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Molecular and Cellular Biology, June 1999, p. 4039-4046, Vol. 19, No. 6
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Accessory Subunit of Xenopus laevis Mitochondrial
DNA Polymerase 
Increases Processivity of the Catalytic Subunit
of Human DNA Polymerase and Is Related to Class II
Aminoacyl-tRNA Synthetases
José A.
Carrodeguas,1
Ryuji
Kobayashi,2
Susan E.
Lim,3
William C.
Copeland,3 and
Daniel
F.
Bogenhagen1,*
Department of Pharmacological Sciences, State University of
New York at Stony Brook, Stony Brook, New York
11794-86511; Cold Spring Harbor
Laboratory, Cold Spring Harbor, New York 117242;
and Laboratory of Molecular Genetics, National Institute of
Environmental Health Sciences, Research Triangle Park, North Carolina
277093
Received 11 December 1998/Returned for modification 20 January
1999/Accepted 10 March 1999
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ABSTRACT |
Peptide sequences obtained from the accessory subunit of
Xenopus laevis mitochondrial DNA (mtDNA) polymerase 
(pol ) were used to clone the cDNA encoding this protein.
Amino-terminal sequencing of the mitochondrial protein indicated the
presence of a 44-amino-acid mitochondrial targeting sequence, leaving a
predicted mature protein with 419 amino acids and a molecular mass of
47.3 kDa. This protein is associated with the larger, catalytic subunit
in preparations of active mtDNA polymerase. The small subunit exhibits
homology to its human, mouse, and Drosophila counterparts.
Interestingly, significant homology to glycyl-tRNA synthetases from
prokaryotic organisms reveals a likely evolutionary relationship. Since
attempts to produce an enzymatically active recombinant catalytic
subunit of Xenopus DNA pol have not been successful, we
tested the effects of adding the small subunit of the
Xenopus enzyme to the catalytic subunit of human DNA pol
purified from baculovirus-infected insect cells. These experiments
provide the first functional evidence that the small subunit of DNA pol
stimulates processive DNA synthesis by the human catalytic subunit
under physiological salt conditions.
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INTRODUCTION |
Mitochondrial DNA (mtDNA) is
replicated by a DNA polymerase, DNA polymerase (pol ), that is
distinct from nuclear DNA polymerases 
, 
, 
, 
, and 
.
Since DNA pol represents only a small fraction of total cellular
DNA polymerase, purification and characterization of the subunit
composition of this enzyme have been difficult. Molecular cloning has
contributed greatly to understanding the structure of DNA pol in
different organisms. The catalytic subunits of DNA pol have been
cloned for several organisms (6, 16, 17, 27, 34) and have
been found to resemble family A of DNA polymerases, related to
Escherichia coli DNA pol I. In Saccharomyces cerevisiae DNA pol is composed of a single polypeptide, while in Drosophila melanogaster DNA pol is comprised of two
different polypeptides, a catalytic subunit of 125 kDa and an accessory subunit of 41 kDa (24, 33). The Drosophila
subunits copurify and have been shown to interact, but the recombinant
proteins have not yet been shown to be functional. The function of the small subunit, which we refer to as pol B, is unknown. It has been
proposed to influence the processivity of the catalytic subunit. Putative mammalian homologs of the Drosophila accessory
subunit have been identified in sequence databases. One published
purification scheme for human DNA pol suggested the existence of a
small subunit (11), but the potential relationship between
this polypeptide and Drosophila pol B has not been
established. Recently, the catalytic subunit of human DNA pol was
expressed in an active form (10, 19). Surprisingly, the
recombinant catalytic subunit alone displayed most of the
characteristics of the enzyme purified from human cells, which did not
appear to contain a stoichiometric amount of a small subunit. Thus, it
is not clear what role, if any, is played by putative human DNA pol
B.
We have previously described the cloning of the large subunit of
Xenopus laevis mtDNA polymerase (34). Here we
describe the cloning and initial characterization of the accessory
subunit of Xenopus mtDNA polymerase, which is homologous to
its Drosophila and human counterparts. The cDNA encodes a
protein of 463 amino acids with a mitochondrial targeting sequence of
44 amino acids. Both the catalytic subunit and the accessory subunit
copurify through several steps of chromatography, and
coimmunoprecipitation assays indicated that they are associated in a
holoenzyme. We were not able to study the effect of the small subunit
on the catalytic subunit of Xenopus pol , since the
recombinant Xenopus catalytic subunit was inactive following
expression in bacteria or insect cells. Therefore, we attempted to
determine whether the Xenopus small subunit would stimulate
the recombinant human catalytic subunit. Our data indicates that the
Xenopus accessory subunit confers processivity on the human
catalytic subunit. A striking similarity found between the accessory
subunit and some class II aminoacyl-tRNA synthetases suggests a common
evolutionary origin.
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MATERIALS AND METHODS |
Purification of Xenopus pol and sequencing of the
small subunit.
Purification of X. laevis DNA pol has been described elsewhere (15, 23). Sodium dodecyl
sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) separated two
polypeptides of 140 and 45 kDa. The smaller polypeptide was gel
purified and subjected to digestion with endoprotease LysC, and
peptides were sequenced by Edman degradation as described previously
(32). The intact small subunit was also subjected to direct
N-terminal sequencing after blotting to a polyvinylidene difluoride
(PVDF) membrane (Immobilon P; Millipore).
Cloning.
Degenerate primers were synthesized from peptide
sequences, and two rounds of nested PCR were carried out. Primer XLGB1
(GARTAYGTICCIWSIATGGA; sense strand, EYVPSME; R is A or G, Y is C
or T, W is A or T, and S is C or G), primer XLGB2
(TTISWIACRTGIATIGTYTC; antisense strand, ETIHVSK), primer XLGB3
(AARGARACIATICAYGT; sense strand, KETIHV), primer XLGB4
(ACRTGIATIGTYTCYTT; antisense strand, KETIHV), and primer XLGB5
(CAYCCIACIYTIGCICC; sense strand, HPTLAP) were used in
"touchdown" PCR (5). Nested PCR with primers XLGB1 and XLGB4 followed by primers XLGB5 and XLGB4 yielded a 281-bp PCR product that was cloned by TA cloning (Invitrogen) and sequenced.
A probe made from this clone was used to screen 500,000 clones from a
lambda gt11 Xenopus cDNA library made with oligo(dT) and
random primers (Clontech). Eight positive clones were identified and
analyzed by PCR. All of them contained inserts of the same size, and
one of them, designated pJAC3, was subcloned in pBluescript KS
(Stratagene). This 1,491-bp cDNA clone was completely sequenced on both
strands and found to contain an open reading frame coding for a protein
of 463 amino acids and including a short 5' untranslated region (UTR)
and a 3' UTR with a putative polyadenylation signal located 25 bp
upstream from the beginning of a poly(A) tail.
Protein expression.
A PCR primer was designed to contain an
NdeI site positioned to permit translation initiation at an
ATG codon immediately preceding the first residue of the mature
mitochondrial protein (without the 44-amino-acid mitochondrial
presequence). Another primer was made with a NotI site at
the position of the stop codon for the protein to permit in-frame
linkage to a C-terminal His tag in the vector. PCR with these primers
and pJAC3 DNA as a template was used to amplify a fragment of DNA that
was subsequently digested with NdeI and NotI and
cloned into pET22b(+) (Novagen) to generate clone pJAC8. Induction of
expression of the protein from this clone in E. coli
BL21(DE3) produced a C-terminally His-tagged version of the
mitochondrial protein containing an extra methionine in the amino
terminus. The majority of this protein was insoluble under native
conditions. When the protein was solubilized with urea, it did not bind
well to an Ni-nitrilotriacetic acid (NTA) column (Qiagen).
Electrophoretic purification with a Prep-Cell 491 apparatus (Bio-Rad)
was used to prepare protein to inoculate a rabbit (13).
For baculovirus expression, the pFastBac I vector (Life Technologies)
was modified to contain a His tag derived from pET22b(+)
in such a way
that a C-terminally His-tagged protein could be
produced. Briefly,
pET22b(+) was digested with
BlpI, an isoschizomer
of
Bpu1102I, and the ends were made blunt by filling in with
Pfu polymerase and then digested with
NotI to
generate a
NotI-blunted
fragment coding for a His tag. In a
similar way, pFastBac I was
digested with
HindIII, and
the ends were made blunt and then cut
with
NotI. The
NotI-blunted fragment from pET22b(+) was then cloned
in
pFastBac I, regenerating the
HindIII site but not the
BlpI
site. An oligonucleotide was used to insert an
NdeI restriction
site following the
BamHI site in
pFastBac I. This step allowed
cloning of the
NdeI-
NotI insert directly from pJAC8 into the
modified
baculovirus expression vector. The resulting clone was named
pJAC30.
Expression in
Spodoptera frugiperda (Sf9) cells was
achieved by
following the procedures recommended by the manufacturer of
the
Bac-to-Bac baculovirus expression system (Life Technologies).
The
C-terminally His-tagged protein was purified by affinity chromatography
on Ni-NTA superflow columns (Qiagen) with phosphate buffer under
native
conditions as recommended by the supplier, followed by
chromatography
on Mono S. The protein was concentrated by a second
round of
chromatography on Ni-NTA resin. The His-tagged recombinant
catalytic
subunit of human DNA pol
was expressed and purified
as described by
Longley et al. (
19). To quantify the recombinant
proteins,
various quantities of the catalytic and accessory subunits
were
subjected to SDS-PAGE along with various amounts of quantitative
protein standards (glutamate dehydrogenase and phosphorylase
b;
Boehringer Mannheim). The protein concentrations were
determined
by densitometry of the Coomassie blue-stained
gel.
Antibody methods.
Proteins were separated by SDS-PAGE with
10% gels, electroblotted onto Immobilon P membranes, and incubated
overnight at room temperature with a 1:20,000 dilution of antiserum in
phosphate-buffered saline containing 0.5% Tween 20. In one experiment
(see Fig. 3), polyclonal sera directed against Xenopus pol
A and pol B were used. In another experiment (see Fig. 4), an
antipeptide serum directed against the sequence TRRAVEPTWLTASN(C)
was used to detect either Xenopus or human DNA pol A
(34). Proteins were detected by successive incubation of the
blots with a commercial alkaline phosphatase-conjugated goat
anti-rabbit secondary antibody and 5-bromo-4-chloro-3-indolylphosphate-nitroblue tetrazolium (Kirkegaard and Perry Laboratories). Affinity-purified antibodies against Xenopus DNA pol B were prepared by adsorption of
antibodies to a column containing a recombinant protein cross-linked to
an Affiprep 10 matrix (Bio-Rad) and elution with 50 mM glycine (pH
2.3)-0.5 M NaCl-0.02% Triton X-100. The solution was quickly
neutralized by the addition of one-fourth volume of 0.2 M
Na2HPO4.
DNA polymerase assays.
DNA pol activity was calibrated
in reactions with poly(rA)-oligo(dT) as described previously
(15). One unit corresponds to the incorporation of 1 pmol of
deoxynucleoside monophosphate into acid-insoluble products in a 30-min
reaction. All polymerase assays with DNA templates were performed at
30°C with a buffer consisting of 10 mM Tris (pH 8); 50 mM KCl; 8 mM
MgCl2; 2 mM dithiothreitol; 25 µM each TTP, dATP, dGTP,
and dCTP; and 100 µg of bovine serum albumin per ml (the KCl and
MgCl2 concentrations were varied as noted in the figures).
For primer extension reactions, oligonucleotide dT24 or
dT16 and the M13 
20 sequencing primer
(GTAAAACGACGGCCAGT) were phosphorylated with
[-32P]ATP and polynucleotide kinase and gel purified
by standard methods (28). Oligo(dT) was annealed to poly(dA)
(estimated chain length, ~1 to 1.2 kb), and the 20 primer was
annealed to M13mp9 DNA in a buffer containing 100 mM NaCl, 10 mM Tris
(pH 7.5), and 1 mM EDTA. Primer extension reactions included a final
concentration of 20 nM primer-template. Samples were withdrawn at
various times, mixed with 2 volumes of formamide DNA loading solution,
boiled, and analyzed by electrophoresis on 20% polyacrylamide gels
containing 8 M urea.
DNA binding assays were conducted by an electrophoretic mobility shift
assay as described by Mikhailov and Bogenhagen (
23)
with 40 fmol of a 44-mer oligonucleotide
(5'-
32P-CCATCTAAGCAGACTCACGAATTCACCTAGTTGTTCTAGGTGAA)
labeled with [
-
32P]ATP and polynucleotide
kinase. This oligonucleotide sequence
is expected to fold into a
partial hairpin with a 9-bp duplex
and a 19-nucleotide single-stranded
tail to provide a primer-template
structure. The quantities of
polymerase used in binding reactions
are specified in the figure
legends.
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RESULTS |
Cloning of the X. laevis mtDNA polymerase accessory
subunit.
DNA pol was purified from X. laevis ovary
mitochondria as previously described (15, 23). SDS-PAGE
analysis of the purified enzyme revealed the presence of two
polypeptides, of approximately 140 and 45 kDa (Fig.
1). The larger polypeptide has been
previously identified as the catalytic subunit of DNA pol (15,
34). The smaller polypeptide was assumed to be the accessory
subunit of X. laevis DNA pol by analogy to the situation
for Drosophila (33). The purified small subunit
was subjected to amino-terminal and internal sequencing. For internal
protein sequencing, the gel-purified protein was digested with
endoprotease LysC, and peptides were separated by high-pressure liquid
chromatography and sequenced. PCR was carried out on first-strand cDNA
(synthesized from Xenopus ovary mRNA) by use of degenerate
primers derived from those peptides. A 281-bp product was amplified,
cloned, and sequenced. A putative open reading frame encoding 93 amino
acids spanned the whole cDNA fragment and showed homology to part of the human and Drosophila homologs. This fragment was used to
screen an X. laevis ovary cDNA library. A positive clone was
completely sequenced. The 1,491-bp long cDNA clone encodes a putative
protein of 463 amino acids and contains short 5' and 3' UTRs.
Amino-terminal sequencing of the purified protein revealed a
mitochondrial targeting sequence of 44 amino acids, leaving a mature
protein of 419 amino acids with a predicted molecular mass of 47.3 kDa,
which agrees with the size of 45 kDa estimated by electrophoresis. An
mRNA of approximately 1,600 nucleotides was identified on a Northern blot (data not shown). We believe that the cDNA clone is a full-length cDNA clone, since it corresponds well with the size of the mRNA and
includes the N terminus of the mature protein with an apparently complete mitochondrial signal sequence. Furthermore, a stop codon is
located in frame six codons before the putative start codon. We refer
to the product of this cDNA clone as the B subunit of DNA pol , or
pol B, while the larger catalytic subunit is referred to as pol
A.
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FIG. 1.
Polypeptide composition of X. laevis pol .
Purified DNA pol was analyzed by SDS-PAGE and Coomassie blue
staining. The solid arrowhead indicates the large subunit, and the open
arrowhead indicates the small subunit. Numbers on the right indicate
the molecular masses in kilodaltons of prestained protein mobility
markers.
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Vertebrate DNA pol B shows primary sequence similarity to
glycyl-tRNA synthetases.
The protein sequence of mature X. laevis DNA pol B, excluding the putative mitochondrial
targeting sequence, was used to search the GenBank database. This
search revealed 21.05% identity and 29.53% similarity (including
identical residues) with Drosophila DNA pol B and 52.69%
identity and 66.13% similarity with a human cDNA product identified as
a likely homolog of Drosophila DNA pol B (33).
Surprisingly, this search also revealed a highly significant similarity
between Xenopus DNA pol B and certain class II
aminoacyl-tRNA synthetases. An alignment of the DNA pol B sequences
and one tRNA synthetase sequence is shown in Fig. 2. X. laevis DNA pol B has
25.54% identity with Thermus thermophilus glycyl-tRNA
synthetase, an identity higher than that reported above with
Drosophila DNA pol B. The significance of these
homologies can be interpreted with reference to the E values
or expectation values reported in a Blast search for matches to
X. laevis pol B, which indicate the probability that the
match could occur by chance. The E values for the match to
human pol B, T. thermophilus glycyl-tRNA synthetase, and
Drosophila pol B are e
109,
4e24, and 2e10, respectively. It is
interesting that Drosophila DNA pol B shows significantly
less similarity to tRNA synthetases, a finding which may account for
the fact that no homology between Drosophila DNA pol B
and tRNA synthetases was noted in the original report by Wang et al.
(33).
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FIG. 2.
X. laevis, Drosophila, and
putative human pol small subunits (XLGB, GenBank accession no.
AF124606; DMGB, accession no. U94702; and HSGB, accession no. U94703,
respectively) were aligned with the glycyl-tRNA synthetase from
T. thermophilus (TTGRS; accession no. P56206) by use of the
program CLUSTAL from PCGene. Amino acids conserved among all four
proteins are indicated by asterisks; similar amino acids are indicated
by dots. Amino acids conserved between XLGB and TTGRS are indicated in
bold. The three conserved motifs identified in class II glycyl-tRNA
synthetases are also labeled under the sequences. Note that the
X. laevis pol B sequence begins with the N terminus of
the mature protein and does not include the signal sequence.
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Xenopus DNA pol is a stable heterodimer.
Bacterially expressed recombinant protein was used to generate a rabbit
antiserum against DNA pol B. The antibodies recognize specifically
both the recombinant protein and the protein purified from ovary
mitochondria. To determine whether the two subunits of DNA pol copurified, column fractions obtained at several steps of the
purification of the polymerase from Xenopus ovaries were
analyzed by Western blotting with antibodies directed against pol A
and pol B. Both proteins were found to copurify on cation-exchange, gel filtration, and hydrophobic interaction chromatography (Fig. 3). Antibodies against the small subunit
were able to coimmunoprecipitate the large subunit from a partially
purified preparation of active DNA pol (data not shown). This
result indicates that the two subunits of X. laevis DNA pol
are associated in a holoenzyme. The molecular mass predicted for a
heterodimer containing one molecule each of X. laevis DNA
pol A and pol B is 181.5 kDa, consistent with the molecular mass
of 185 kDa determined for native X. laevis pol by
sedimentation and gel filtration (15).
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FIG. 3.
Copurification of large and small subunits of pol .
Protein fractions collected during different steps of the purification
of DNA pol were separated by SDS-PAGE and electroblotted onto PVDF
membranes. The membranes were cut into upper and lower halves by use of
prestained protein molecular weight markers as a guide. The upper part
of each membrane was probed with an antibody directed against the DNA
pol large subunit (L). The lower part of each membrane was probed
with an antibody directed against the small subunit (S). Fractions are
indicated by numbers, and FT denotes the flowthrough fraction. (A)
Cation exchange (S Sepharose). (B) Gel filtration (Superdex 200 HiLoad). (C) Hydrophobic interaction (Poros PH).
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We expressed both subunits of
X. laevis DNA pol
in
insect cells by using baculovirus vectors in an attempt to characterize
the potential effects of the small subunit on the catalytic subunit.
We
were unable to recover polymerase activity from preparations
of the
recombinant catalytic subunit, so we were not able to assess
the effect
of the small subunit on polymerase activity. We also
did not observe
enzyme activity when insect cells were coinfected
with baculoviruses
carrying the genes encoding both the catalytic
and the accessory
subunits. A deleterious mutation in the recombinant
catalytic subunit
could account for these results. During the
course of experiments
carried out to characterize the DNA pol
gene, we identified several
discrepancies in the amino-terminal
portion of the sequence of the
catalytic subunit of
X. laevis pol
initially reported by
Ye et al. (
34), who had cloned the
cDNA by using 5' rapid
amplification of cDNA ends. To correct
these errors, most of the cDNA
was recloned with a Marathon cDNA
kit (Clontech; synthesized from
Xenopus mRNA by use of
Pfu polymerase).
The
revised cDNA sequence was found to agree with the genomic
DNA sequence
at each site at which the original cDNA sequence
differed from the
genomic DNA sequence. Nevertheless, the proteins
expressed from clones
constructed from the revised sequence exhibited
no polymerase activity.
We currently suspect that recombinant
protein misfolding or the absence
of required posttranslational
modifications may be responsible for the
lack of
activity.
The Xenopus small subunit stimulates the human
catalytic subunit at physiological salt concentrations.
As all our
attempts to recover activity from the Xenopus recombinant
catalytic subunit failed, we decided to determine whether the
Xenopus small subunit would alter the properties of the
active recombinant human catalytic subunit purified from insect cells (19). Figure 4A shows an
immunoblot analysis of samples of Xenopus pol [lane 1;
1,500 U of enzyme, as assayed by TMP incorporation on
poly(rA)-oligo(dT)] and recombinant human pol A (lane 2; 180 U of
enzyme, 14 ng of polypeptide). Both proteins were detected with an
antibody raised against a peptide sequence that is identical in the two
proteins. We estimate that on a per-molecule basis, recombinant human
pol A is 10- to 20-fold less active than the native
Xenopus protein in the poly(rA)-oligo(dT) assay at low salt
concentrations. It is possible that all of the recombinant protein is
not correctly folded or that posttranslational modifications of the
authentic mitochondrial protein increase its activity. Nevertheless, we
judged that the quality of the recombinant human DNA pol A
preparation was sufficient for some mechanistic studies. Figure 4B
shows a Coomassie blue-stained SDS-polyacrylamide gel used for analysis
of the recombinant Xenopus small subunit.
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FIG. 4.
Characterization of recombinant pol A and pol B
and analysis of primer-template binding by electrophoretic mobility
shift assays (EMSA). (A) Xenopus pol (1,500 U) and
recombinant human pol A (180 U) were run in lanes 1 and 2, respectively, of an SDS-6% polyacrylamide gel, blotted to a PVDF
membrane, and detected with a 1:20,000 dilution of antipeptide
antibodies directed against the sequence TRRAVEPTWLTASN(C), contained
in both human and Xenopus pol A. (B) Coomassie blue-stain
SDS-PAGE analysis of Xenopus pol B purified from
baculovirus-infected cells as described in Materials and Methods.
Numbers at the right in panels A and B indicate the mobilities of
protein mass standards in kilodaltons. (C) Autoradiogram of EMSA
results. A 5'-32P-labeled 44-mer hook oligonucleotide was
incubated either alone (lane 1) or in the presence of either pol purified from X. laevis ovaries (lanes 2 and 6; labeled X)
or recombinant subunits of pol , either human pol A (lanes 3 and
7; labeled A), human pol A plus Xenopus pol B (lanes 4 and 8; labeled AB), or Xenopus pol B alone (lanes 5 and
9; labeled B). Following incubation for 10 min at 25°C,
affinity-purified antibodies raised against Xenopus DNA pol
B were added to reaction mixtures in lanes 6 through 9. Incubation
was continued for an additional 10 min. Samples were subjected to
electrophoresis under nondenaturing conditions, and 32P
radioactivity in the dried gel was detected by phosphorimager
analysis.
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Electrophoretic mobility shift assays were conducted with these
proteins to examine their ability to bind a 44-mer hook oligonucleotide
primer-template. We have previously shown that authentic
Xenopus DNA pol
binds to oligonucleotide substrates and
retains polymerase
activity following native gel electrophoresis
(
23). Binding
of the human catalytic subunit alone produced
a shifted complex
with a more rapid gel mobility than that observed for
the
Xenopus pol
holoenzyme (compare lanes 2 and 3 in
Fig.
4C). When the
Xenopus accessory subunit was included in
the binding reactions,
a complex with the same electrophoretic mobility
as that of the
authentic
Xenopus holoenzyme was observed
(compare lanes 2 and
4 in Fig.
4C). However, we were not able to
convert all of the
complexes containing pol
A to this more slowly
migrating form.
X. laevis pol
B was not able to bind to
DNA in the absence of
the catalytic subunit (lane 5). These results
suggest that
Xenopus DNA pol
B does not have appreciable
DNA binding ability on its
own but is able to associate with the human
catalytic subunit
in a complex with a primer-template. To test this
hypothesis,
parallel binding reactions in which affinity-purified
antibodies
directed against
Xenopus pol
B were added
after 10 min were performed.
The addition of antibodies resulted in a
clear supershift of the
complex containing
Xenopus pol
(lane 6), suggesting that the
small subunit is present in this complex.
The addition of anti-DNA
pol
B antibodies did not affect the complex
with recombinant
human pol
A (lane 7). The addition of antibodies to
a reaction
mixture containing both human pol
A and
Xenopus pol
B (Fig.
4C, lane 8) provided mixed results
consistent with the possibility
that some complexes were disrupted by
the antibodies while a minor
fraction was supershifted. As expected,
the anti-pol
B antibodies
did not affect the complexes in lane 8 which did not appear to
contain DNA pol
B.
A variety of primer-template constructs were used to test whether
X. laevis DNA pol
B influences the catalytic activity of
human DNA pol
A. Figure
5 shows the
results of an experiment
in which recombinant human DNA pol
A was
assayed on a poly(dA)-oligo(dT)
substrate with and without
Xenopus DNA pol
B. In this experiment,
we varied the
monovalent salt concentration in reactions performed
with either 2 or 8 mM MgCl
2 to encompass the range of physiological
salt
concentrations. Human pol
A was found to be very sensitive
to
increasing ionic strength when assayed at either 2 or 8 mM
MgCl
2, as reported previously for reactions performed with
1 mM
MgCl
2 (
19). The addition of the
Xenopus small subunit markedly
stimulated the activity of
the human catalytic subunit at higher,
more physiological KCl
concentrations, but not under low-salt
conditions. Heating the factor
to 80°C for 10 min completely abolished
this stimulation (data not
shown).
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FIG. 5.
Stimulation of human DNA pol A by Xenopus
DNA pol B. Polymerase activity of human DNA pol A ( ) and an
equimolar mixture of human pol A plus Xenopus pol B
( ) on oligo(dT)-poly(dA) was measured with reaction mixtures
containing 2 (A) or 8 (B) mM MgCl2. All reaction mixtures
contained 0.2 pmol of the indicated polymerase subunits.
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To determine whether
Xenopus pol
B increased the
processivity of DNA synthesis by human pol
A, primer extension
assays were
conducted under conditions of excess primer-template with
two
different kinds of templates, singly primed M13 DNA and
poly(dA)-oligo(dT).
The homopolymeric template was included in this
analysis since
we have previously shown that this is a preferred
template for
DNA pol
because it does not present the enzyme with
blocks to
replication that require a mitochondrial single-stranded DNA
binding
protein (SSB) as an additional processivity factor
(
23). In
each case, the primers were
32P end
labeled for the primer extension assays. In order to compare
the
activities of the authentic
Xenopus and recombinant human
enzymes in these experiments, it was necessary to consider that
the
polypeptide concentration of the
Xenopus enzyme could not
be
determined accurately due to the small amount of material available
and
that its specific activity was certainly higher than that
of the
recombinant human enzyme, as noted in Fig.
4. Conducting
these
reactions with excess primer-template tends to make the
precise
polymerase concentration a secondary concern.
Xenopus pol
generated products as long as 500 nucleotides in the first
minute
of incubation, with a progressive accumulation of products
as the
reaction continued. Recombinant human pol
A alone synthesized
only
short products under these relatively high-salt conditions.
The
addition of the
Xenopus small subunit significantly
increased
the processivity of the human polymerase on both templates,
permitting
the synthesis of DNA chains nearly as long as those
synthesized
by the
Xenopus DNA pol
holoenzyme (Fig.
6). These results strongly
suggest a role
for the small subunit as a processivity factor
for pol
in both
Xenopus and humans.
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FIG. 6.
Xenopus DNA pol B confers increased
processivity on human pol A. Primer extension assays with singly
primed M13 single-stranded DNA (A) or oligo(dT)-poly(dA) (B) were
carried out as described in Materials and Methods. Each reaction
included 200 fmol of annealed primer. In both panels, the reaction
mixture in lane 1 contained no enzyme; those in lanes 2, 3, and 4 contained 75 U of Xenopus pol and were incubated for 1, 3, and 10 min, respectively. Reaction mixtures in lanes 5, 6, and 7 contained 9 U of recombinant human pol A, equivalent to 50 fmol of
polypeptide, and were incubated for the same periods of time; reactions
in lanes 8, 9, and 10 were carried out with 50 fmol of recombinant
human pol A plus 50 fmol of recombinant Xenopus pol B,
incubated as in the previous reactions. Lane M shows DNA molecular
weight markers of 32P-labeled MspI fragments of
plasmid pUC18, with the sizes, in nucleotides, indicated to the right
of each panel. Due to space constraints, the larger marker fragments,
of 501, 489, 404, and 353 nucleotides, are not individually labeled.
|
|
| |
DISCUSSION |
mtDNA pol has a variable subunit structure in different
organisms, with or without a processivity factor.
Our results
indicate that X. laevis DNA pol is a heterodimer. Both
polypeptides copurify through several steps of chromatography and can
be coimmunoprecipitated. The mass predicted for a heterodimer containing one molecule each of pol A and pol B agrees very well
with the native size determined by sedimentation and gel filtration.
This situation is similar to what has been described for
Drosophila mtDNA pol, in which both subunits appear to be tightly associated (33). It appears that not all DNA pol enzymes are stable heterodimers. No small subunit has been reported for DNA pol in the yeast S. cerevisiae (6). The
complete S. cerevisiae genome sequence does not appear to
contain a homolog for Xenopus or Drosophila DNA
pol B. The sequences of the DNA pol catalytic subunits have been
reported for other yeasts (26, 34), but it is currently not
known whether these organisms use a small subunit of DNA pol . The
understanding of the machinery involved in the maintenance of mtDNA in
yeasts is still incomplete, so other accessory factors that assist
yeast DNA pol may remain to be identified. In light of our
observation that vertebrate DNA pol B is related to tRNA
synthetases, it is interesting that tyrosyl-tRNA synthetase (a class I
aminoacyl-tRNA synthetase) has been found to rescue a defect in mtDNA
maintenance caused by a mutation in another uncharacterized gene,
MGM104 (12). It has been proposed that
tyrosyl-tRNA synthetase may have a biochemical function that enhances
the activity of the MGM104 gene product.
It is not clear whether human DNA pol
exists in vivo as a monomer
or a heterodimer. The evidence bearing on this point has
been discussed
above. The possibility that the human proteins
are not tightly
associated as a heterodimer would not preclude
the association of these
two proteins during DNA replication.
We do not yet know whether pol
B influences any of the template
binding properties of DNA pol
in a way that would contribute
to the regulation of mtDNA replication.
In
Drosophila and
Xenopus,
the two subunits in
the pol
heterodimer contain complete or
nearly complete leucine
zipper domains that may mediate their
dimerization. The human pol
subunits lack complete leucine zipper
motifs. Experiments are under way
to define the sequences in pol
subunits responsible for their
interaction.
Our observation that
Xenopus pol
B can act as a
processivity factor for the human catalytic subunit suggests that it is
likely
to play the same role for its homologous partner. It has been
suggested that the accessory subunit of
Drosophila pol
is a
processivity factor, but this suggestion could not be demonstrated
because the recombinant enzyme was inactive (
33). We have
also
been unable to recover activity from the recombinant
Xenopus catalytic
subunit.
Xenopus pol
resembles its
Drosophila counterpart in
that both enzymes
seem to be composed of tightly associated A
and B subunits. It is
interesting that the only pol
enzymes
that have been expressed in
an active form to date, the human
and yeast enzymes, can also be
purified from mitochondria without
a tightly associated small subunit.
It is tempting to suggest
that the
Xenopus and
Drosophila enzymes may require the coordinate
assembly of
the large and small subunits in a mitochondrial
environment.
The current view of DNA pol
is reminiscent of the situation for the
prokaryotic DNA polymerases
E. coli DNA polymerase I
and T7
DNA polymerase. The latter polymerase is well known to
use host
thioredoxin as an accessory processivity factor (
30).
E. coli DNA polymerase I acts as a rather distributive
polymerase
without an accessory factor and functions mainly as a repair
polymerase.
Interestingly, grafting the thioredoxin interaction
domain of
T7 DNA polymerase onto the Klenow fragment of
E. coli DNA polymerase
I permits the engineered polymerase to
interact with thioredoxin
and improves its processivity (
1).
Clearly, further experiments
are required to explore the relationship
between the two subunits
of pol
in a variety of organisms to arrive
at a similarly sophisticated
model for their
interaction.
DNA pol B is related to aminoacyl-tRNA synthetases.
Several
key enzymes involved in mtDNA maintenance are closely related to their
prokaryotic counterparts; these include mitochondrial RNA polymerase
(3, 21), SSB protein (4, 9, 20, 31), the
high-mobility-group-box DNA binding protein mitochondrial transcription
factor A (25), and the catalytic subunit of DNA pol (17, 26, 27, 34). Nevertheless, the observation that the
small subunit of DNA pol is related to prokaryotic aminoacyl-tRNA
synthetases is surprising, since the latter represent a different class
of enzyme involved in nucleic acid metabolism. Other systems have
provided some evidence for structural similarities between components
of the replication machinery and tRNA synthetases. Bochkarev et al.
(2) have shown that the structure of the single-stranded DNA
binding domain of human replication protein A is related to that of
yeast aspartyl-tRNA synthetase bound to tRNA. However, the similarity
that we describe is unusual in that it is apparent at the level of
primary sequence homology.
The closest homolog that we have identified for
X. laevis
DNA pol
B (other than human DNA pol
B) is the glycyl-tRNA
synthetase
from
T. thermophilus and
Mycobacterium
tuberculosis. This glycyl-tRNA
synthetase is similar to the
archaeal and eukaryotic enzymes,
which are dimeric, but is highly
divergent from other prokaryotic
enzymes, such as that of
E. coli, which are tetrameric (
22).
E. coli
glycyl-tRNA synthetase can charge only prokaryotic tRNA
Gly,
whereas the enzyme from
T. thermophilus is also able to
charge
eukaryotic tRNA
Gly. We have not tested whether
X. laevis DNA pol
B has tRNA synthetase
activity. In the
experiment shown in Fig.
3, we did not detect
a pool of the small
subunit free of the large subunit, a result
which may have been
expected if the small subunit served a secondary
role as a tRNA
synthetase.
The tRNA synthetases closely related to the pol
accessory subunit
belong to the class II family of aminoacyl-tRNA synthetases,
which
differ from the class I enzymes in the aminoacylation site
in the tRNA
(3' OH versus 2' OH of the terminal ribose, with the
exception of
phenylalanyl-tRNA synthetase) (
7,
14,
29)
as well as in the
structure of the active site of the enzyme.
Class II enzymes contain a
seven-stranded antiparallel
-sheet
motif (
18). Three
sequence motifs have been identified in class
II aminoacyl-tRNA
synthetases. These are believed to be involved
in multimerization,
recognition of the tRNA, and catalysis of
the aminoacylation reaction
(for a review, see reference
8).
As shown in Fig.
2,
the sequence conservation between
X. laevis DNA pol
B and
T. thermophilus glycyl-tRNA synthetase is relatively
high
near the three motifs that form the active site of class
II
aminoacyl-tRNA synthetases, as well as in the C-terminal portion
of
each protein, which has been implicated in tRNA recognition.
The fact
that the regions of aminoacyl-tRNA synthetases that are
conserved as
functional domains show the greatest similarity to
pol
B is striking
and may suggest a functional relationship between
both types of
proteins. It will be interesting to determine whether
the structure of
X. laevis DNA pol
B is similar to the known
structure of
T. thermophilus glycyl-tRNA synthetase (
18).
| |
ACKNOWLEDGMENTS |
This work was supported by NIH grant GM29681 and NIEHS grant
P01-04068 to D.F.B.
We thank Kevin Pinz for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-8651. Phone: (516) 444-3068. Fax: (516) 444-3218. E-mail: dan{at}pharm.sunysb.edu.
| |
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Molecular and Cellular Biology, June 1999, p. 4039-4046, Vol. 19, No. 6
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Top
Abstract
Introduction
