From the
Escherichia coli single-stranded DNA-binding protein
( Eco SSB) has been shown previously to display several DNA
binding modes depending on the ionic conditions. To determine what
effect these various binding modes have on DNA replication, we have
studied DNA synthesis by the T7 DNA polymerase under ionic conditions
where Eco SSB interacts with either 72 or 91 nucleotides of
M13 DNA. These forms presumably correspond to the previously described
(SSB)
Escherichia coli single-stranded DNA-binding protein
( Eco SSB)(
The
Eco SSB tetramer has been shown to interact with ssDNA in a
number of different binding modes (Lohman and Ferrari, 1994), which
differ in the number of nucleotides occluded by a single Eco SSB tetramer. These binding modes are referred to as the binding
size (SSB)
Comparison of the physical properties of the three
Eco SSB binding modes shows substantial differences. Eco SSB can exhibit a very high cooperativity when mixed with DNA at
the low concentration of NaCl required to form the (SSB)
Because of the
multiple roles that Eco SSB plays in the life cycle of E.
coli, it has been suggested that the different properties of the
SSB binding modes might allow it to selectively participate in this
wide range of processes (Lohman et al., 1986b, 1988; Lohman
and Bujalowski, 1990). Studies to support this suggestion have been
limited to the effect of Eco SSB binding modes on the
activities of E. coli RecA protein and these have shown that
the (SSB)
This paper is a
continuation of our systematic study of the mechanisms by which SSB
stimulates DNA synthesis. The goal of the present study is to determine
if the different binding modes of Eco SSB differentially
effect DNA synthesis and the mechanisms by which any differences occur.
To address these questions we have used a chloride-free potassium
glutamate buffer (KGB) system (McClelland et al., 1988) that
did not inhibit DNA synthesis at the high concentrations required to
induce the highest binding site modes. We incorporated these buffer
conditions into an in vitro system, comprising a
single-stranded viral DNA template hybridized to an oligonucleotide
pentadecamer primer, which serves as a model for lagging strand DNA
synthesis. In this study we show that the magnitude of stimulation of
the T7 DNA polymerase and molecular mechanism which induces this
stimulation is dependent on the binding site size with which Eco SSB interacts with the single-stranded template.
Materials
The fluorescence
measurements were corrected for dilution, photobleaching, and inner
filter effects using the method of Lohman and Mascotti (1992). The
fluorescence signal from a blank containing the standard KGB buffer
solution was subtracted from all data. Photobleaching of SSB was
determined under each set of buffer conditions that were used in the
experimental titrations and was found to be less than 2%. The SSB
concentration used in these experiments was 1.11
To determine if the binding site size affected the stimulation
induced by Eco SSB, we have measured DNA synthesis by the T7
DNA polymerase on a primed single-stranded M13 template under
conditions that span the transition from (SSB)
To
determine the effect of Eco SSB binding mode on strand
displacement, DNA synthesis was carried out on a primed single-stranded
M13mp9 template under conditions where either (SSB)
The extent of dissociation of the T7
DNA polymerase from performed polymerase-DNA complexes was determined
by measuring the amount of DNA synthesis on a primed single-stranded
DNA competitor (Fig. 7). The polymerase was allowed to associate with a
pentadecamer-primed single-stranded M13mp9 template in the absence of
dNTPs. After this preincubation, synthesis was initiated by addition of
dNTPs. After 1 min EcoRI-linearized single-stranded M13mp7L1
primed with a 5`-end labeled primer 26 bases from the 3` terminus was
added to the actively replicating complex to trap any dissociating
polymerase. The extent of 42 base-long runoff synthesis product on the
second template provides a measure of dissociation of the polymerase
from the preformed polymerase-ssM13mp9 complexes after partially or
completely copying it. At various time intervals after the addition of
the second template, aliquots were withdrawn from the reaction mixture
and the production of 42-mer was analyzed by polyacrylamide gel
electrophoresis. Since the dissociation rate of preformed
polymerase-template complexes is strongly dependent on competitor DNA
concentration, probably due to direct transfer of the polymerase
between DNA (Huber et al., 1987), we selected the
concentration of competitor DNA to be used by increasing its
concentration until a plateau run-off synthesis level was reached while
unused challenger DNA remained (data not shown).
Autoradiographs of
the gels were used to visualize the 42-mer synthesis products, and the
amount of radioactivity in each band was quantified (Fig. 7). The
average percent transfer of polymerase was determined from a standard
curve of 42-mer production in the presence of only competitor DNA and
amounts of T7 DNA polymerase used for the transfer experiment. At a low
polymerase to template ratio, approximately 7% of the polymerase
dissociates from the template when a 0.8
Using native ssM13 DNA and a potassium glutamate
buffer system, we find two distinct Eco SSB binding modes with
sizes of 72 and 91 nucleotides. It is likely that these binding modes
correspond to the two largest Eco SSB binding modes determined
for Eco SSB-poly(dT) complexes (Bujalowski and Lohman, 1986)
of n = 56 and 65 nucleotides/ Eco SSB tetramer.
Several pieces of evidence support this conclusion. First, these are
the two highest binding site sizes we have observed; we saw none higher
even at salt concentrations as high as 2.5
The two
Eco SSB binding modes show substantial differences in some of
the mechanisms of stimulation of T7 DNA polymerase, while some of the
properties of each form are shared. For example, both (SSB)
The ability of Eco SSB to stimulate DNA
synthesis on templates that do not form base-paired secondary structure
has been shown before and cited as evidence that the mechanism for
stimulation involves properties other than destabilizing secondary
structure (Myers and Romano, 1988). One property was identified as an
ability to increase the binding strength of the polymerase for the
template (Myers and Romano, 1988). In this present study, we find that
when the template is present in large molar excess, (SSB)
On
the other hand, (SSB)
Other studies have suggested differences in the biological
properties of the Eco SSB binding modes. Recent in vitro studies with RecA protein have suggested that the higher Eco SSB binding modes might specifically be involved in recombination.
The higher binding modes displays a limited cooperativity, which may
result in the formation of clusters of dimers of Eco SSB
tetramers leaving regions where other proteins may interact with the
DNA (Lohman et al., 1988; Lohman and Ferrari, 1994). In
addition, solution conditions that favor the formation of the higher
Eco SSB binding modes have been shown to be more favorable for
formation (Griffith et al., 1984) and stabilization (Morrical
and Cox, 1990) of RecA protein filaments on ssDNA than solution
conditions that favor the lowest Eco SSB binding mode
(Griffith et al., 1984; Bujalowski and Lohman, 1986; Lohman
and Bujalowski, 1990). Finally, in the lowest binding mode Eco SSB ((SSB)
At present it is
not known whether these various binding modes play a specific role in
DNA replication in general and T7 replication specifically. However, it
is tempting to speculate how the presence of either (SSB)
A proposal for recycling of DNA
polymerase for lagging strand synthesis was not included in these
models (Nakai and Richardson, 1988a). Myers and Romano (1988) have
suggested that the ability of Eco SSB to increase binding of
the T7 DNA polymerase to a single-stranded template at low polymerase
concentrations may provide this mechanism. We have shown that
(SSB)
Is it possible
that either or both of these Eco SSB binding modes might
specifically form in vivo? The intracellular ionic environment
of E. coli includes a mixture of organic and inorganic species
but the primary ionic osmolytes are K
We thank Dr. Thomas M. Lohman for helpful discussions
and Mubashir Sabir and Shuaib Malik for invaluable technical
assistance.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and (SSB)
(Lohman and Ferrari, 1994)
that were determined using the binding of SSB to homopolymers. Here we
report the stimulation induced by (SSB)
to be 4-fold
greater than that produced by (SSB)
under conditions where
the template is in large excess. Surprisingly, when the polymerase
level is raised so that it is in molecular excess, (SSB)
no longer stimulates synthesis while (SSB)
affords a
4-fold stimulation, which is the same level of stimulation as when the
template was in excess. Both SSB forms increase the rate of DNA
synthesis and were found to stimulate synthesis by relieving template
secondary structures. However, (SSB)
specifically
increases strand displacement synthesis, while (SSB)
stimulates synthesis by increasing the affinity of the polymerase
for the template.
)
binds tightly and
cooperatively to single-stranded DNA to produce a complex that has been
shown to participate in DNA replication, repair, and recombination
(Chase and Williams, 1986; Meyer and Laine, 1990). This protein is
essential for the replication of the E. coli chromosome as
well as several other coliphages, including bacteriophage T7 (Meyer and
Laine, 1990). Eco SSB exists as a stable homotetramer over a
wide range of protein concentrations and solution conditions, and it is
this tetrameric form of Eco SSB that binds to single-stranded
DNA (Lohman and Ferrari, 1994). Electron microscopy, nuclease digestion
(Chrysogelos and Griffith, 1982; Griffith et al., 1984),
nuclear magnetic resonance experiments (Greipel et al., 1987),
and the stoichiometry of oligonucleotide binding (Krauss et
al., 1981; Bujalowski and Lohman, 1989) support the idea that the
ssDNA is wound around an Eco SSB core in a structure
reminiscent of a eukaryotic nucleosome and that, depending on the
solution conditions, several complexes can be formed, which produce
different percent decreases in the contour length of ssDNA.
, where n indicates the average
number of nucleotides bound by the Eco SSB tetramer in that
mode. Variation in early estimates of the binding site sizes (Krauss
et al., 1981; Weiner et al., 1975) was explained by a
series of experiments showing that the different complexes of Eco SSB and ssDNA which form depend on the salt concentrations, cation
and anion valence or type, temperature, pH (Lohman and Overman, 1985;
Bujalowski et al., 1988), and protein binding density
(Bujalowski et al., 1988; Griffith et al., 1984;
Lohman et al., 1986b). Lohman and co-workers showed that by
increasing the ionic strength of the medium, the Eco SSB
tetramer can form three distinct binding modes in complexes with
poly(dT) at 25 °C, pH 8.1, where n = 35, 56, or 65
(Lohman and Overman, 1985), and one additional binding mode of n = 42 at 37 °C (Bujalowski and Lohman, 1986). Although
complexes with native ssDNA appear to undergo the same salt-dependent
size transitions, reported values of n have been larger than
those for poly(dT) (Lohman and Overman, 1985; Morrical and Cox, 1990).
These larger values may be due to stabilization of secondary structures
in the native ssDNA as salt levels are raised, excluding protein from
these regions.
binding mode. In this low salt mode, ssDNA interacts with only
two subunits of the Eco SSB and seems to form unlimited
clusters (Lohman and Ferrari, 1994). This may be the binding mode of
smooth contoured complexes visualized in the electron microscope, where
approximately 60% compaction of M13 occurs (Griffith et al.,
1984; Bujalowski and Lohman, 1986). However, (SSB)
exhibits a transient or ``metastable'' unlimited
cooperativity, which is not a property of the binding mode at
equilibrium (Lohman et al., 1986b). In both the (SSB)
and (SSB)
modes, all four subunits of the Eco SSB tetramer interact with ssDNA (Lohman and Ferrari, 1994), and
it is thought that these forms produce the beaded SSB-ssDNA morphology
observed in the electron microscope. Conditions that favor formation of
both the (SSB)
and the (SSB)
binding modes
are associated with approximately 80% compaction of M13 DNA (Griffith
et al., 1984; Lohman and Bujalowski, 1990).
mode selectively inhibits reactions of this
protein (Griffith et al., 1984; Morrical and Cox, 1990;
Muniyappa et al., 1990). The fact that (SSB)
displays unlimited cooperative binding has been used as evidence
to support the suggestion that this mode might be present at the
replication fork since this form can completely coat the ssDNA (Lohman
and Bujalowski, 1990; Lohman et al., 1986b). There are no
experimental data that address this possibility.
Bacterial Strains and Bacteriophages
E.
coli JM103 and bacteriophage M13mp9 and M13mp7 have been
described (Messing and Vieira, 1982). E. coli
M72SmlacZam
bio-uvrB
trpEA2
(
Nam7-Nam53cI857
H1/pTL119A-5) was obtained from Dr.
Timothy M. Lohman (Texas A & M University) and has been described
previously (Lohman et al., 1986a).
DNA
ssM13 DNA was prepared as described by Hayes
and LeClerc (1983).
Nucleotides
Unlabeled nucleotides and (dT)were purchased from Pharmacia Biotech Inc. The (dT)
and (dA)
were purchased from the
Midland Certified Reagent Co. The 15-mer and 17-mer primers
(5`-TCCCAGTCACGACGT-3` and 5`-GTTTTCCCAGTCACGAC-3`) were purchased from
New England Biolabs. [
H]TTP (80 Ci/mmol) was from
DuPont NEN, [
-
S]dATP (1000 Ci/mmol) was
from Amersham Corp., and [
-
P]dATP (25
Ci/mmol) and [
-
P]dATP (7000 Ci/mmol) were
from ICN Corp.
Proteins
T7 DNA polymerase Form II (95% pure) was
either prepared by Dr. William Clay Brown (Brown and Romano, 1989) or
purchased from U. S. Biochemical Corp. The specific activity of these
preparations ranged 3000 to 7000 units/mg. E. col SSB (99%
pure) was prepared from E. coli
M72SmlacZam
bio-uvrB
trpEA2
(
Nam7-Nam53cI857
H1/pTL119A-5) (Lohman et
al., 1986a). The concentration of Eco SSB was determined
spectrophotometrically by using the extinction coefficient of
= 1.5 ml mg
cm
(1.13
10
M
cm
tetramer) in
a standard buffer (pH 8.1) of 10 m
M Tris, 0.1 m
M Na
EDTA, and 0.20
M NaCl (Lohman and Overman,
1985). The purity of the above proteins was determined by
polyacrylamide gel electrophoresis in the presence of SDS. T4
polynucleotide kinase and proteinase K were obtained from U. S.
Biochemical Corp.
Other Materials
DE81 filter discs were obtained
from Whatman. Methods
Buffers
Potassium glutamate buffers were prepared
essentially as described by McClelland et al. (1988), where
2.0 KGB denotes a buffer
(K
Glutamate
Buffer) that contains
200 m
M potassium glutamate, 50 m
M Tris acetate (pH
7.5), 20 m
M magnesium acetate. 1.5
, 1.25
, 1.0
, and 0.8
are dilutions of 2
KGB made with
distilled water, except that in 1.0
and 0.8
the
concentration of Tris acetate was raised to 30 m
M in each.
Fluorescence Measurements
Reverse titrations of
E. col SSB protein with ssM13mp9 were monitored by the
quenching of intrinsic tryptophan fluorescence as described by Lohman
and Overman (1985) using a Spex Spectramate 1680. The excitation
wavelength of 300 nm and excitation band pass = 0.9 nm (0.5-mm
slit width) were used, while the emission at 347 nm was monitored;
emission bandpass = 4.5 nm (2.5-mm slit width). All titrations
were carried out in a cell compartment maintained at 37.0 ± 0.2
°C using a thermostated cuvette holder and a circulating water
bath. A 3.0-ml solution of SSB in a 4.0-ml quartz fluorescence cell
(Starna, 10 10
4.5 cm) was stirred constantly with a
Teflon-coated magnetic stir bar (8
8 mm) while concentrated
ssM13mp9 DNA was titrated in 2-5-µl aliquots at 2-min
intervals, with a 5-s acquisition time.
10
M (tetramer) (8.47 µg/ml). At a
given salt concentration, the SSB binding sizes are constant over a
5-fold range of SSB concentration (6.3-29.7 µg/ml). Using the
method of Lohman and Overman (1985), no precipitation of SSB was
detected at the salt and SSB concentrations used in these studies. The
relationship between the corrected fluorescence intensity and
concentration of SSB (Lohman and Mascotti, 1992) was determined in
total reaction buffer over the range of 1.0 to 2.0
KGB at 37.0
± 0.2 °C. A linear response was observed over a range of
[SSB] from 0.41 to 20.5 µg/ml, which corresponds to 5.0%
to greater than 200% of the concentration of SSB (8.47 µg/ml) used
in binding site size determinations.
DNA Synthesis Assays
M13 single-stranded DNA
(either 95 or 150 µg/ml) and 7-fold molar excess 15-mer primer
(5`-TCCCAGTCACGACGT-3`) or (dA)(65
µg/ml) and a 1:1 molar ratio of the hexadecamer (dT)
were annealed in 100 m
M potassium glutamate by heating
to 65 °C for 3 min, then allowing the solutions to cool for 1 h.
The indicated amounts of primed DNA were added to a reaction containing
the indicated level of KGB, 5 m
M 2-mercaptoethanol, 200
µ
M dATP, dCTP, dGTP, and [
H]dTTP
(approximately 100 cpm/pmol). Reactions using poly(dA) as a template
contained only 300 µ
M [
H]dTTP. T7
DNA polymerase and Eco SSB were added to the reaction mix as
indicated. The reaction mixtures (100 µl) were incubated at 37
°C for 30 min and stopped by the addition of 3 ml of ice-cold 100
m
M Na
P
O
in 1
M HCl. DNA synthesis was determined by measuring the amount of
acid-insoluble radioactivity by a modification of the procedure
described by Richardson (1971). The precipitate was collected on
Whatman GF/C filter discs, then rinsed three times with 3 ml of the
acid solution and once with 95% ethanol. Acid-precipitable counts were
measured in 5 ml of a nonaqueous scintillation mixture on a Beckman LS
7500 liquid scintillation counter.
Formation of Linear Single-stranded M13mp7L1
DNA
Linear single-stranded M13mp7L1 DNA was prepared as
described (Banerjee et al., 1990).
Polyacrylamide Gel Electrophoresis
Primer-template
annealing reactions were carried out as described except that the
pentadecamer primer was labeled at the 5` end with P. The
reaction mixture (80 µl) contained 50 m
M Tris-Cl, pH 7.6,
10 m
M MgCl
, 10 m
M 2-mercaptoethanol, 0.48
µg (96 pmol) of primer, 5 µ
M [
-
P]dATP(7000 Ci/mmol), and 20 units
of T4 polynucleotide kinase. Incubation was for 90 min at 37 °C,
followed by 10 min at 75 °C to inactivate the kinase. The DNA
synthesis reactions were carried out as described above except the dTTP
was unlabeled. The reactions were then incubated with proteinase K (80
µg) at 65 °C for 20 min. This step was repeated twice, to
insure maximum removal of Eco SSB from DNA synthesis products.
To remove the possibility that different levels of Eco SSB
affected the electrophoresis or sample loading, either the Eco SSB concentration was adjusted so that all reactions had the same
concentration of Eco SSB prior to loading the gel, or it was
verified that reactions were identical except for their Eco SSB level appeared the same on the gel. Reactions were diluted 1:1
with a mixture of 100% formamide, 0.1% bromphenol blue, and 0.1% xylene
cyanol, then boiled for 5 min and quick cooled in ice water and
immediately loaded onto a 10% wedge denaturing polyacrylamide gel. Gels
were autoradiographed at -70 °C using Kodak XAR-5 film and a
DuPont Cronex Lighting Plus intensifying screen. Gels were also scanned
using a Molecular Dynamics PhosphorImager SF and radioactivity
quantitated using ImageQuant software.
Alkaline Agarose Gel Electrophoresis
DNA synthesis
reactions (50 µl) were carried out as described above except that
dTTP was unlabeled and 300 µ
M [-
S]dATP (1000 Ci/mmol) was used.
Eco SSB was present as indicated. To determine the extent of
strand displacement DNA synthesis, DNA synthesis reactions (50 µl)
were carried out as described above except that 300 µ
M dATP was present and the pentadecamer primer labeled at the 5` end
with
P as described above. The reactions were stopped by
addition of 5 µl of 0.2
M EDTA (pH 8). Eleven µl of
300 m
M NaOH, 6 m
M EDTA (pH 8), 18% Ficoll type 400
(in water), 0.15% bromcresol green, and 0.25% xylene cyanol was added
to each reaction and the samples were loaded onto a 0.7% alkaline
agarose gel prepared as described (Sambrook et al., 1989).
Gels were soaked in 7% trichloroacetic acid for 15 min, then dried and
autoradiographed at room temperature using Kodak XAR-5 film. To
determine the extent of strand displacement DNA synthesis,
radioactivity in the dried gels was quantitated using a Molecular
Dynamics PhosphorImager SF.
Single Round of DNA Synthesis on M13mp9
M13
single-stranded DNA (270 µg/ml) and 7-fold molar excess of the
15-mer primer (5`-(TCCCAGTCACGACGT-3`) or (dA)(average size 1400 bases) (260 µg/ml) and a 3-fold molar
excess of the hexadecamer (dT)
were annealed in 100 m
M potassium glutamate by heating to 65 °C for 3 min, and then
allowing the solutions to cool for 1 h. All incubations were carried
out at 25 °C. The preincubation mixture (120 µl) contained
indicated amounts of primed DNA, the indicated level of KGB, and 5
m
M 2-mercaptoethanol. Eco SSB was present or not, as
indicated. Preincubation from 2 to 15 min gave identical results. A
standard preincubation time of 2 min was chosen. The reaction was
started by the addition of a 80-µl start mix containing dCTP, dGTP,
dTTP, [
-
P]dATP (3000 Ci/mmol), and the
indicated amount of primed (dA)
in the
indicated level of KGB to give final concentrations of 50 µ
M dCTP, dGTP, [
-
P]dATP (3000 Ci/mmol),
and 150 µ
M dTTP. Eco SSB (13.2 µg) was
present as indicated. Reactions were stopped by addition of 60 µl
of 0.5
M EDTA (pH 8) at the indicated times. The reactions
containing Eco SSB were ethanol-precipitated as described
(Sambrook et al., 1989). [
-
P]dAMP
incorporation was measured on DE81 filter discs as described (Bryant
et al., 1983).
Identification of Eco SSB-ssDNA Binding Modes in
KGB
Reverse titrations, where a lattice of ssM13mp9 DNA was
added to the Eco SSB ligand, were used to monitor the
quenching of the intrinsic tryptophan fluorescence induced upon binding
(Lohman and Overman, 1985; Lohman and Mascotti, 1992) (data not shown).
This procedure was performed over a range of KGB concentrations, with
at least four replicate determinations at each salt concentration
(Table I). Two binding site sizes were obtained for which n = 72 ± 4 in 0.8-1.0 KGB, corresponding
to 80-100 m
M potassium glutamate and 8-10 m
M magnesium acetate, and n = 91 ± 4 in
2-2.5
KGB, corresponding to 200-250 m
M potassium glutamate and 20-25 m
M magnesium acetate.
We believe that these binding modes correspond to the two largest
Eco SSB binding modes determined for Eco SSB-poly(dT)
complexes (Bujalowski and Lohman, 1986) of n = 56 and
65 nucleotides/ Eco SSB tetramer (see Discussion). The
relationship between fluorescence and concentration of Eco SSB
was verified to be linear in both 1
and 2
KGB over a
range of Eco SSB concentrations from 5% to greater than 200%
of the concentration used in fluorescence determinations of Eco SSB binding mode. At a given salt level, the Eco SSB
binding sizes are constant over a 5-fold range of Eco SSB
concentration (6.3-29.7 µg/ml) (data not shown), suggesting
that our measurements of binding site size are not influenced by
Eco SSB affinity (Lohman and Mascotti, 1992). We also found
that the fluorescence of Eco SSB by ssM13mp9 was dependent on
salt concentration, and the fluorescence of free Eco SSB was
independent of salt concentration (not shown).
Stimulation of DNA Synthesis by Eco SSB Is Dependent on
the Binding Site Size
Prior studies have shown that Eco SSB can stimulate DNA synthesis by the T7 DNA polymerase and gene
4 protein on a duplex template (Scherzinger et al., 1977;
Romano and Richardson, 1979a), and this stimulation is greatest for
RNA-primed lagging strand synthesis (Romano and Richardson, 1979a,
1979b). It has also been shown that on single-stranded templates the T7
DNA polymerase can use short RNA primers synthesized by the T7 gene 4
protein to initiate DNA synthesis (Romano and Richardson, 1979a,
1979b). For this study, we used oligonucleotide-primed single-stranded
DNA templates as a model for T7 gene 4 protein-primed lagging strand
synthesis. Addition of Eco SSB to this system (Myers and
Romano, 1988) and to many in vitro replication systems (Meyer
and Laine, 1990) results in a substantial stimulation of DNA synthesis.
to
(SSB)
in both the absence and presence of Eco SSB
(Fig. 1). In the absence of Eco SSB, synthesis levels remained
in the 10-20 pmol range, showing that this change in salt
concentration has little or no effect on synthesis when Eco SSB is not present (Fig. 1). However, in the presence of
Eco SSB, we obtained a dramatic increase in the level of
synthesis when the binding site size was increased from 72 to 91 (Fig.
1). These increased levels of synthesis correspond to a 4-fold
stimulation of DNA synthesis by (SSB)
, while (SSB)
provided a 14-fold stimulation.
Figure 1:
Stimulation of
T7 DNA polymerase by Eco SSB depends on SSB binding mode. DNA
synthesis by the T7 DNA polymerase (0.005 units) was performed on a
primed M13mp9 DNA template (0.60 µg) in the absence or presence of
Eco SSB (15.8 µg) using the indicated concentration of KGB
as described under ``Experimental Procedures.'' DNA synthesis
levels were measured by the incorporation of
[H]dTTP into acid-precipitable
DNA.
The data presented in
Fig. 1
used conditions where the template was in large molar
excess, conditions that provide the largest effect by Eco SSB.
Prior studies (Myers and Romano, 1988) have shown that under these
conditions Eco SSB stimulates synthesis by increasing the
affinity of the polymerase for the template. It was also found that
when the polymerase is present at high concentrations, it binds stably
to the template in the absence of Eco SSB and the stimulation
in this situation was the result of other Eco SSB properties.
Therefore, it is important to explore the effect Eco SSB
binding site size has on synthesis under conditions where the template
is limiting. We carried out a similar set of experiments to that shown
in Fig. 1, but used a 40-fold higher level of polymerase. Under
these conditions, the binding site size had an effect but,
surprisingly, it was the reverse of that observed when the template was
in excess (Fig. 2). Thus, at low binding site size ( n =
72), we observed a 3-4-fold stimulation by Eco SSB,
while at high binding site size ( n = 91), we obtained
no stimulation by Eco SSB. Note also that even at the high
polymerase level the KGB concentrations had no effect on synthesis in
the absence of Eco SSB (Fig. 2).
Figure 2:
Stimulation by Eco SSB binding
mode depends on the T7 polymerase:template ratio. DNA synthesis by the
T7 DNA polymerase (0.2 units) was performed on a primed M13mp9 DNA
template (0.38 µg) in the absence or presence of Eco SSB
(10.1 µg) using the indicated concentration of KGB as described
under ``Experimental Procedures.'' DNA synthesis levels were
measured by the incorporation of [H]dTTP into
acid-precipitable DNA.
What mechanisms
might account for these intriguing differences? Prior studies have
suggested that Eco SSB stimulates by at least three
mechanisms. One of these involves the relief of secondary structures
that inhibit the progress of the polymerase (Myers and Romano, 1988;
Kowalczykowski et al., 1981; Tabor et al., 1987). A
second mechanism suggests that Eco SSB specifically increases
strand displacement synthesis (Nakai and Richardson, 1988b). Finally,
Eco SSB has been shown to increase the affinity of the
polymerase for the DNA template (Myers and Romano, 1988). To more fully
characterize the effect of Eco SSB binding site size on
polymerase stimulation, we attempted to determine how each binding mode
affected each mechanism of stimulation.
Eco SSB Stimulation by Removal of Template Secondary
Structures
As a first step in determining the mechanism by which
Eco SSB binding site size is involved in the stimulation of T7
DNA polymerase, we determined the effect on synthesis through a stable
22-base perfect homology hairpin. In the absence of Eco SSB,
the majority of the pause sites are on the proximal side of the hairpin
over the range of KGB concentrations in which the (SSB)and (SSB)
binding modes have been identified (Fig.
3, lanes a-e). The presence of Eco SSB
(Fig. 3, lanes f-j) results in a dramatic decrease
in the number of hesitation sites and a substantial increase of
full-length product over the entire range of KGB concentrations. The
extent of polymerase pausing was quantified by calculating the
percentage of product formed that extended beyond the hairpin. In the
absence of Eco SSB, between 15 and 20% of the synthesis was
observed to synthesize past the hairpin no matter which KGB level was
present. Similarly, between 70 and 76% of the synthesis bypassed the
hairpin if Eco SSB was present in either binding site mode.
These data strongly suggest that both (SSB)
and
(SSB)
aid the T7 DNA polymerase by allowing it to
synthesize through this extremely stable secondary structure. Moreover,
any differences in stabilization of secondary structure over the range
of KGB concentrations needed to study the Eco SSB binding
modes are not sufficiently large to inhibit the ability of Eco SSB to melt secondary structures.
Figure 3:
Both (SSB) and
(SSB)
binding modes increase synthesis by T7 DNA
polymerase through the hairpin structure of M13mp7. DNA synthesis by
the T7 DNA polymerase (0.20 units) in the absence ( lanes
a-e) or presence ( lanes f-h) of Eco SSB (10.1 µg) was carried out on an M13mp7 template (0.38
µg) in the indicated concentration of KGB. DNA synthesis reaction
conditions are described under ``Experimental Procedures.''
The primer-template was the
P-labeled 15-mer primer
annealed to M13mp7 as described under ``Experimental
Procedures.'' The labeled DNA products were run on a 10%
denaturing polyacrylamide wedge gel and were visualized by
autoradiography.
Stimulation of T7 DNA Polymerase by Eco SSB on a Poly(dA)
Template
Because no differences were observed in relieving
secondary structure inhibition by the two Eco SSB binding
modes, other mechanisms must be involved, which lead to the observed
differences in synthesis levels. This was confirmed by carrying out
synthesis reactions on a poly(dA) template that lacks the ability to
form secondary structures. When the template is present in large
excess, we obtained a result using this template similar to the result
obtained using M13. Thus (SSB)afforded no stimulation of
synthesis, while (SSB)
produced a large stimulation
(3-5-fold) (Fig. 4). Interestingly, when the template was not
present in excess, Eco SSB did not stimulate under any
conditions (data not shown). The implication of this latter result is
that at high polymerase to template ratios, (SSB)
might be
stimulating on an M13 template by the relief of secondary structures
but on a poly(dA) template (where no secondary structure can exist) no
stimulation is obtained. Alternatively, other differences between the
M13 and poly(dA) templates, such as the potential for strand
displacement synthesis on the circular M13 template via a rolling
circle model, may play a role.
Effect of Eco SSB Binding Mode on Strand Displacement DNA
Synthesis
On a circular M13 template, once the leading strand
has progressed once around the template, strand displacement will yield
a (-) strand product that is larger than the 7.2 kb (+) M13
DNA. This process is referred to as a rolling circle model in which the
leading strand synthesis continually displaces the newly synthesized
(-) strand DNA so that very large products are produced. In the
absence of other proteins, the native form of T7 DNA polymerase (Form
II) lacks the ability to catalyze leading strand DNA synthesis by a
rolling circle mechanism of strand displacement. The lack of strand
displacement activity of native T7 DNA polymerase has been attributed
to its exonuclease activity, which results in high nucleotide turnover
as it stalls when synthesizing at a nick, or region of strong secondary
structure (Engler et al., 1983; Tabor and Richardson, 1989).
However, in the presence of Eco SSB, native T7 DNA polymerase
can catalyze strand displacement synthesis on a primed ssM13 DNA
template, and it has been suggested that this is the major mechanism by
which Eco SSB stimulates DNA synthesis on a primed
single-stranded circular template (Nakai and Richardson, 1988b).
or
(SSB)
formed and the products analyzed on an alkaline
agarose gel (Fig. 5). In the absence of Eco SSB, no strand
displacement synthesis was observed, regardless of the KGB
concentration (Fig. 5). However, in the presence of Eco SSB, we found that the extent of strand displacement synthesis was
very much dependent upon the Eco SSB binding site size. Under
conditions where (SSB)
was induced (2.25
KGB), we
could not observe any strand displacement synthesis, even when
polymerase was present in large excess (Fig. 5). But when the
(SSB)
binding mode formed, extensive strand displacement
synthesis was observed, even when the polymerase concentration was
limiting (Fig. 5, lanes a, e, and i).
The extent of strand displacement synthesis was measured in a separate
experiment using a 5` end labeled primer (not shown) so that the amount
of radioactivity incorporated would be independent of the length of the
product. This experiment indicated that approximately 3-fold more
strand displacement synthesis was produced over full-length product
when 3 polymerase/template were used. Finally, to verify that strand
displacement synthesis was not being prohibited by the (SSB)
mode due to an increase in exonuclease activity with increasing
salt levels, we repeated this study using Sequenase version 2.0 T7 DNA
polymerase, which lacks any 3` to 5` exonuclease activity (Tabor and
Richardson, 1989). Using this polymerase, (SSB)
was still
the only binding mode able to stimulate strand displacement synthesis
(data not shown).
Figure 5:
Effect of Eco SSB binding mode on
strand-displacement DNA synthesis by T7 DNA polymerase. DNA synthesis
was performed in the indicated concentration of KGB with 0.474 units
( lanes a-d), 0.158 units ( lanes e-h), or
0.003 units ( lanes i-l) of T7 DNA polymerase on 0.19
µg ( lanes a-h) or 0.24 µg ( lanes
i-l) of a primed M13mp9 template in the absence or presence
of 5.05 µg ( lanes a-h) or 6.36 µg ( lanes
i-l) of Eco SSB as described under
``Experimental Procedures.'' Synthesis was labeled with
[-
S]dATP with lanes i-l having 5 times the specific activity of lanes a-h.
DNA products were denatured and resolved on a 0.7% alkaline agarose
gel.
Interestingly, at high polymerase to template
ratios, the amount of strand-displacement synthesis as a function of
Eco SSB concentration correlated with the stimulation afforded
by Eco SSB when it was in the (SSB)binding mode
(Fig. 6). Fig. 6 A shows that strand displacement
synthesis increases as the amount of Eco SSB present was
increased, while the amount of full-length product (7.2 kb) remained
constant. The radioactivity present in this gel was quantified, and
these data are presented in Fig. 6 B. Total DNA synthesis
as a function of Eco SSB concentration was then measured for
both the (SSB)
and (SSB)
binding modes
(Fig. 6 C). As expected, at a high polymerase to template
ratio, the (SSB)
binding mode produced no stimulation as
the Eco SSB concentration was increased (Fig. 6 C).
However, as the concentration of (SSB)
was increased, a
stimulation of total synthesis was observed that closely paralleled the
stimulation of strand displacement synthesis that occurred
(Fig. 6 B) with both reaching plateaus when 4 µg of
Eco SSB was present. This is additional support for the idea
that a significant mechanism by which (SSB)
stimulates the
T7 DNA polymerase is specifically through a stimulation of strand
displacement synthesis.
Figure 6:
(SSB) stimulation of DNA
synthesis by T7 DNA polymerase follows same pattern as
(SSB)
stimulation of strand displacement synthesis. DNA
synthesis by the T7 DNA polymerase on a uniquely primed M13mp9 DNA
template using approximately 3 polymerase molecules:1 template molecule
in the presence of increasing levels of Eco SSB was carried
out as described under ``Experimental Procedures.''
A, autoradiograph of the products of DNA synthesis in 0.8
KGB containing either 0, 0.86, 2.0, 4.1, 5.1, 6.3, or 8.0
µg of SSB ( lanes a-g, respectively) and labeled with
[
-
S]dATP. The products were denatured, run
on a 0.7% alkaline agarose gel, and the bands visualized by
autoradiography. B, the gel in panel A was scanned
with a Molecular Dynamics PhosphorImager using ImageQuant software.
Full length indicates products having a length
corresponding to one round of synthesis on the M13 template (7.2 kb).
Strand displacement indicates products that are
larger then the M13 template. C, DNA synthesis in either 0.8
or 2.25
KGB by 0.2 units of T7 DNA polymerase on 0.38 µg of
M13mp9 containing the indicated levels of SSB. Synthesis levels were
measured by incorporation of [
H]dTTP into
acid-precipitable DNA. All synthesis re-actions were carried out as
described under ``Experimental
Procedures.''
Effect of Eco SSB Binding Mode on the Binding of T7 DNA
Polymerase to DNA
If (SSB)does not stimulate
strand displacement, then what might be the basis for the differential
stimulation observed by this binding mode at low polymerase to template
ratios? Prior studies have shown that Eco SSB can increase the
affinity of T7 DNA polymerase for the template (Myers and Romano,
1988), and therefore this might be a potential mechanism for this
binding mode. To determine whether the (SSB)
and
(SSB)
binding modes differ in their ability to alter the
binding affinity of T7 DNA polymerase to the template, we measured the
extent of disassociation of the T7 DNA polymerase in the presence and
absence of both Eco SSB binding modes at both high and low
polymerase to template ratios.
KGB buffer system is
used (Fig. 7). However, in 2
KGB, approximately 70% of
the polymerase dissociates from the template (Fig. 7). Repeating
these experiments in the presence of Eco SSB shows that
(SSB)
caused only 20% of the polymerase molecule to
dissociate from the template, suggesting that this binding mode is
increasing the affinity of the polymerase for the template under these
high salt conditions. Interestingly, at high polymerase to template
ratios, Eco SSB had no effect on the polymerase transfer in
either of the binding modes. Under all conditions, approximately
15-19% of the polymerase molecules were free to transfer. These
results are in good agreement with prior studies, which showed that
Eco SSB increased the affinity of the T7 DNA polymerase for
the template only when the polymerase was limiting (Myers and Romano,
1988).
Figure 7:
Effect of Eco SSB binding mode on
transfer of T7 DNA polymerase to single-stranded competitor DNA. Stage
I, reaction mixtures (43 µl) contained the indicated concentration
of KGB, either 0 µg (% free) or 0.15 µg of pentadecamer-primed
M13mp9, either 0 µg (-SSB) or 3.19 µg (+SSB) of
Eco SSB, and either 0.083 units (2pol:1 mp9) or 0.0028 units
(1pol:15 mp9) of T7 DNA polymerase. Incubation was at 4 °C for 3
min, allowing a polymerase-template complex to form. Stage II, 2 µl
containing dATP, dCTP, dGTP, and dTTP were added to each reaction,
yielding a concentration of dNTPs of 250 µ
M each.
Incubation was at 25 °C for 1 min, to initiate synthesis. Stage
III, 5-µl mixtures containing 0.019 µg (1pol:15 mp9), 0.076
µg (2pol:1 mp9 +SSB), or 1.15 µg (2pol:1 mp9-SSB) of
EcoRI-cut P-labeled pentadecamer-primed M13mp7L1
DNA, either 0 µg (-SSB), 0.44 µg (1pol:15 mp9+SSB),
or 0.87 µg (2pol:1 mp9+SSB) Eco SSB, and dATP, dCTP,
dGTP, and dTTP were added to each reaction. Final concentrations of
dNTPs were 500 µ
M each. Incubation was at 25 °C.
10-µl aliquots were removed over a range of time points. Reactions
were stopped by addition of 2.5 µl of 0.5
M EDTA (pH 8).
A, autoradiogram of the DNA synthesis products after they were
digested with proteinase K, denatured, and resolved on a 15% denaturing
polyacrylamide gel. The control reactions (% free) were without
pentadecamer-primed M13mp9 DNA, using the indicated percentage of total
T7 DNA polymerase added to each reaction in presence of primed M13mp9
DNA. The 42-mer bands present at each time point were quantitated using
Molecular Dynamics PhosphorImager SF, as described under
``Experimental Procedures.'' For each percentage transfer
reaction, synthesis was plotted versus time. An average of
plateau synthesis values was then used to generate a standard curve for
synthesis in each reaction condition, and this standard curve used to
determine the percentage of free polymerase in the presence of M13mp9
template and M13mp7L1 trap. The percentage of free polymerase
represents the average of at least three
experiments.
Effect of Eco SSB Binding Mode on T7 DNA Polymerase
Synthesis Rate
Finally, we determined if the Eco SSB
binding modes have a differential effect on the rate of synthesis by
the T7 DNA polymerase. To measure this property, T7 DNA polymerase was
preincubated with a primed ssM13 template to allow a DNA-protein
complex to form under conditions of limiting polymerase. A level of
approximately 1 polymerase molecule/15 template molecules was chosen.
Although some strand displacement synthesis does occur in 0.8
KGB at this polymerase per template level, it comprises only a small
percent of total synthesis (Fig. 5). dNTPs were added to initiate
DNA synthesis simultaneously with excess oligo(dT)-primed poly(dA)
challenger DNA. Polymerase dissociating from the template after
partially or completely copying the M13 DNA is trapped by the
challenger DNA and does not reinitiate synthesis on a second M13
template. This is indicated by a plateau of synthesis reached in
presence of trap. In the absence of trap synthesis continues to
increase over the time course of the experiment. When Eco SSB
is not present (Fig. 8, -SSB) a plateau synthesis
level is reached within 4 min at both ionic strengths (Fig. 8).
This is additional evidence that polymerase sensitivity to changes in
ionic strength necessary to form the different Eco SSB binding
modes is not playing a significant role in the effects that are
measured. The elongation rate was calculated assuming that the full
7200 nucleotides of M13mp9 circular DNA was replicated. Even in the
absence of Eco SSB the majority of synthesis products on
M13mp9, which lacks strong secondary structure, are full-sized (data
not shown). In the presence of Eco SSB (Fig. 8,
+SSB) a plateau is reached within 1 min in the presence
of both (SSB)
and (SSB)
indicating that the
Eco SSB binding mode does not have a significant effect on the
ability of Eco SSB to stimulate the rate of DNA synthesis.
Figure 8:
Effect of Eco SSB binding mode on
rate of DNA synthesis by T7 DNA polymerase. T7 DNA polymerase was
preincubated at 25 °C for 1 min with primed M13mp9 at the indicated
concentration of KGB in the presence or absence of Eco SSB to
form a polymerase-template complex. DNA synthesis reactions at 25
°C were initiated by simultaneous addition of dATP, dCTP, dGTP, and
dTTP. Aliquots were removed at the indicate time points and synthesis
stopped with excess EDTA. A 10-fold excess of oligo(dT)-primed poly(dA)
challenger DNA molecules was either present or absent during the DNA
synthesis reactions. Polymerase-template association reactions
contained either 0.0078 units or T7 DNA polymerase, 0.72 µg of
primed M13mp9 DNA, and 19.0 µg of Eco SSB (+SSB) or
0.023 units of T7 DNA polymerase, and 2.16 µg of primed M13mp9 DNA
(-SSB). Challenge reactions contained 0 µg of poly(dA)
(-trap), 1.4 µg of poly(dA) and 13.2 µg Eco SSB
(+trap, +SSB), or 4.19 µg of poly(dA) (+trap,
-SSB). Synthesis was labeled with
[-
P]dATP, and measured on DE81 filter discs
as described under ``Experimental
Procedures.''
Eco SSB Binding Site Sizes
The distinct binding
modes for the interaction of Eco SSB and single-stranded DNA
are the first example of multiple types of interactions for a single
protein binding to single-stranded DNA (Bujalowski and Lohman, 1986).
Although the salt concentration range in which these binding modes were
observed encompasses that routinely used for in vitro studies
of DNA replication, recombination, and repair (Bujalowski and Lohman,
1986; Morrical and Cox, 1990; Greip and McHenry, 1989; Richey et
al., 1987; Leirmo et al., 1987) as well as the ranges
estimated for E. coli in vivo (Bujalowski and Lohman, 1986;
Leirmo et al., 1987; Kuhn and Kellenberger, 1985), there are
no reports of differential effects of Eco SSB binding modes on
DNA replication.
KGB. Second, the
quenching of intrinsic tryptophan fluorescence measured for the two
highest binding Eco SSB-poly(dT) complexes was 89 ± 2%
(Bujalowski and Lohman, 1986, 1988; Lohman and Overman, 1985), in
agreement with the values we determined for (SSB)
and
(SSB)
. Third, others have seen higher values for the
binding site sizes using native (non-homopolymer) DNA (Lohman and
Overman, 1985; Morrical and Cox, 1990) and suggested that these
differences are due to the fact that native DNA has duplex regions that
do not bind Eco SSB resulting in a smaller pool of bound
nucleotides and therefore a larger average calculated binding site
size. Fourth, others have reported that there are ion-specific effects
on the transition between Eco SSB binding modes, and that the
transitions depend on the identity and charge of cations and anions, as
well as electrostatic interactions (Bujalowski and Lohman, 1986; Lohman
and Overman, 1985; Overman et al., 1988). Finally, we have
also verified that Eco SSB-poly(dT) complexes undergo a
transition in binding site size over the same range of KGB
concentrations as Eco SSB-M13mp9 (Table II) and exhibit larger
binding site sizes, but approximately the same percentage of
fluorescence quenching as observed for Eco SSB-M13mp9
complexes at these concentrations of KGB. In addition, our
determinations of binding sites sizes with both poly(dT) and
single-stranded M13mp9 in NaCl were in agreement with previous reports
(). Taken together, these results suggest that the larger
binding site sizes we observe for Eco SSB-M13mp9 complexes in
KGB are due to two effects: the use of native DNA rather than
homopolymers, and ion-specific effects because of our having used a
potassium glutamate buffer system.
Effect of Binding Site Size on DNA Synthesis
In
this study we have determined that the two highest Eco SSB
binding modes have different effects on DNA synthesis by the T7 DNA
polymerase, a polymerase that is one of the best studied replication
enzymes. For these experiments we have used uniquely primed
single-stranded DNA as a model for RNA-primed lagging strand synthesis
in the bacteriophage T7 replication system. Addition of Eco SSB to a T7 in vitro replication system has been shown to
result in a substantial stimulation of DNA synthesis by the T7 DNA
polymerase (Scherzinger et al., 1977; Romano and Richardson,
1979a; Fuller and Richardson, 1985; Myers and Romano, 1988).
and (SSB)
reduce polymerase pausing at secondary
structures (Fig. 3), and both provide a 4-fold enhancement in the
rate of DNA synthesis (Fig. 8). The differences include the ability of
(SSB)
to stimulate DNA synthesis on a poly(dA) template,
which lacks the ability to form hairpin structures, while (SSB)
does not stimulate on this template, and the fact that only
(SSB)
is able to stimulate strand displacement synthesis
(Fig. 5).
can increase the affinity of the polymerase for the template
(Fig. 7), resulting in a greater than 3-fold decrease in its
dissociation from a primed ssDNA template over that observed in the
absence of Eco SSB. Interestingly, it is only under these
conditions of limiting polymerase that (SSB)
can stimulate
DNA synthesis ( cf. Figs. 1 and 2). suggesting that this
increased affinity of the polymerase for the template may be
responsible for the stimulation observed for (SSB)
.
is able to stimulate T7 DNA
polymerase synthesis even though we cannot show an increased affinity
of the polymerase for the template when Eco SSB is present in
this binding mode. The fact that (SSB)
is unable to
stimulate synthesis on poly(dA) is also evidence that the effect of
this form of Eco SSB has a fundamentally different effect on
the T7 DNA polymerase. Interestingly, (SSB)
induces a
4-fold stimulation of DNA synthesis, which is independent of the
polymerase concentration (Figs. 1 and 2). Prior studies have shown that
the effect of Eco SSB on the polymerase template interaction
is very dependent on the polymerase concentration, with no differences
observed at high polymerase to template ratios. Taken together, these
facts suggest that (SSB)
is not altering the affinity of
the polymerase for the template. Therefore, the mechanism of
stimulation afforded by (SSB)
most likely involves either
its effect on secondary structure or on strand displacement synthesis.
) has been shown to displace RecA from
ssDNA (Griffith et al., 1984), presumably because the
unlimited cooperativity associated with this binding mode allows it to
saturate a region of single-stranded DNA, even at low protein
concentrations (Lohman et al., 1988). Unfortunately the lowest
binding mode of Eco SSB requires MgCl
concentrations less than 1 m
M (Bujalowski and Lohman,
1986), and therefore we were unable to study this binding mode because
of requirement of the T7 DNA polymerase for Mg
concentrations greater than this. However, the free
Mg
concentration in vivo has been estimated
to be in the range of 1-4 m
M (Lusk et al.,
1968; Alatossava et al., 1985), while the total (bound +
free) intracellular concentration of Mg
in E.
coli has also been shown to be higher than this (Kuhn and
Kellenberger, 1985). Therefore, it is unlikely that this form plays a
significant role in vivo, since even in 2 m
M Mg
and the absence of other ions the
intermediate binding mode of Eco SSB has been shown to be
induced on poly(dT) (Bujalowski and Lohman, 1986).
or (SSB)
might fit in the model proposed for T7
RNA-primed DNA synthesis (Nakai and Richardson, 1986a, 1986b, 1988a).
The T7 in vitro replication system requires only three
proteins to catalyze the basic reactions necessary for leading and
lagging strand synthesis on a duplex DNA molecule (Richardson, 1983).
These proteins are the T7 gene 4 protein and a 1:1 complex of T7 gene 5
protein and E. coli thioredoxin, which function together as a
high processivity complex referred to as T7 DNA polymerase (Richardson,
1983). The gene 4 protein binds to single-stranded DNA and translocates
5` to 3`, unwinding duplex DNA (Matson and Richardson, 1983) for T7 DNA
polymerase to catalyze leading strand synthesis (Lechner and
Richardson, 1983). Both of these reactions are highly processive (Nakai
and Richardson, 1988a, 1988b). In lagging strand synthesis, the gene 4
protein primase activity catalyzes the synthesis of tetraribonucleotide
primers at recognition sites on single-stranded DNA, and these primers
are extended by T7 DNA polymerase (Romano and Richardson, 1979a).
Lagging strand synthesis is not processive, requiring multiple
association and dissociation steps by the gene 4 protein (Nakai and
Richardson, 1988a, 1988b).
is able to increase polymerase-template binding, and
we present evidence that (SSB)
does not. Alternatively,
(SSB)
is specifically able to stimulate strand
displacement synthesis by the T7 DNA polymerase. These differences
suggest that (SSB)
may be more effective at recycling the
T7 DNA polymerase for lagging strand synthesis by promoting a tighter
binding of the polymerase at the primer terminus. On the other hand,
the presence of (SSB)
at a replication fork, with its
ability to promote strand-displacement DNA synthesis, could result a
more efficient progression of the leading strand.
and glutamate
(Richey et al., 1987; Measures, 1975). The levels of these
ions in E. coli vary widely enough in response to external
osmotic strength so one potassium glutamate concentration has not been
assigned as physiological (Leirmo et al., 1987; Kuhn and
Kellenberger, 1985). Moreover, the intracellular ionic environment of
both prokaryotes and eukaryotes is known to vary in response to changes
in the external environment, the rate of cell growth, or the
developmental stage of the cell (Epstein and Schultz, 1965; Cameron
et al., 1980; Epel, 1982; Steinhardt, 1982; Richey et
al., 1987); therefore, it is possible that any of the binding
modes may form and that some may be used preferentially in different
DNA metabolic processes. Whether both forms of Eco SSB could
form in close proximity to a replication fork is less likely, although
it is possible that other factors, such as interactions with other
replication proteins or the release of ions caused by the binding of
these proteins (Record et al., 1985) to the DNA in close
proximity to the Eco SSB-DNA complex, could play a role.
Table: Eco SSB binding site size and quenching
determined in KGB
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.