(Received for publication, September 22, 1994)
From the
Mitochondrial DNA polymerase from Drosophila embryos
has been characterized with regard to its mechanism of DNA synthesis in
the presence of single-stranded DNA-binding protein from Escherichia coli. The rate of DNA synthesis by DNA polymerase
was increased nearly 40-fold upon addition of single-stranded
DNA-binding protein. Processivity of mitochondrial DNA polymerase was
increased
2-fold, while its intrinsic rate of nucleotide
polymerization was unaffected. Primer extension analysis showed that
the rate of initiation of DNA strand synthesis by DNA polymerase
was increased 25-fold in the presence of single-stranded DNA-binding
protein. Our results indicate that the stimulation of Drosophila DNA polymerase
by single-stranded DNA-binding protein
results primarily from an increased rate of primer recognition and
binding. Concurrent achievement of maximal activity and processivity by
mitochondrial DNA polymerase in the presence of binding protein
suggests that DNA polymerase
, like other replicative DNA
polymerases, associates with accessory factors in vivo to
catalyze efficient and processive DNA synthesis.
Single-stranded DNA-binding protein (SSB) ()from Escherichia coli has numerous roles in the replication of the
bacterial DNA genome. It enhances helix destabilization by DNA helicase
by preventing renaturation of unwound duplex (1) and protects
single-stranded DNA from nuclease digestion(2) . SSB increases
the fidelity of DNA polymerase, likely by increasing the rigidity of
the DNA template, promoting increased steric hindrance between template
and incorrect nucleotides(3, 4) . Furthermore, SSB
increases the processivity of DNA polymerase by destabilizing the DNA
secondary structure that causes enzyme pausing and
dissociation(5, 6) . The presence of E. coli SSB in sufficient amounts to saturate the single-stranded DNA
(ssDNA) template results in the stimulation of E. coli DNA
polymerases II and III holoenzyme(7, 8) ,
bacteriophage T7 DNA polymerase(9) , herpes simplex virus-1 DNA
polymerase(10) , and human DNA polymerase
holoenzyme(11) . In contrast, SSB has no effect on E. coli DNA polymerase I (12) or on bacteriophage T4 DNA
polymerase(13) .
The mitochondrial counterpart of E.
coli SSB, mitochondrial SSB (mtSSB), has been purified from rat
liver(14) , Xenopus laevis(15) , and Saccharomyces cerevisiae(16) . The gene encoding mtSSB
from S. cerevisiae(16) and the corresponding cDNAs
from X. laevis(17) and rat and human tissues (18) have been cloned and sequenced. Mitochondrial SSB
comprises a 13-15-kDa polypeptide with an estimated native
molecular mass of 56 kDa, suggesting that it is a tetramer in
solution(14, 16, 18, 19) . It binds
cooperatively to DNA with a binding site size of 8-9 nucleotides
and has an affinity for DNA that is similar to that displayed by E.
coli SSB(15, 20) , to which it is
25%
identical and 50% similar by deduced amino acid
sequence(16, 18, 19) .
Mitochondrial SSB
is required for mtDNA maintenance in yeast, in which it was identified
as a suppressor of mtDNA thermosensitivity associated with disruption
of a mtDNA helicase gene, PIF1(16) . mtSSB has been
shown to coat the displaced ssDNA that is the template for lagging DNA
strand synthesis in mtDNA replication(21) , and its apparent
role in preventing the renaturation of displacement loops likely
enhances DNA helicase activity(22) . Furthermore, mtSSB is
proposed to aid in the prevention of nonspecific lagging DNA strand
initiation by mtDNA primase(23, 24) . However, the
effect of mtSSB on the function of mitochondrial DNA polymerase (pol
) is unclear. Rat mtSSB stimulates the activity of partially
purified rat pol
10-fold on the synthetic homopolymeric
substrate poly(dT)
oligo(dA), but the activity of rat pol
was unaffected on poly(dA)
oligo(dT), for which rat mtSSB exhibits
a low DNA binding affinity(20) . X. laevis mtSSB was
shown to stimulate severalfold a partially purified form of X.
laevis pol
on poly(dA)
oligo(dT) and on
poly(rA)
oligo(dT)(25) . In contrast, X. laevis mtSSB had no effect on DNA synthesis in a mitochondrial lysate and
was completely inhibitory to mitochondrial DNA polymerase activity on
singly primed single-stranded viral DNA(25) .
The nearly
homogeneous mitochondrial DNA polymerase from Drosophila
melanogaster embryos is a heterodimeric enzyme (26) whose
mechanism of DNA strand synthesis has been well characterized
biochemically(27, 28, 29) . It is capable of
the highly processive DNA strand synthesis characteristic of bacterial,
bacteriophage, and nuclear replicative DNA polymerases, but only under
conditions that are suboptimal for DNA synthetic rate(29) .
Although mtDNA replication in vivo is slow (4.5
nucleotides/s/DNA strand)(30) , Drosophila pol
catalyzes DNA synthesis at an even lower rate when carrying out highly
processive DNA synthesis in vitro (
0.5 nucleotides/s/DNA
strand) (27, 29) . Considering the effects of E.
coli SSB on bacterial DNA replication and because mitochondrial
SSB both is critical for mtDNA replication in vivo and affects
the activity of partially purified forms of pol
, we have
investigated the effect of E. coli SSB on Drosophila pol
. The high amino acid sequence conservation between
mtSSBs and E. coli SSB and their physical and biochemical
similarities suggest that theeffects of E. coli SSB on
mitochondrial DNA polymerase will reflect those of mtSSB as well.
Recombinant and wild-type M13 viral DNAs (10,650 and 6407 nucleotides, respectively) were prepared by standard laboratory methods. Synthetic oligodeoxynucleotides (15 nucleotides) complementary to the M13 viral DNAs were synthesized in an Applied Biosystems Model 477 oligonucleotide synthesizer.
Figure 1:
E. coli SSB stimulates the rate of DNA synthesis by Drosophila pol . DNA synthesis was measured on singly primed M13 DNA as
described under ``Methods'' in the presence of the indicated
amounts of E. coli SSB and 30 (closedcircles) or 120 (opencircles) mM KCl. STD, standard.
We found previously that the mechanism of DNA synthesis by Drosophila pol is dependent on the KCl concentration at
which it was assayed(29) : at 120 mM KCl, pol
is
most active (standard activity), yet only moderately processive
(average processive unit of 45 nucleotides); at 30 mM KCl, pol
is less active (25% of standard activity), but highly processive
(average processive unit of 2500 nucleotides). Therefore, we
investigated the effect of KCl concentration on the stimulation of Drosophila pol
by E. coli SSB. The stimulation
of mitochondrial DNA polymerase by SSB was maximal from
20-90 mM KCl (Fig. 2), thus lowering the KCl
concentration required to achieve optimal DNA polymerase activity as
much as 6-fold. Furthermore, SSB inhibited completely pol
when
the concentration of KCl was >150 mM. This shift in KCl
optimum may reflect a change in DNA binding mechanism with varying salt
concentration both by SSB (31) and by pol
in the presence
of SSB and/or a change in protein-protein interactions.
Figure 2:
E. coli SSB lowers the KCl
optimum of Drosophila pol . DNA synthesis was measured on
singly primed M13 DNA as described under ``Methods'' in the
presence of the indicated amounts of KCl and in the absence (closedcircles) or presence (opencircles) of E. coli SSB (1 µg). STD,
standard.
Figure 3:
Time course of stimulation of Drosophila pol by E. coli SSB. DNA synthesis
was measured on singly primed M13 DNA over a time course of incubation
as described under ``Methods'' in the presence of 30 mM KCl and in the absence (closedcircles) or
presence (opencircles) of E. coli SSB (1
µg). A, nucleotide incorporation by pol
was
determined after incubation at 30 °C for 0, 1, 2, 4, 8, 16, 30, 45,
and 60 min. In the absence of SSB, 1% of the input DNA template was
copied after 60 min of incubation versus 17% in its presence. B, the data from A were replotted to show the ratio
of nucleotide incorporation by Drosophila pol
in the
presence versus absence of E. coli SSB at each time
point.
Surprisingly, only 17% of the template DNA was copied
after 60 min of DNA synthesis in the presence of SSB, corresponding to
approximately the amount expected if each DNA polymerase molecule
copied only a single DNA molecule and did not cycle to an unused primer
terminus. That at least 70% of the input DNA could serve as template
for DNA replication was shown by readdition of pol after 60 min
of incubation (data not shown). Furthermore, the extent of DNA
synthesis was proportional to the amount of DNA polymerase added: in
enzyme titrations, the percentage of DNA copied, ranging from 4 to 70%,
reflected that expected for a single binding and polymerization cycle
(data not shown).
Figure 4:
E.
coli SSB increases the processivity of Drosophila pol .
DNA synthesis was performed on singly primed M13 DNA (6407 nucleotides)
at 30 mM KCl as described under ``Methods,'' and the
DNA product strands were isolated, denatured, and electrophoresed on a
1.5% denaturing agarose gel. Reactions were performed in the absence (lanes1 and 2) or presence (lanes3 and 4) of SSB (2 µg) and incubated at 30
°C for 4 (lanes1 and 3) or 8 (lanes2 and 4) min. Numbers at left indicate the positions and sizes (in nucleotides) of HindIII restriction fragments of
-DNA that were
electrophoresed in an adjacent lane.
Figure 5:
E. coli SSB 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 ``Methods.'' DNA product strands were
isolated, denatured, and electrophoresed on an 18% denaturing
polyacrylamide gel. Reactions were performed in the absence (lane2) or presence (lane3) of SSB (2
µg). Lane1 represents a control reaction lacking
both pol
and SSB.
Quantitation of the DNA product strands in the
presence and absence of SSB revealed that SSB stimulates primer
extension 25-fold at 30 mM KCl following 5 min of
incubation at 30 °C (Fig. 5). This corresponds closely to
the 30-fold stimulation of polymerase activity at this time of
incubation (Fig. 3B), indicating that
80% of pol
stimulation by SSB is due to an increase in the rate of
initiation of DNA synthesis by mitochondrial DNA polymerase. The
stimulation may result from SSB-facilitated primer recognition
accomplished by elimination of nonproductive binding of DNA polymerase
to excess single-stranded DNA or by increasing the affinity of unbound
polymerase molecules for DNA. In support of this hypothesis is the
observation that SSB stimulates to a similar extent the 3`
5`-exonuclease activity of DNA polymerase
on M13 DNA containing
mispaired primers. (
)
Figure 6:
Gel analysis of the effect of E. coli SSB on a time course of DNA synthesis by Drosophila pol
. A time course of DNA synthesis was performed on singly primed
recombinant M13 DNA (10,650 nucleotides) at 30 mM KCl as
described under ``Methods.'' DNA product strands were
isolated, denatured, and electrophoresed on a 1.5% denaturing agarose
gel. Reactions were performed in the absence (lanes 1-8)
or presence (lanes 9-16) of SSB (1 µg) and were
incubated at 30 °C for 1 (lanes1 and 9), 2 (lanes2 and 10), 4 (lanes3 and 11), 8 (lanes4 and 12), 16 (lanes5 and 13),
30 (lanes6 and 14), 45 (lanes7 and 15), and 60 (lanes8 and 16) min. The arrow indicates the position of
full-length products. Numbers at left indicate the
positions and sizes (in nucleotides) of HindIII restriction
fragments of
-DNA that were electrophoresed in an adjacent
lane.
While SSB has no effect on the intrinsic rate of DNA
synthesis by pol and only a modest effect on pol
processivity, the extents of DNA synthesis by pol
varied
10-20-fold over the time course of incubation in the absence and
presence of SSB: in the absence of SSB, 0.06% of the DNA template was
copied after 1 min and 1% after 60 min of incubation, as compared with
the presence of SSB, where the extents of DNA synthesis were 0.6 and
17% after 1 and 60 min, respectively. Interestingly, the
10-20-fold variation in the extent of DNA synthesis by pol
in the absence versus presence of SSB correlates with a
5-10-fold variation in the fraction of pol
molecules
actively engaged in DNA synthesis, which can be estimated using the
average DNA strand length to calculate an average rate of nucleotide
polymerization at each time point. That the extent of stimulation of
pol
by SSB is generally proportional to the number of active pol
molecules supports the hypothesis that the mechanism of
stimulation of mitochondrial DNA polymerase by E. coli SSB is
via an increase in the rate of initiation.
Figure 7:
Drosophila SSB and bacteriophage
T4 gp32 stimulate DNA synthesis by Drosophila pol . The
rate of DNA synthesis was determined at 30 mM KCl over a time
course of incubation at 30 °C as described under
``Methods'' in the absence (closedcircles)
or presence of saturating Drosophila SSB (1 µg; closedtriangles) or gp32 (1 µg; opencircles).
Replication of procaryotic, eucaryotic, and viral DNA genomes requires DNA polymerase in conjunction with its accessory factors and single-stranded DNA-binding protein. Mitochondrial single-stranded DNA-binding proteins homologous to E. coli SSB have been identified in X. laevis, S. cerevisiae, and rat and human tissues(14, 15, 18, 19) . The substantial homology between mtSSBs and E. coli SSB in both sequence and proposed subunit structure and the requirement for mtSSB in yeast mtDNA replication led us to examine the effects of E. coli SSB on Drosophila mitochondrial DNA polymerase.
E.
coli SSB stimulates Drosophila pol at both 30 and
120 mM KCl. KCl affects greatly the mode and cooperativity of
SSB binding even though it has little effect on the affinity of SSB for
DNA: below 10 and above 200 mM NaCl, E. coli SSB
binds to ssDNA in two modes that have site sizes of 33 and 65
nucleotides/tetramer, respectively (31) . In the range between
these salt concentrations, SSB tetramers bind in both modes, resulting
in apparent binding site sizes between the two
limits(31, 33, 34) . Consistent with the
known binding properties of E. coli SSB, we found that maximal
stimulation of mitochondrial DNA polymerase at both 30 and 120 mM KCl occurs at the SSB concentrations required to saturate the
ssDNA template.
The ability of E. coli SSB to increase the
processivity of E. coli DNA polymerases II and III and
bacteriophage T7 DNA polymerase (5, 6, 9) suggested that it might also
increase the processivity of mitochondrial DNA polymerase. We found
that the presence of SSB increases the processivity of Drosophila pol
2-fold at 30 mM KCl (this report) and
3-fold at 120 mM KCl, (
)reducing or eliminating
pausing of DNA polymerase at specific sites and increasing the fraction
of completed DNA product strands. Interestingly, the increase in
processivity corresponds closely with the 2-3-fold stimulation of
pol
at 120 mM KCl, but cannot account for the 37-fold
stimulation observed at 30 mM KCl.
The stimulation of several DNA polymerases by E. coli SSB has been documented and, in some cases, appears to be due to the ability of SSB to increase primer recognition by either preventing nonproductive binding of DNA polymerase to or enhancing the affinity of DNA polymerase for single-stranded DNA. SSB stimulation of herpes simplex virus-1 DNA polymerase occurs by the former mechanism(10) , while that of bacteriophage T7 DNA polymerase is proposed to occur by the latter(32) . The elimination of nonproductive binding by DNA polymerase may result from a decreased affinity of DNA polymerase for SSB-coated ssDNA, causing multiple binding and dissociation events that continue until the primer terminus is recognized and bound. Alternatively, the presence of SSB may maintain or enhance DNA binding and facilitate a sliding or looping mechanism by which DNA polymerase locates efficiently the primer terminus. Our primer extension data show that the stimulation of mitochondrial DNA polymerase by E. coli SSB is due primarily to an increased rate of primer recognition and binding, consistent with either mechanism.
By determining the
extents of DNA synthesis and the average lengths of DNA product strands
over a time course of incubation, we could estimate the number of
active DNA polymerase molecules at each time point. In doing so, we
conclude that in the presence of SSB, nearly all of the pol
molecules were polymerizing DNA after 16 min of incubation. In
contrast, in the absence of SSB, only
12% of the pol
molecules were active after 16 min and 25% after 60 min of incubation.
In fact, the stimulation of pol
by SSB at each time point was
generally proportional to the increase in the estimated number of pol
molecules actively synthesizing DNA in the presence and absence
of SSB, providing further support for the hypothesis that SSB
stimulates pol
by increasing the rate of primer recognition.
Given the proposed mechanism of stimulation of Drosophila pol by E. coli SSB, it is not surprising that Drosophila SSB and bacteriophage T4 gp32 also stimulate
mitochondrial DNA polymerase, albeit to different extents. While the
three proteins bind to DNA with approximately equal affinity, they may
impose different conformations onto single-stranded
DNA(35, 36) . Thus, mitochondrial DNA polymerase may
bind to DSSB- and gp32-coated DNA nonproductively with higher affinity
than it binds to E. coli SSB-coated DNA. Alternatively, pol
may displace E. coli SSB more effectively than DSSB or
gp32 from the DNA template during polymerization. Because of its
homology to the DNA-binding domain of E. coli SSB, mtSSB might
be expected to impose a conformation onto ssDNA similar to that of E. coli SSB: mtSSBs are 50-67% identical to several
regions of the amino-terminal two-thirds of E. coli SSB that
is involved in DNA binding (16, 18, 19, 37) . In addition, two
amino acids known to be involved in DNA binding by E. coli SSB
are identical in all mtSSBs identified to date, and a third amino acid
is identical in all mtSSBs except that in
yeast(18, 38, 39, 40) . Hence, the
effects of E. coli SSB on DNA polymerase
may be expected
to represent more closely those of mtSSB than those of bacteriophage
SSBs or eucaryotic SSBs of nuclear origin.
Primer recognition and
processivity factors from E. coli failed to alter the activity
of mitochondrial DNA polymerase. This may be due to a lack of stable
protein-protein associations. Stimulation of pol by E. coli SSB is likely mediated by the interaction of SSB with DNA and not
through specific pol
-SSB interactions. In contrast, primer
recognition and processivity factors associate with DNA polymerase to
tether it onto the DNA template(41) . Because the polymerase
catalytic subunits of mitochondrial DNA polymerase and E. coli DNA polymerase III holoenzyme lack regions of significant
homology(42) , specific association of the procaryotic
accessory proteins with Drosophila pol
is perhaps
unlikely.
Animal mtDNA is replicated by a highly asymmetric
mechanism(30, 43) . Leading DNA strand synthesis
occurs on the double-stranded parental DNA template, while lagging DNA
strand synthesis, which occurs on the displaced parental DNA strand,
involves replication of a predominantly (or entirely) single-stranded
DNA template. Thus, replication of the singly primed single-stranded
viral DNA template used in this study provides an excellent model of
lagging strand mtDNA synthesis. We have found previously that Drosophila mitochondrial DNA polymerase is capable of highly
processive DNA synthesis in vitro only under reaction
conditions that are suboptimal for DNA synthetic rate(29) .
Here, we find that the addition of E. coli SSB enhances
greatly the rate of DNA synthesis while maintaining high processivity,
providing further support for an important role for SSB in
mitochondrial DNA replication. That previous studies examining the
effects of both E. coli SSB and homologous mtSSBs on pol
did not yield a uniform conclusion (20, 25) may
reflect both the purity of the pol
preparation and the reaction
conditions employed. We have found that both KCl concentration and
enzyme purity have a substantial effect on the stimulation of Drosophila pol
by E. coli SSB. For example, in
Fractions I-V of our standard pol
purification(26) , which vary from
0.04 to 20% in purity,
SSB stimulation may vary from 0.5- to 20-fold. (
)In any
case, the SSB-induced ability of Drosophila pol
to copy
efficiently and processively long stretches of ssDNA is characteristic
of a DNA polymerase that catalyzes continuous DNA strand synthesis, as
proposed in current models of mtDNA
replication(30, 43) .
This paper is dedicated to I. Robert Lehman on the occasion of his 70th birthday.