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INTRODUCTION |
Single-stranded DNA-binding proteins
(SSBs)1 serve critical roles
in DNA replication, repair and recombination (1). Whereas high affinity
DNA binding by SSBs can occur independently of other proteins, both
functional and physical interactions between SSBs and a variety of
enzymes involved in the above processes have been documented. In
particular, interactions between SSBs and replicative DNA polymerases
have been demonstrated in bacterial, nuclear, and viral systems (1).
The near-homogeneous mitochondrial DNA polymerase from
Drosophila embryos catalyzes relatively efficient DNA
synthesis on both predominantly double- and single-stranded DNA
templates (2, 3), yet its activity and processivity are greatly
affected by reaction conditions (4). Mitochondrial SSBs share similar physical and biochemical properties with Escherichia coli
SSB (5-10), with which they exhibit a high degree of amino acid
sequence conservation (10-12). Considering the roles served by
E. coli SSB in bacterial replication in helix
destabilization (13) and in enhancing DNA polymerase processivity (14,
15) and fidelity (16, 17), we purified Drosophila mtSSB and
studied its effects in in vitro DNA synthesis by pol
, in
an assay that mimics lagging DNA strand synthesis in mitochondrial
replication (9). These studies allowed the first demonstration of
stimulation by a mtSSB of DNA synthesis by a near-homogeneous pol
.
Our biochemical data are consistent with an important role for mtSSB in
mitochondrial DNA replication that has been documented genetically by
the fact that a null mutation in the gene for the yeast homolog
(RIM1) results in complete loss of mitochondrial DNA
in vivo (7). Furthermore, we found that
Drosophila mtSSB stimulates pol
by a mechanism highly
similar to that which we found for E. coli SSB (9, 18). Here
we demonstrate a dual role for mtSSB in initiation and elongation of
DNA strand synthesis catalyzed by pol
, and evaluate for the first
time the effects of mtSSB on the mispair-specific 3'
5' exonuclease
in pol
.
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EXPERIMENTAL PROCEDURES |
Materials
Nucleotides and Nucleic Acids--
Unlabeled deoxy- and
ribonucleotides were purchased from Amersham Pharmacia Biotech.
[3H]dTTP, [
-32P]dATP, and
[
-32P]ATP were purchased from ICN Biochemicals.
Recombinant M13 DNAs (M13trx22, 10,650 nucleotides (nt); M13mp19, 7249 nt; and M13mp7, 7238 nt) were prepared by standard laboratory methods.
Oligodeoxynucleotides complementary to the M13 viral DNAs were
synthesized in an Applied Biosystems oligonucleotide synthesizer.
Primers for primer extension and 3'
5' exonuclease assays were 17 nt in length to produce a 3'-terminal base pair (dGMPtemplate:dCMPprimer) and 15 nt in length
to produce a 3'-terminal mispair (dGMP:dGMP) on M13trx22 DNA. The
sequences of the 17-mer and 15-mer are 5'-AGGATCGCCCCGTCCGC-3' and
5'-GATCGCCCCGTCCGG-3', respectively. The sequence of the 49-nt primer
used in the DNase I footprinting experiments is complementary to
positions 993-1041 in M13mp19 DNA; this primer was the gift of Dr.
Charles McHenry (University of Colorado Health Sciences Center). The
sequence of the 38-nt primer used in the template-primer binding and
idling experiments is complementary to positions 6291-6329 in M13mp7 DNA.
Enzymes and Proteins--
Drosophila DNA polymerase
(Fraction VI, >90% homogeneous) was prepared as described by
Wernette et al. (3). Drosophila mtSSB (>90%
homogeneous) was prepared from embryonic mitochondria essentially as
described by Thommes et al. (9). Bovine pancreatic DNase I
(Type IV) and T4 polynucleotide kinase were purchased from Sigma and
Roche Molecular Biochemicals, respectively. Sequenase, version 2.0, was
purchased from United States Biochemical Corp.
Methods
Bacterial Subcloning, Overexpression, and Purification of
Recombinant mtSSB--
The 372-base pair coding sequence of
Drosophila mtSSB was engineered by polymerase chain reaction
amplification of a full-length cDNA clone (12) to contain
NdeI restriction endonuclease sites at its ends; on the
amino-terminal end, an NdeI site was created to contain an
ATG at amino acid position 16, corresponding to the residue prior to
the first amino acid in the mature Drosophila mtSSB (9), and
on the carboxyl-terminal end, the NdeI site was positioned
at a site 11 base pairs distal to the termination codon. The resulting
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 analyses.
For overexpression, pET-11a recombinant plasmid-containing BL21
(
DE3) cells (400 ml) were grown at 37 °C with aeration, in Luria
broth containing 100 µg/ml ampicillin. When the bacterial cells
reached an optical density of 0.6 at 595 nm,
isopropyl-1-thio-
-D-galactopyranoside was added to 0.3 mM, and the culture was incubated further for 2 h.
Cells were harvested by centrifugation, washed in 50 mM
Tris-HCl, pH 7.5, 10% sucrose, recentrifuged, frozen in liquid
nitrogen, and stored at
80 °C.
For preparation of cell extracts and purification of recombinant mtSSB,
frozen cells were thawed on ice, and all further steps were performed
at 0-4 °C. Cells were suspended in
volume of original
cell culture in 50 mM Tris-HCl, pH 7.5, 10% sucrose, 2 mM EDTA, 5 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM
sodium metabisulfite, 2 µg/ml leupeptin. Cells were lysed by
incubation for 30 min in the presence of 0.3 mg/ml final concentration
of hen egg white lysozyme (Roche Molecular Biochemicals) and 20 mM spermidine, 0.25 M NaCl, followed by
freezing in liquid nitrogen and thawing on ice. The suspension was then centrifuged at 17,500 × g for 30 min. The supernatant
fluid was recovered for use as the soluble protein fraction for
Cibacron blue agarose chromatography.
The soluble extract (~70 mg of total protein, ~4 mg of mtSSB) was
diluted to an ionic equivalent of 100-120 mM NaCl and
loaded onto a Cibacron blue agarose column (1.9 × 3.5 cm)
equilibrated with buffer containing 30 mM Tris-HCl, pH 7.5, 10% glycerol, 100 mM NaCl, 2 mM EDTA, 2 mM DTT, 1 mM PMSF, 10 mM sodium
metabisulfite, 2 µg/ml leupeptin. The column was washed with 40 ml of
the same buffer containing 800 mM NaCl, and the bound
protein was eluted with 40 ml each of buffer lacking NaCl and
containing 0.5, 1.0, and 1.5 M NaSCN. Recombinant mtSSB
eluted at 1.5 M NaSCN; fractions were pooled; dialyzed
against buffer containing 50 mM KPO4, pH 7.6, 200 mM (NH4)2SO4, 2 mM EDTA, 0.015% Triton X-100, 2 mM DTT, 1 mM PMSF, 10 mM sodium metabisulfite, 2 µg/ml
leupeptin; and then concentrated in a Centricon-30 spin concentrator
(Amicon) treated with Tween 20 as described by the manufacturer. The
dialyzed and concentrated fraction was loaded onto two preformed
12-30% glycerol gradients as described by Wernette and Kaguni (2). Fractions containing recombinant mtSSB in tetrameric form were pooled
and concentrated as described above.
DNA Polymerase
Assay--
Reaction mixtures (0.05 ml)
contained 50 mM Tris-HCl, pH 8.5, 4 mM
MgCl2, 10 mM DTT, 0-180 mM KCl as
indicated, 400 µg/ml bovine serum albumin, 20 µM each
of dGTP, dATP, dCTP, and [3H]dTTP (1000 cpm/pmol), 10 µM (as nt) singly primed recombinant M13 DNA, and 0.1 unit of Fraction VI enzyme (6-fold excess of primer ends over pol
molecules). mtSSB (0.8 µg) was added as indicated in the figure
legends. Incubation was at 30 °C for 30 min. Specific modifications
are described in the figure legends. One unit of activity is that
amount that catalyzes the incorporation of 1 nmol of
deoxyribonucleoside triphosphate into acid-insoluble material in 60 min
at 30 °C using DNase I-activated calf thymus DNA as the substrate.
Preparation of 5'-32P-Labeled M13 DNA Substrates for
Product Analysis--
Synthetic oligodeoxyribonucleotides (15, 17, or
38 nt) as described under "Experimental Procedures" were
5'-end-labeled. The kinase reaction (0.04 ml) contained 50 mM Tris-HCl, pH 8.3, 10 mM MgCl2,
0.1 mM EDTA, 5 mM DTT, 0.1 mM
spermidine, [
-32P]ATP (0.2 µM, 4500 Ci/mmol), 28 or 84 pmol (as nt) of oligonucleotide (15-mer and 17-mer
or 38-mer, respectively), and 10 units T4 polynucleotide kinase.
Incubation was for 30 min at 37 °C. Recombinant M13 DNA (M13trx22
DNA for 15- and 17-nt primers and M13mp7 DNA for 38-nt primer) was
added to a concentration of ~70 mM (as nt, in 4-fold molar excess over homologous oligonucleotide), and the DNA mixture was
precipitated with ethanol. The pellet was resuspended in a buffer (0.1 ml) containing 10 mM Tris-HCl, pH 8.0, 0.3 M
NaCl, and 0.03 M sodium citrate and was incubated at
65 °C for 1 h, followed by incubation at 37 °C for an
additional 1 h in order to anneal the primer to the template.
3'
5' Exonuclease Assay--
Reaction mixtures (0.05 ml)
contained 50 mM Tris-HCl, pH 8.5, 4 mM
MgCl2, 10 mM DTT, 0-180 mM KCl as
indicated, 400 µg/ml bovine serum albumin, 4 µM
5'-end-labeled singly primed recombinant M13 DNA containing a
3'-terminal mispair, and 0.1 unit of Fraction VI enzyme. mtSSB (0.4 µg) was added as indicated in the figure legends. Incubation was for
30 min at 30 °C. Samples were then made 1% in SDS and 10 mM in EDTA, heated for 10 min at 65 °C, and precipitated
with ethanol in the presence of 1 µg of sonicated salmon sperm DNA as
carrier. The ethanol precipitates were resuspended in 80% formamide
and 90 mM Tris borate. Aliquots were denatured for 2 min at
100 °C, chilled on ice, and electrophoresed in an 18%
polyacrylamide slab gel (13 × 23 × 0.075 cm) containing 7 M urea in 90 mM Tris borate, pH 8.3, and 25 mM EDTA. After electrophoresis, the gel was washed in 15%
glycerol for 20 min and exposed to a PhosphorImager screen (Molecular
Dynamics). The data were analyzed using the ImageQuant version 4.2a software.
Primer Extension Assay--
Reaction mixtures (0.05 ml)
contained 50 mM Tris-HCl, pH 8.5, 4 mM
MgCl2, 10 mM DTT, 30 mM KCl, 400 µg/ml bovine serum albumin, 30 µM each of dGTP, dATP,
dTTP, 20 µM (as nt) 5'-end-labeled singly primed
recombinant M13 DNA, and 0.35 unit of Fraction VI enzyme (3.5-fold
excess of primer ends over pol
molecules). mtSSB (2.0 µg) was
added as indicated in the figure legends. Incubation was at 30 °C
for the indicated times. Samples were then made 1% in SDS and 10 mM in EDTA, heated for 10 min at 65 °C, and precipitated with ethanol in the presence of 1 µg of sonicated salmon sperm DNA.
Samples were electrophoresed, the gel was processed, and the data were
quantitated as described above.
DNase I Footprinting--
Pol
interactions at the
template-primer terminus were examined by DNase I footprinting on
singly primed M13 DNA in an experimental scheme modified from Reems and
McHenry (19).
A 49-nt primer was annealed to M13mp19 DNA (at a position corresponding
to 993-1041 in the latter) and radiolabeled at its 3'-end essentially
as per Reems and McHenry (19), except that the 3'-end was extended by 2 nt with Sequenase, version 2.0, in the presence of
[
-32P]dATP (3000 Ci/mmol, 0.13 µM) and
ddTTP (0.8 µM). The radiolabeled template-primer DNA was
separated from excess unannealed primer and unincorporated nucleotide
by gel filtration on a Sephadex G-50 column equilibrated with 10 mM Tris-HCl, pH 8.0, 1 mM EDTA.
The DNase I digestion reactions were performed in a reaction mixture
(40 µl) containing 50 mM Tris-HCl, pH 8.5, 4 mM MgCl2, 5 mM DTT, 30 mM KCl, 5% glycerol, 17 fmol of 3'-end-labeled
template-primer DNA, and varying levels of pol
Fraction VI and
recombinant mtSSB (0.2 µg) as indicated. The reaction mixtures were
incubated for 5 min at 30 °C. DNase I was then added, and the
mixtures were incubated further for 2 min at 37 °C. The digestions
were terminated by addition of 150 µl of stop solution (0.5% SDS,
0.2 M sodium acetate, 30 mM EDTA, 25 µg/ml
tRNA) and then extracted with an equal volume of phenol/chloroform
(1:1) and precipitated with ethanol. The ethanol precipitates were
washed with 70% ethanol and resuspended in 95% formamide containing
10 mM NaOH, 1 mM EDTA, and 0.3 mg/ml each of
bromphenol blue and xylene cyanol. Aliquots were denatured for 2 min at
100 °C, chilled on ice, and electrophoresed in a 10% polyacrylamide
slab gel (31 × 38.5 × 0.04 cm) containing 7 M
urea in 90 mM Tris borate, pH 8.3, and 25 mM
EDTA. After electrophoresis, the gel was processed, and the data were
quantitated as described above.
DNA Synthesis Assay for the Stability of Pol
·DNA
Complexes--
The stability of pol
·DNA complexes in
template-primer binding and enzyme idling was determined in an
experimental scheme modified from Hacker and Alberts (20). Reaction
mixtures contained 50 mM Tris-HCl, pH 8.5, 4 mM
MgCl2, 10 mM DTT, 400 µg/ml bovine serum
albumin, 30 or 120 mM KCl, and 4 µM singly
primed M13mp7 DNA (as nt). For experiments to examine the effect of
mtSSB on the stability of primer binding, mtSSB (0.4 µg/200 pmol of
DNA as nt) was added to the reaction mixture and incubated for 1 min at
30 °C. Pol
Fraction VI (0.2-0.7 unit) was then added, and the
incubation was continued for 1 min. After incubaton with pol
, a
50-µl aliquot was removed and terminated as described below. DNase
I-activated calf thymus DNA (180-600 µM as nt) was added to the remainder of the reaction mixture to serve as a DNA trap. After
DNA trap addition, 50-µl aliquots were removed at varying times to
tubes containing dGTP, dATP, and dTTP at a final concentration of 0.3 mM each. Reactions were incubated for an additional 2 min at 30 °C to allow primer extension to the GGG trinucleotide
position. Reactions were terminated and samples were processed and
analyzed as described for the 3'
5' exonuclease assay.
Experiments to examine pol
idling were conducted essentially as
above. Pol
, in the presence or absence of mtSSB was preincubated with the M13 DNA substrate for 1 min at 30 °C. dGTP, dATP, and dTTP
were added to a final concentration of 0.5 µM, and
samples were incubated for 25 s at 30 °C, to allow extension to
the GGG trinucleotide position. The DNase I-activated calf thymus DNA trap was then added as above, and aliquots were removed at varying times to tubes containing dGTP, dATP, dTTP, and dCTP at a final concentration of 0.3 mM. The reactions were incubated for
an additional 2 min at 30 °C to allow primer extension to the
hairpin by pol
molecules still associated with the template-primer
DNA. To ensure that no further primer extension was initiated on
unreacted M13 DNA substrate molecules after addition of the DNA trap,
control reactions were prepared and incubated as described above,
except that they were terminated immediately after the variable time incubation period. Reactions were terminated and analyzed as above. Products resulting from DNA synthesis past the hairpin were analyzed in
a 6% polyacrylamide, 7 M urea slab gel (13 × 30 × 0.15 cm).
 |
RESULTS |
Bacterial Overexpression and Purification of Recombinant Drosophila
mtSSB--
DrosophilamtSSB was purified previously to homogeneity
from both whole embryos and embryonic mitochondria with a high yield of
0.5-1.0 µg/g of embryos (9). To pursue further biochemical and
physical studies of the interaction of mtSSB with Drosophila mitochondrial DNA polymerase, we sought a more amenable source and a
simpler purification. To that end, we employed bacterial overexpression
and developed a two-step purification scheme. Having determined the
amino-terminal sequence of the protein purified from
Drosophila embryos (9), we engineered a plasmid construct in
the T7 promoter-based vector pET-11a from a full-length cDNA clone,
to produce the mature protein lacking the 16 amino acid presequence.
Overexpression upon
isopropyl-1-thio-
-D-galactopyranoside induction of
plasmid-containing BL21 (
DE3) cells yielded ~16 µg of mtSSB/ml
of cell culture, ~60% of which remained in the soluble fraction upon
cell lysis. Protein analysis by SDS-polyacrylamide gel electrophoresis,
followed either by silver staining or by immunoblot analysis with
rabbit antiserum against native mtSSB from Drosophila
embryos, identifies the overexpressed polypeptide of ~18 kDa as
recombinant mtSSB (Fig. 1). We then
purified the recombinant mtSSB from soluble cell extracts by a single
chromatographic step in which the protein bound to Cibacron blue
agarose is washed under stringent conditions (in the presence of 0.8 M NaCl), and eluted with increasing concentrations of
sodium thiocyanate. The 1.5 M sodium thiocyanate eluate
containing the recombinant mtSSB represents a yield of ~60% of
homogeneous protein; the native tetrameric form was recovered upon
glycerol gradient sedimentation of a dialyzed and concentrated blue
agarose fraction. Overall, the yield of D. melanogaster
mtSSB is 12-fold greater from 400 ml of induced bacterial culture than
from 200 g of Drosophila embryos.

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Fig. 1.
Bacterial overexpression and purification of
Drosophila mtSSB. Protein fractions were
denatured and electrophoresed in a 17% SDS-polyacrylamide gel as
described under "Experimental Procedures." Proteins were detected
by silver staining (A) or by immunoblotting using the goat
anti-rabbit IgG-alkaline phosphatase method with rabbit antiserum
against D. melanogaster mtSSB at a 1:1000 dilution
(B). A, lane 1, molecular mass markers, the sizes
of which are indicated in kDa at left; lane 2, E. coli SSB (0.5 µg); lane 3, blank; lane 4, induced BL21 DE3 cells; lane 5, soluble extract;
lanes 6 and 7, Cibacron blue agarose pool (0.25 and 0.5 µg, respectively; see under "Experimental Procedures").
B, lane 1, E. coli SSB (0.25 µg); lane 2, D. melanogaster mtSSB (0.05 µg); lane 3,
uninduced BL21 DE3 cells; lane 4, induced cells;
lane 5, soluble extract; lanes 6 and
7, Cibacron blue agarose pool (0.05 and 0.3 µg,
respectively).
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Mitochondrial SSB Stimulates Both the DNA Polymerase and 3'
5'
Exonuclease Activities of Drosophila DNA Polymerase
--
We showed
previously that SSB stimulates the DNA polymerase activity of
Drosophila DNA polymerase
on singly- primed M13 DNA, in
an assay that mimics lagging DNA strand synthesis in mitochondrial replication (9, 18). To extend these results and provide a comparative
analysis for the native and recombinant proteins, we assayed both forms
in the DNA synthesis reaction, and also evaluated their effects in
mispair hydrolysis by pol
on the same DNA substrate containing
3'-terminal mispaired primers. Native and recombinant
Drosophila mtSSB stimulate similarly the DNA polymerase activity of pol
over a broad range of KCl concentrations (Fig. 2). DNA polymerase activity is stimulated
18- and 21-fold, respectively, at 15 mM KCl, thereby
lowering the KCl concentration required to achieve optimal DNA
synthetic rate 8-fold relative to the 120 mM KCl
concentration that is optimal in the absence of SSB.

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Fig. 2.
mtSSB stimulates DNA synthesis and mispair
hydrolysis by Drosophila pol over a broad KCl range. DNA synthesis (A) and
mispair hydrolysis (C) were measured on singly-primed M13
DNA as described under "Experimental Procedures," in the presence
of the indicated KCl concentrations and in the absence (closed
circles) and presence (open circles) of
Drosophila or recombinant (open triangles) mtSSB.
B and D, the data from A and
C were replotted to show the ratio of nucleotide
incorporation and mispair hydrolysis by Drosophila pol in the presence versus absence of mtSSB at each KCl
concentration.
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Likewise, the native and recombinant Drosophila mtSSBs
stimulate the 3'
5' exonuclease activity of pol
. Both the
maximal stimulation, 16- and 13-fold, respectively, and the KCl
titration curves are highly similar to those for the DNA synthetic
reaction. This suggests the likelihood of effective coordination of the two activities in native pol
under the fluctuating ionic conditions present in the mitochondrial matrix (21, 22). Notably, the mispair
specificity of the 3'
5' exonuclease is unchanged in the presence
of mtSSB: over the entire KCl range, less than 10% of the paired
termini generated by 3'-terminal mispair hydrolysis were hydrolyzed
(data not shown). Thus, although we have shown previously that mtSSB
enhances the processivity of pol
in nucleotide polymerization,
contributing severalfold to the overall stimulation of DNA synthetic
rate (9), the mechanism of stimulation of the 3'
5' exonuclease is
clearly unrelated to enhanced processivity. At the same time, it may be
likely that mtSSB would enhance the processivity of pol
in
hydrolysis of multiple 3'-terminal mispairs.
Our working hypothesis for the mechanism of pol
stimulation is that
mtSSB increases the rate of initiation on single-stranded substrates
for both DNA synthesis and exonucleolytic hydrolysis. We examined the
former experimentally using a time course analysis of DNA synthesis
that involves limited primer extension on M13 DNA in the absence of
dCTP, such that DNA strand termination occurs after polymerization of 8 or 11 deoxynucleotides. We find that mtSSB stimulates the production of
short nascent strands 10-30-fold from 5-120 s of incubation (Fig.
3). This stimulation results in 40 versus 1.3% of the substrate being utilized at 2 min of incubation in the presence versus the absence of mtSSB,
respectively.

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Fig. 3.
mtSSB increases the rate of initiation of DNA
synthesis by Drosophila pol
. Primer extension was performed at 30 mM KCl in the absence of dCTP as described under
"Experimental Procedures." Reactions were performed in the absence
(A, closed circles, and B) or presence
(A, open circles, and C) of
recombinant mtSSB. B and C, DNA product strands
were isolated, denatured, and electrophoresed in 18% polyacrylamide
gels; quantitation of the data obtained in three experiments is shown
in A. Lane 1 (B and C) represents
controls lacking both pol and mtSSB. Lanes 2-5 and
6-8 represent time points of 5, 10, 20, and 40 s and
1, 2, and 4 min, respectively.
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Assuming a binding site size of 68 nt per tetramer (9), mtSSB was used
in this analysis at a level 2.5-fold in excess of that required to
saturate the ssDNA and was preincubated with the DNA substrate in the
presence of reaction mix containing dNTPs for 5 min prior to pol
addition. Thus, the lag before maximal stimulation of DNA strand
synthesis likely relates to the time required for formation of
productive pol
complexes.
mtSSB Enhances Primer Binding by Pol
--
mtSSB may stimulate
the rate of initiation of DNA synthesis by enhancing primer recognition
and binding, by enhancing formation of stable and/or productive pol
·template-primer DNA complexes, and/or by stimulating nucleotide
polymerization per se. To evaluate these possibilities, we
began with a DNase I footprinting analysis of template-primer DNA
binding. Because pol
has a high affinity for ssDNA, we examined
primer-terminus interactions directly using an M13 DNA substrate to
which was annealed an oligonucleotide primer (49 nt) labeled at its
3'-terminus. We find that in the absence of accessory proteins, the pol
heterodimer forms a stable complex with template-primer DNA that
results in DNase I protection of 20 nt of the primer DNA strand (Fig.
4A). Whereas addition of mtSSB
sufficient to saturate the ssDNA did not alone result in any observable
footprint, mtSSB reduced 3-4-fold the amount of pol
required to
detect a stable footprint on the primer terminus, to a level
corresponding to a single pol
molecule per template-primer DNA
(Fig. 4B). Thus, mtSSB enhances primer recognition and
binding, yet the 3-4-fold increase can account only partially for the
30-fold stimulation of the rate of initiation observed under similar
low salt reaction conditions (Fig. 3). In this regard, the DNase I footprinting analysis does not relate directly substrate DNA binding to
catalysis and so does not measure the potential contribution of mtSSB
to productive complex formation, which might exhibit a shorter
half-life than that required to observe a stable footprint. The
footprinting analysis of primer binding may therefore represent an
underestimate of the effect of increased binding on catalysis.

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Fig. 4.
DNase I footprinting of pol
on template-primer DNA. DNase I footprinting
was performed at 30 mM KCl on singly primed M13 DNA as
described under "Experimental Procedures." A, reactions
were performed in the absence of mtSSB and in the presence of 0, 7, 14, 27, 55, 83, 135, or 0 fmol of Drosophila pol (lanes 1-8, respectively). B, reactions were
performed in the presence of saturating recombinant mtSSB and 0, 7, 14, 27, 55, 83, or 0 fmol Drosophila pol (lanes
2-8, respectively). Lane 1 represents a control
reaction lacking pol and mtSSB. The sizes of the DNA fragments were
determined relative to a DNA sequencing ladder electrophoresed in a
parallel lane (not shown). 7 fmol of Drosophila pol corresponds to 1.1 ng and 0.09 units.
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Stability of Pol
·DNA Complexes in Template-Primer Binding and
Enzyme Idling--
mtSSB may stimulate primer recognition and
template-primer binding indirectly, by eliminating nonproductive
binding of pol
to ssDNA regions. Alternatively or in addition,
mtSSB may stabilize enzyme-DNA interactions, increasing the lifetime
rather than the formation of productive complexes. We examined the
latter by measuring dissociation rates using a strategy developed by
Hacker and Alberts (20) to study the bacteriophage T4 holoenzyme. The
experimental scheme is shown in Fig. 5.
M13 DNA primed with a 5'-end-labeled oligonucleotide is used as the
substrate for DNA binding and catalysis. By staging the addition of a
DNA trap either before or after dNTP addition, we measured by DNA
product strand analysis the rate of dissociation of pol
, in the
presence or absence of mtSSB, either from the primer terminus or after
the incorporation of eight nucleotides, where the enzyme pauses in the
absence of dCTP at the position of the three consecutive template dGMP
residues.

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Fig. 5.
Scheme for assay of template-primer DNA
binding and enzyme idling. A, template-primer DNA
structure. The 5' 32P-labeled oligonucleotide primer (38 nt) is annealed to M13mp7 DNA at a site corresponding to map positions
6291-6329. Downstream from the primer are eight C, A, or T nucleotides
followed by three G nucleotides (GGG) in M13mp7 DNA; 32 nt farther
downstream is a 22-base pair DNA hairpin helix. B,
experimental scheme. In template-primer DNA binding experiments, dGTP,
dATP, and TTP are added after incubation periods with radiolabeled DNA
substrate followed by the DNA trap (see under "Experimental
Procedures"). Product DNA strands are measured at the position of the
GGG trinucleotide. In enzyme idling experiments, dGTP, dATP, and TTP
are added in the first incubation with the radiolabeled DNA substrate.
dCTP is added after the DNA trap to measure DNA product strand
extension from the GGG trinucleotide to the DNA hairpin helix. The
experimental scheme was modified from that described by Hacker and
Alberts (20).
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We find that mtSSB has a modest effect on the dissociation rate of pol
bound at the primer terminus and measured in terms of productive
complexes, that is, enzyme bound with the capacity for nucleotide
polymerization (Fig. 6). At 30 mM KCl, where stimulation by mtSSB is 30-fold at 2 min in
the initiation assay, the half-life of pol
primer binding is
13.2 ± 2.3 and 4.6 ± 0.82 min in the absence and presence
of mtSSB, respectively. Because the half-life for dissociation in
either case is longer than the time required to observe the effect on
initiation, the negative contribution of mtSSB on complex stability is
minimized. The relatively long half-life of the pol
·DNA complexes
suggests that formation rather than the stability of productive
complexes is rate-limiting in the overall replication scheme. The
apparently negative effect of mtSSB on complex stability at low salt is
not present at 120 mM KCl, where stimulation is less than
3-fold, and pol
activity alone is highest. Notably, the
dissociation rate at 120 mM KCl is 15-fold higher than at
30 mM KCl, with a half-life of 0.87 ± 0.15 min. This
likely reflects a salt-stimulated rapid recycling of pol
upon
nonproductive DNA binding to ssDNA regions.

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Fig. 6.
Stability of template-primer DNA binding by
Drosophila pol .
Reactions were performed using the radiolabeled substrate depicted in
Fig. 5 at 30 mM KCl (A and B) and 120 mM KCl (C and D), as described under
"Experimental Procedures," in the absence (A and
C, closed circles; B and D, lanes
1-7) or presence (A and C, open
circles; B, lanes 8-15; D, lanes 8-14) of
recombinant mtSSB. B and D, DNA product strands
were isolated, denatured, and electrophoresed in 18% polyacrylamide
gels; quantitation of the data to show the rate of pol dissociation
is plotted in A and C. Lanes 1 and 8, no enzyme controls; lanes 2 and 9, no DNA trap
added. B, lanes 3-7, time points of 2, 4, 10, 30, and 60 min, respectively; lanes 10-15, time points of 0.33, 0.67, 1, 2, 4, and 10 min. D, lanes 3-7 and 10-14,
time points of 0.33, 0.67, 1, 2, and 4 min. The positions of the primer
and the product terminated prior to the first G residue
(triG) are indicated.
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As with template-primer DNA binding, we find that mtSSB has only a
modest effect on the dissociation rate of pol
upon enzyme idling
after polymerization of 8 nt (Fig. 7).
The dissociation rate of pol
upon idling is 16-40-fold slower at
30 mM than at 120 mM KCl, with half-life values
of 17.9 ± 3.2 and 1.08 ± 0.18 min, respectively, in the
absence of mtSSB, and 33.8 ± 6.0 (30 mM KCl) and
0.84 ± 0.15 min (120 mM KCl) in its presence. The stabilizing effect of mtSSB on the idling complex likely reflects the
structural difference between an elongation complex versus an initiation complex. The functional or physical interactions between
the enzyme and mtSSB may also change once pol
engages in processive
DNA synthesis. Notably, whereas DNA synthesis is stalled at the
position of the 22-base pair hairpin helix in the DNA substrate (see
Fig. 5) in the absence of mtSSB, pol
polymerizes 3 nt into the
helix at 120 mM KCl (Fig. 7D) and completely
through it at 30 mM KCl (Fig. 7B) in the
presence of mtSSB. This and the longer half-life of the stalled complex
at 30 mM KCl are likely a reflection of both the
substantially higher processivity of pol
at low salt (18), and the
further enhancement of enzyme processivity by the helix destabilizing
function of mtSSB (9).

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Fig. 7.
Stability of template-primer DNA binding upon
idling in DNA synthesis by Drosophila pol
. Reactions were performed using the
radiolabeled substrate depicted in Fig. 5 at 30 mM KCl
(A and B) and 120 mM KCl
(C and D), as described under
"Experimental Procedures," in the absence (A and
C, closed circles; B and D,
lanes 1-14) or presence (A and C,
open circles; B and D, lanes 15-21)
of recombinant mtSSB. B and D, DNA product
strands were isolated, denatured and electrophoresed in 18%
(B, lanes 1-14, and D) or 6%
(B, lanes 15-21) polyacrylamide gels;
quantitation of the data to show the rate of pol dissociation are
plotted in A and C. Lanes 1, 8, and 15 (B) and lanes 1, 14 and 21 (D) represent no enzyme controls; lanes 9 and
16 (B), and lanes 2, 8 and
15 (D), no DNA trap added. Lanes 3-7
(B and D) represent control reactions in which
the incubation with dCTP was omitted to evaluate the effectiveness of
the DNA trap (see under "Experimental Procedures"). B, lanes
3-7 and 10-14 represent time points of 1, 3, 10, 20, and 40 min, respectively; lanes 17-21 represent time points
of 4, 12, 20, 40, and 60 min. D, lanes 3-7, 9-13, and
16-20 represent time points of 0.33, 0.66, 1, 2, and 4 min.
The positions of the primer, the product terminated prior to the first
G residue (triG), and the hairpin helix are indicated.
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Taken together, the dissociation experiments indicate that whether
bound at the primer terminus in the absence of nucleotides, or stalled
in the polymerization mode by a single nucleotide omission, pol
·DNA complexes are remarkably stable. Thus, the major contribution of mtSSB in stimulating the rate of initiation of DNA synthesis is most
likely in complex formation.
 |
DISCUSSION |
We have evaluated mechanistically the effects of mtSSB on the
catalytic activities of pol
under in vitro conditions
that mimic lagging DNA strand synthesis in mitochondrial replication. Our finding that mtSSB stimulates similarly both the DNA polymerase and
3'
5' exonuclease activities of pol
over a broad range of KCl
concentrations suggests functional coordination of the two activities
at the replication fork. In evaluating 3'
5' exonuclease activity,
we measured specific mispair hydrolysis on oligonucleotide-primed
ssDNA. At the replication fork, the growing 3'-terminus is paired to
the template strand that is coated with mtSSB (23). Thus, coordination
of DNA polymerase and 3'
5' exonuclease in pol
is anticipated
in the catalysis of proofreading DNA synthesis that is required to
ensure replication fidelity, and our results support this. In fact,
that mispair hydrolysis is specifically stimulated by mtSSB over base
pair hydrolysis may reflect a lower cost of editing in mitochondrial
DNA replication as compared with bacterial and bacteriophage systems
with replicative DNA polymerases that exhibit high nucleotide turnover
(1).
We have shown previously that SSB increases severalfold the
processivity of nucleotide polymerization by pol
(18).
Nevertheless, neither the major contribution of mtSSB to increasing the
rate of nucleotide polymerization nor its stimulation of 3'
5'
exonuclease are apparently a consequence of it. We have shown here that
in stimulating the rate of initiation of DNA strand synthesis by pol
, mtSSB increases the fraction of substrate molecules utilized 30-fold and thus likely recruits pol
to the primer terminus. Whether this occurs by an active or passive mechanism remains to be
elucidated, but our DNase I footprinting analysis supports the
recruiting model. Remarkably, the footprinting data show that unlike
most replicative DNA polymerases, the native pol
heterodimer forms
stable complexes with template-primer DNA, and template-primer binding
is enhanced in the presence of mtSSB. mtSSB alone does not protect the
primer from digestion, nor does it alter the footprinting pattern of
pol
, which protects two helical turns of the DNA template strand at
the primer terminus.
Mikhailov and Bogenhagen (24) found mtSSB to inhibit binding of
Xenopus pol
to template-primer DNA, in a gel mobility shift analysis on short oligonucleotides. On such substrates, we would
anticipate efficient binding of pol
alone because the ssDNA present
is less than 50 nt per substrate molecule. Indeed, we find that
oligonucleotide template-primer binding by Drosophila pol
is not stimulated by mtSSB nor is the initiation of DNA synthesis
on such substrates.2 We
suggest that initiation of lagging DNA strand synthesis in mitochondrial replication should require mtSSB-facilitated primer recognition and binding by pol
where, as in our model M13 assay in vitro, the displaced lagging DNA strand template is
thousands of nucleotides in length (25). Our data show clearly that the rate of initiation of DNA strand synthesis to produce an 8-11-mer on a
long ssDNA is stimulated up to 30-fold by mtSSB.
What factors mediate the functional interactions of pol
with mtSSB?
Functional interactions may occur upon DNA binding and/or by specific
physical interactions. E. coli SSB is known to bind DNA in
several modes depending on ionic conditions (26). Studies of mtSSBs
also show salt-dependent effects on DNA binding and, in
particular, on binding site size and in cooperativity of DNA binding
(5, 8, 9). Ionic conditions likely affect both DNA conformation and SSB
structure, and these may vary on different template primers. Likewise,
pol
binding to the template DNA at the primer terminus may differ
from that in long ssDNA regions. Our data show that mtSSB stimulates
DNA synthesis by pol
on M13 DNA over a broad range of salt
concentrations, under which the effects on mtSSB on template-primer
binding and dissociation by pol
vary substantially. This suggests
flexibility in presumptive protein-protein interactions and perhaps in
the mode of mitochondrial DNA replication under the fluctuating ionic
conditions that occur in vivo (21, 22).
Specific physical interactions have been demonstrated between
replicative DNA polymerases and SSB proteins in several systems. Both
bacteriophage T4 and T7 DNA polymerases interact physically with their
cognate SSBs, the gene 32 and 2.5 proteins, respectively (27-30). That
T7 DNA polymerase is stimulated in DNA synthesis by both the T7 and
E. coli SSBs (29, 31-33) suggests that whereas specific
physical interactions may facilitate polymerase activity, they are not
essential for functional interactions, at least as measured by in
vitro DNA synthesis. In that regard, E. coli DNA pol
III holoenzyme has recently been shown to interact physically with
E. coli SSB (34, 35). This interaction involves the
subunit of the former and the acidic carboxyl-terminal tail of the
latter and has a dual effect on in vitro DNA synthesis under elevated salt conditions in DNA strand initiation and in chain elongation. Our functional data resemble those studies: mtSSB stimulates both primer recognition by pol
and enhances the
processivity of nucleotide polymerization. Interestingly, although
mtSSB is a homolog of bacterial SSB, sharing significant sequence (7, 10-12) and structural similarity (36), it lacks the acidic carboxyl terminus required for physical interaction in both E. coli
SSB and in T7 gene 2.5 protein (30). In fact, the carboxyl terminus appears as a disorganized loop in the crystal structure of the human
mtSSB (36). Thus, future studies to link functional with physical
interactions between pol
and mtSSB have the potential to reveal
novel interaction domains.