plasmid-containing BL21(
DE3) cells were grown at 37 °C with
aeration in Luria broth containing 100 µg/ml ampicillin. When the
cells reached an absorbance of 0.6 at 595 nm,
isopropyl-
-D-thiogalactopyranoside was added to 0.3 mM, and the culture was incubated further for 45 min. Cells
were harvested by centrifugation; washed in 50 mM Tris-HCl,
pH 7.5, and 10% sucrose; recentrifuged; frozen in liquid nitrogen; and
stored at
80 °C.
Preparation of bacterial cell extracts was as described for the catalytic subunit by Lewis et al. (7), except that the lysis buffer contained 0.1 M NaCl, and detergents (0.5% (w/v) deoxycholic acid and 0.5% (v/v) Nonidet P-40) were added to the cell extract prior to centrifugation to recover the soluble fraction. The resulting pellet was resuspended in 0.025 volume of original cell culture in 0.5% Triton X-100 and 1 mM EDTA; then the sample was recentrifuged for 10 min, and the resulting supernatant fluid was removed and discarded. The previous step was repeated three times, and the final "detergent-washed, insoluble pellet" was extracted with 7 M urea as described by Lewis et al. (7).
For antibody production, the recombinant - and
subunits obtained
from urea-extracted insoluble fractions were electrophoresed on a 10%
SDS-polyacrylamide gel, and the protein-containing bands were excised.
The gel slices were minced in 10 mM NaPO4, pH
7.0, and 154 mM NaCl (PBS) and emulsified with an equal
volume of Freund's complete adjuvant. Immunizations of virgin female
New Zealand White rabbits were performed by subcutaneous injections of
15-30 µg of protein. Booster immunizations were administered in
Freund's incomplete adjuvant at 2-4-week intervals.
SDS-polyacrylamide gel electrophoresis was performed according to Laemmli (12). Proteins were transferred to nitrocellulose membranes (BA85, Schleicher & Schuell) and probed by immunoblotting using the goat anti-rabbit IgG-alkaline phosphatase (Bio-Rad) method as described by Olson et al. (6).
Immunoprecipitation of Drosophila DNA PolymerasePol
Fraction VI (~1 µg) was diluted 1:1 with PBS, incubated for 10 min at 65 °C, and then incubated with preimmune serum or
subunit-specific rabbit antiserum overnight on ice. Immune complexes
were precipitated by incubation with preswollen protein A-agarose (50 µl of a 50% slurry) for 2 h with gentle rotation. The
precipitates were collected by centrifugation, washed three times in
PBS, suspended in Laemmli sample buffer, heated for 10 min at 85 °C,
and recentrifuged. The supernatant fractions were then subjected to
immunoblotting analysis as described by Olson et al.
(6).
D. melanogaster mitochondrial DNA polymerase
holoenzyme was purified to near homogeneity from embryonic mitochondria
as described by Wernette and Kaguni (5). The enzyme derived from
1120 g of embryos was subjected to SDS-polyacrylamide gel
electrophoresis, and the subunit polypeptide was transferred to
polyvinylidene difluoride membrane. Following tryptic digestion and
fractionation of the resulting peptides by microbore HPLC, five
amino-terminal sequences were obtained. Degenerate oligonucleotides
were constructed that corresponded to amino- and carboxyl-terminal
amino acid residues of one of the peptides and were used to amplify by
PCR D. melanogaster cDNA fragments as described under
"Methods." A correctly sized PCR product was recovered by gel
purification, and DNA sequence analysis confirmed that it corresponded
to the amino acid sequence of the tryptic peptide. A 23-nucleotide DNA
probe was then synthesized and used to screen a
gt11 cDNA
library derived from D. melanogaster ovarian mRNA.
Tertiary-screen positive clones (seven) were obtained at a frequency of
~1.5 × 10
6 phage screened, a frequency comparable
to that found in screening the same library for catalytic subunit
cDNA clones (~5 × 10
6) (7). The largest
cDNA was 1.7 kilobase pairs in length, and its nucleotide sequence
was determined in its entirety on both DNA strands. We also determined
the sequence of the 5
- and 3
-ends of four additional independently
isolated cDNAs. All contained an identical 3
-end sequence and
differed only in the length of the 5
-untranslated region.
An open reading frame of 1083 nucleotides was identified, encoding a
subunit polypeptide of 361 amino acid residues with a predicted
molecular mass of 41 kDa (Fig. 1). This is consistent with the size of 35 kDa determined for the small subunit in purified pol
(5). The amino-terminal sequences of all five tryptic peptides
derived from the
subunit of the native enzyme are located within
the deduced amino acid sequence of the
subunit cDNA. Because of
the limited amount of
subunit polypeptide derived from native pol
, we did not obtain the amino-terminal sequence of the mature
subunit. However, because the amino terminus of one of the tryptic
peptides is located at position +19 in the deduced amino acid sequence,
the mitochondrial presequence peptide is no longer than 18 amino acid
residues. No other in-frame translational initiation codon is present
between the 5
-end of the cDNA and the ATG codon at position +1,
although there are multiple in-frame stop codons.
No
homolog of the subunit was identified upon searching the latest
releases of the GenBankTM and EMBL data bases (Releases 98 and 49, respectively) or the Saccharomyces genome data base. However, we detected a partial human cDNA sequence (GenBankTM
accession number H05453[GenBank]) in the dbEST (expressed sequence tag) data
base of the National Center for Biotechnology Information in a BLAST
search using the deduced amino acid sequence of the Drosophila
subunit as query. We obtained the human
cDNA (I.M.A.G.E. Consortium (LLNL) Clone ID number 44673),
determined the nucleotide sequence in its entirety on both DNA strands
(1606 bp), and found it to contain a complete open reading frame.
The human subunit homolog has a deduced amino acid
sequence of 372 residues, comparable to that of 361 residues for the Drosophila polypeptide. Alignment of the
Drosophila and human sequences using the Gap program of the
Genetics Computer Group program package (Version 7) shows 31% amino
acid sequence identity and 60% sequence similarity overall. The
C-terminal regions contain a leucine zipper motif with the sequences
L(X)6L(X)6L(X)6L
(Drosophila, positions +321-342) and
V(X)6L(X)6L(X)6M
(human, positions +328-349) (Fig. 2). This 22-amino
acid stretch represents the most highly conserved sequence in the
polypeptide, with 55% sequence identity and 86% similarity. The
Drosophila
subunit also contains a zinc finger motif of
the C2H2 class on the amino-terminal side of the leucine zipper motif
(positions +257-285). Although the human homolog lacks this motif, it
contains a zinc-binding signature sequence (LGDHELLHMY) found in
neutral zinc metallopeptidases (16), near the middle of the polypeptide
at positions +193-202.
Genomic DNA Structure of the
The data base search revealed an unidentified genomic
sequence corresponding to the subunit cDNA that was derived
from a D. melanogaster P1 genomic DNA library (GenBankTM
accession numbers L39626[GenBank] and L39627[GenBank]). The genomic sequence encoding the
subunit is located on the left arm of chromosome 2 (subdivision 34D-34E) near the region containing the alcohol dehydrogenase gene
(subdivision 35B). Remarkably, the same P1 clone contains the catalytic
subunit gene of D. melanogaster mitochondrial DNA polymerase
(mtPolA) (7). Furthermore, the genes encoding the two
subunits of the native holoenzyme are separated only by an interval
sequence of 3.8 kilobase pairs, with the
subunit gene located 5
to
the catalytic subunit gene (Fig. 3).
Comparison of the genomic and cDNA sequences indicates that the subunit gene contains only one small intron of 54 nucleotides that
splits the codon specifying Ala231 in the deduced amino
acid sequence. The intron/exon boundaries are GTAAGT (donor site) and
TAG (acceptor site), respectively. The coding sequence of the exons in
the genomic clone is 99% identical to that of the corresponding
regions of the cDNA. There are two single nucleotide deletions in
the genomic sequence that disrupt the reading frame and 11 mismatches
between the genomic and cDNA sequences. Of the latter, only five
would result in amino acid substitutions. These may represent genetic
variation among the different fly strains or derive from errors in
sequencing of the genomic clones.
A search for RNA polymerase II promoter elements in the genomic DNA
sequence in the region upstream of the putative transcriptional initiation site (5-end of the longest cDNA) did not reveal a consensus TATA sequence. A sequence matching the consensus
transcriptional initiator TCAGT (17) is located in the genomic sequence
16 bp upstream from the 5
-end of the cDNA sequence, and the
translational initiation codon is located at a position 459 bp
downstream (Fig. 3). Two additional consensus transcriptional initiator
sequences are located within 500 bp in the upstream region.
Interestingly, a Drosophila promoter-activating element,
DRE, previously identified in a number of genes involved in nuclear DNA
replication (18), is located 230 bp upstream from the putative
transcriptional start site. Furthermore, potential binding sites for
nuclear respiratory factor 1 (NRF-1) (19) and the transcription factor
E2F (20) are located at positions
807 and
295, respectively.
Notably, the consensus DRE (position
2028) and potential NRF-1
(positions
53,
494, and
860)- and E2F (position
899)-binding
sites are also present in the upstream region of the catalytic subunit
gene of Drosophila pol
.
The 3-end of the cDNA clone contains an 18-nucleotide poly(A)
sequence. Surprisingly, the poly(A) sequence begins within the
translational termination codon (TAA). Based on the comparison of the
cDNA and genomic sequences, the polyadenylation site is located no
more than six nucleotides downstream from the termination codon. This
feature is common in mitochondrially encoded genes and may be of
evolutionary significance. As with the catalytic subunit cDNA (7),
no match to the consensus poly(A) signal sequence (AATAAA) is present
in the
subunit cDNA, although 23 out of 24 of the nucleotides
immediately upstream of the termination codon are deoxyadenylate or
thymidylate residues. Interestingly, both the catalytic and
subunit
cDNAs contain the sequence AATATA (at positions
51 and
16,
respectively, relative to the polyadenylation site), which might serve
as the poly(A) signal. This alternative signal has been implicated in
directing polyadenylation of developmentally regulated transcripts in
several Drosophila genes (21-24). Taken together, the
presence and locations of common transcriptional and
post-transcriptional regulatory signals in the catalytic and accessory
subunit genes of Drosophila pol
suggest the possibility of coordinate gene regulation of the heterodimeric mitochondrial DNA
polymerase.
To elucidate
structure-function relationships in Drosophila pol , we
pursued bacterial overexpression and purification of the
subunit
and prepared rabbit antisera to both subunits. To do so, we engineered
by PCR an NdeI site surrounding the ATG codon at position +1
of the
subunit and at a site distal to the termination codon. We
then inserted the coding region fragment into the NdeI site
of the bacteriophage T7 promoter-based expression vector pET-11a.
Overexpression upon isopropyl-
-D-thiogalactopyranoside induction of plasmid-containing BL21(
DE3) cells yielded ~7
µg of
subunit polypeptide/ml of cell culture. Despite the use of a variety of induction and lysis conditions, only ~5% of the
overproduced protein remained in the soluble fraction upon cell lysis.
Thus, we pursued the purification of the
subunit from insoluble
fractions. We used the detergent washing procedure of Nagai and
Thogersen (25) to obtain inclusion body fractions, which were then
extracted with 7 M urea.
Protein analysis by SDS-polyacrylamide gel electrophoresis, followed
either by silver staining or by immunoblot analysis with rabbit
antiserum against native pol from Drosophila embryos, identifies the overexpressed polypeptide of 35 kDa as the intact
subunit (Fig. 4, A and B,
respectively). The homogeneous
subunit (lane 5) was
gel-purified and used to develop a highly specific rabbit antiserum. A
similar purification procedure was used to prepare the catalytic
subunit for antiserum production (7). The subunit-specific rabbit
antisera recognize exclusively the
- and
subunits of
Drosophila pol
. In an immunoblot analysis, the antisera
detect only the 125- and 35-kDa subunits of native pol
in the
Fraction III enzyme, which is only ~7% pure (Fig. 5A) (5).
Association of the
Despite extensive efforts to renature the catalytic and subunit polypeptides derived from urea-extracted insoluble fractions, we were unable to reconstitute a recombinant form of the pol
heterodimer. Likewise, we were unable to observe enzyme assembly upon
bacterial coexpression of the two subunits2
under conditions similar to those employed to reconstitute the heterotrimeric human replication factor A (26). Thus, we sought to
demonstrate the association of the
subunit in native pol
. To do
so, we performed an immunoprecipitation analysis using the
subunit-specific antisera developed against the recombinant subunits.
While neither the
- nor
subunit antisera were able to inhibit
the DNA polymerase activity of Drosophila pol
(data not
shown), both were able to immunoprecipitate the native mitochondrial enzyme. Immunoprecipitation of pol
Fraction III with either the
catalytic or
subunit antiserum yields the same two polypeptides upon subsequent immunoblotting with pol
antiserum (Fig.
5B, lanes 3 and 4, respectively). The
data demonstrate that both of the subunit-specific antisera recognize
the native enzyme and that the catalytic and
subunit polypeptides
form a physical complex in native pol
. These data are consistent
with subunit dissociation studies of native pol
that showed that
subunit separation by gel filtration or velocity sedimentation in the presence of denaturants likely occurs only upon partial denaturation of
the enzyme (6).
The redundant nature of mitochondrial DNA does not eliminate the
requirement for high fidelity DNA replication because the accumulation
of mutations in mtDNA is linked to degenerative diseases in humans (1).
The key replicative enzyme in mitochondria is pol . Mitochondrial
DNA polymerases from several species including chicken, pig, fly, frog,
yeast, and man have been demonstrated to contain dual enzymatic
activities (27-32). They possess both highly accurate DNA polymerase
and mispair-specific 3
5
exonuclease activities. While we have
shown that Drosophila pol
is a heterodimer of 125- and
35-kDa subunits (5), the structure of other mitochondrial DNA
polymerases remains unresolved.
In a recent report, we presented physical and immunological evidence
that the 35-kDa subunit of Drosophila pol is intact and
structurally distinct from the 125-kDa catalytic subunit (6). Here we
demonstrated that the
subunit is encoded by a distinct nuclear gene
and bears no similarity to the catalytic subunit in its amino acid
sequence. Furthermore, we found that rabbit antisera directed against
recombinant polypeptides recognize specifically and immunoprecipitate
the native enzyme. We conclude that the 35-kDa polypeptide is a
bona fide subunit of Drosophila pol
.
The nuclear gene encoding the subunit is located at a distance only
3.8 kilobase pairs from the catalytic subunit gene, on the left arm of
chromosome 2. That the genes encoding the two subunits of the
mitochondrial holoenzyme are linked in the nuclear genome may be of
evolutionary significance given current hypotheses regarding the
bacterial origin of mitochondria. At the same time, the phenomenon may
relate to the present day expression of the genes. The genomic
location, similar gene structure (i.e. TATA-less promoter,
presence of consensus transcriptional initiator and alternative poly(A)
signal sequences, small introns, and short 3
-untranslated region), and
the presence of the DRE and potential binding sites for transcription
factors NRF-1 and E2F in the 5
-upstream region suggest that the two
genes share common regulatory mechanisms and the possibility that they
are coordinately regulated. The DRE is the recognition sequence for an
80-kDa DNA-binding protein, DREF (18, 33), that is responsible for
activating the promoters of nuclear replication genes in
Drosophila, including proliferating cell nuclear antigen-,
cyclin A-, and DNA polymerase
-encoding genes (34, 35). Functional
NRF-1 sites have been identified in many mammalian nuclear genes that
encode mitochondrial proteins. These include cytochrome c,
mitochondrial transcription factor A, and at least one subunit of each
of the respiratory complexes III, IV, and V (19, 36). Eight members of
the E2F family in mammals have been shown to form heterodimeric
complexes that are crucial for transcriptional activation of genes
regulating S phase entry (c-myc and cyclin E) and genes
functioning to engage DNA synthesis (dihydrofolate reductase, thymidine
kinase, proliferating cell nuclear antigen, and pol
) (20).
Drosophila E2F-1 is essential for activation of pol
gene
expression (37) and is required for the G1 to S phase
transition during embryogenesis (38). That the DRE, NRF-1, and E2F
sequence elements are found in the Drosophila pol
genes
is consistent with the facts that mitochondrial DNA polymerase is
required for mtDNA replication and hence for cell proliferation and
that pol
activity varies greatly during development and is highest
in embryos (5).
Molecular cloning, bacterial overexpression, and biochemical analysis
of the catalytic subunit allowed us to assign both 5
3
DNA
polymerase and 3
5
exonuclease functions to the large subunit of
Drosophila pol
(7). Likewise, Foury (31) had shown, via
its overexpression in mitochondrial extracts, that the 140-kDa product
of the MIP1 gene contains both activities in
Saccharomyces cerevisiae pol
. Cloning and sequence
alignments have also shown that a large polypeptide of 115-140 kDa
contains both the conserved DNA polymerase and exonuclease domains in
pol
from several yeasts, Xenopus, man, and mouse
(8-11). However, pol
purified from Xenopus, pig, and
human cells has been shown to contain a large polymerase catalytic
subunit and several smaller polypeptides, leaving unresolved the issue
of the native structure of these enzymes (28, 32, 39). Our
identification of a human homolog of the accessory subunit of
Drosophila pol
provides evidence for a common structure
in animal mitochondrial DNA polymerases.
The accessory subunit of Drosophila pol and its human
homolog represent novel proteins of 41 and 43 kDa, respectively. They contain a single highly conserved amino acid sequence, a leucine zipper
motif in the C terminus of the polypeptides. Notably, a single leucine
zipper motif is also present in the catalytic subunit sequences of
Drosophila, Xenopus, man, and mouse (7, 10, 11).
In each case, the leucine zipper motif lies between the conserved DNA
polymerase and exonuclease domains. We suggest that the putative
leucine zipper domains in the catalytic and accessory subunits
represent the subunit interaction domain. If so, we would propose that
both the heterodimeric composition and the structural basis for subunit
interaction are conserved among animal pol
holoenzymes.
What then is the role of the accessory subunit in Drosophila
pol ? To date, we have been unable to assign it a biochemical function. We have shown that Drosophila pol
is both
highly processive and highly accurate in nucleotide polymerization (40,
41), but have been unable to achieve subunit separation without nearly complete loss of catalytic activity (6). We have, however, demonstrated
DNA polymerase and 3
5
exonuclease activities in the bacterially
expressed catalytic subunit, albeit at ~20-fold reduced specific
activity (7). Because the leucine zipper motif lies between the DNA
polymerase and exonuclease domains in the catalytic subunit and
structural studies on enzyme-DNA cocrystals identified a role for this
region in template-primer DNA binding by E. coli DNA
polymerase I Klenow fragment (42), we speculated that the small subunit
may be involved in pol
processivity (7). Remarkably, Richardson and
co-workers (43) have recently shown that the processivity subunit of
bacteriophage T7 DNA polymerase, E. coli thioredoxin, can
also confer high processivity on an E. coli DNA polymerase I
recombinant that was engineered to contain, between its exonuclease and
DNA polymerase domains, the thioredoxin-binding domain of the T7
catalytic polypeptide. Notably, although each of these DNA polymerases
is a member of the family A DNA polymerase group (44), a heterodimeric
holoenzyme structure extends beyond this group. For example, herpes
simplex virus DNA polymerase and nuclear pol
are members of the
family B group (44). In herpesvirus replication, the virus-encoded UL42
protein functions as a processivity factor for the herpes DNA
polymerase (45). The small subunit of animal pol
apparently
functions to recruit its processivity factor, proliferating cell
nuclear antigen (46-49). Inasmuch as Drosophila pol
is
highly processive in its heterodimeric form, we would suggest that the
subunit is itself the processivity factor. Further biochemical
characterization of the recombinant catalytic subunit and
reconstitution studies of the holoenzyme form should allow us to
resolve this issue and to determine the structural basis for holoenzyme
assembly.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) U94702[GenBank] and U94703[GenBank].
We thank Dr. David L. Lewis for contributions
to the amino acid sequence analysis of the subunit polypeptide. We
also thank Anthony T. Lagina III for efforts in the construction and
analysis of a recombinant plasmid for bacterial coexpression of the pol
subunits and Li Fan for helpful discussions.