From the Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005
Received for publication, October 14, 2002, and in revised form, November 22, 2002
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ABSTRACT |
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The organization and proper assembly of
proteins to the primer-template junction during DNA replication is
essential for accurate and processive DNA synthesis. DNA replication in
RB69 (a T4-like bacteriophage) is similar to those of eukaryotes and
archaea and has been a prototype for studies on DNA replication and
assembly of the functional replisome. To examine protein-protein
interactions at the DNA replication fork, we have established solution
conditions for the formation of a discrete and homogeneous complex of
RB69 DNA polymerase (gp43), primer-template DNA, and RB69
single-stranded DNA-binding protein (gp32) using equilibrium
fluorescence and light scattering. We have characterized the
interaction between DNA polymerase and single-stranded
DNA-binding protein and measured a 60-fold increase in the overall
affinity of RB69 single-stranded DNA-binding protein (SSB) for
template strand DNA in the presence of DNA polymerase that is the
result of specific protein-protein interactions. Our data further
suggest that the cooperative binding of the RB69 DNA polymerase and SSB
to the primer-template junction is a simple but functionally important
means of regulatory assembly of replication proteins at the site of
action. We have also shown that a functional domain of RB69
single-stranded DNA-binding protein suggested previously to be the site
of RB69 DNA polymerase-SSB interactions is dispensable. The data
from these studies have been used to model the RB69 DNA polymerase-SSB
interaction at the primer-template junction.
The replisome is a dynamic macromolecular machine that must
constantly respond to changes within the cell that may affect the rate
and accuracy of DNA replication (1). The complex topology of the
chromosome, together with the fact that DNA is constantly accessed for
transcription, recombination, and repair, suggests that the replication
of DNA cannot be static, but rather must maintain a great deal of
structural flexibility (1). The interaction of the replicative DNA
polymerase with its cognate
SSB1 is an example of this
dynamic process. During DNA replication, ssDNA is cooperatively and
non-specifically bound by the SSB family of proteins to protect
template strand DNA from nucleases and facilitate the removal of
adventitious secondary structures (2). The length of available template
strand during lagging strand synthesis can be variable and depends, in
part, on the particular organism and its metabolic state (3). The
problem of how to efficiently bind available ssDNA is solved by the
highly cooperative and non-sequence-specific interaction of the SSB
family proteins for single-stranded DNA. In contrast, the interaction
of the replicative DNA polymerase is, of necessity, confined to the
primer-template junction. The polymerase has a high affinity for this
unique structure over either ssDNA or double-stranded DNA (4,
5). The processivity of the polymerase is further enhanced by the
sliding clamp family of proteins that tether the polymerase to the DNA
(6, 7). Taken together, the SSB, DNA polymerase, and sliding clamp
(once the sliding clamp has been loaded) are able to extend DNA at
nearly in vivo rates (8), and thus, efficient DNA
replication depends on making specific protein-protein contacts within
the context of the expanding DNA replication bubble. We have determined
conditions that allow us to accurately measure the association of a
replicative DNA polymerase for its cognate SSB and apply this
information to our understanding of the functional replisome.
T4 bacteriophage has proven to be a productive system to study DNA
replication because of its simplicity and organizational similarity to
eukaryotic and archaeal systems (9, 10). Bacteriophage RB69 is closely
related to T4, and thus, insights from the well characterized T4 system
are readily applicable to RB69. RB69 and T4 DNA polymerases (gp43)
share 74% amino acid similarity and are members of the pol In bacteriophage T4, specific association of gp32 with gp43 has been
qualitatively demonstrated using affinity chromatography of
radiolabeled crude extracts (17, 18). Limited proteolysis has shown
that the T4/RB69 gp32 can be divided into three distinct domains. The
N-terminal B-domain (residues 1-21) is essential for the
cooperative binding of RB69 SSB to ssDNA. Loss of this domain reduces
affinity for ssDNA by over a thousand-fold (19). The C-terminal
A-domain (residues 255-301 for T4 and residues 255-299 for
RB69) is highly acidic and mediates interactions with other proteins
involved in DNA replication, recombination, and repair (18, 20). The
core domain (residues 22-254) is the DNA-binding domain and has the
same intrinsic affinity as the intact protein for short single-stranded
DNA (19). Based upon proteolysis studies performed in the presence or
absence of DNA, it has been suggested that T4 SSB undergoes a
conformational change upon binding to ssDNA (21). Current models
suggest the conformational changes during binding facilitate T4 SSB
interactions with other proteins in replication, recombination, and repair.
To date, quantitative studies on the interaction between DNA polymerase
and SSB protein have been limited because of difficulties in
establishing conditions that favor formation of a discrete homogeneous
complex (22). The recent determination of co-crystal structures of RB69
polymerase·primer-template DNA complex (13, 14) and core
domain of T4 SSB (16) have allowed us to design conditions and DNA
substrates to form a discrete RB69 DNA polymerase, a primer-template
DNA, and RB69 SSB complex for quantitative analysis. The composition
and stoichiometry of the complexes were validated using light
scattering. Once we had established solution conditions that favored
formation of a 1:1:1 RB69 DNA polymerase·SSB·primer-template DNA complex, we were then able to study the interactions among RB69 DNA
polymerase, RB69 SSB, and primer-template DNA by equilibrium fluorescence methods where the tryptophan signal from the RB69 DNA
polymerase and SSB is quenched upon binding to the DNA. We have found
that the protein-protein cooperativity between RB69 DNA polymerase and
SSB causes a roughly 60-fold increase of affinity of SSB to the
adjoining single-stranded template region. These data have allowed us
to construct testable models for the formation of the DNA
polymerase-SSB interaction as they occur in replication forks of all organisms.
Materials
Enzymes were purchased from New England Biolabs, and chemicals
were from either Sigma or Fluka. Chromatography resins were purchased
from Whatman and Amersham Biosciences. The ssDNA cellulose column was prepared in the laboratory using the protocols of Alberts and Herrick (23). Oligonucleotides were purchased from
Integrated DNA Technologies.
Methods
Cloning and Overexpression of RB69 Gene 32--
The original
clone encoding RB69 gene 32 (pLIA5) was a gift from Dr. J. Karam
(Tulane University School of Medicine). pLIA5 was not efficient in
making the RB69 SSB, because an upstream autogenous regulatory element
was present in the clone (24). gp32 was subcloned into the
overexpression vector pKC30 without the native 5' autoregulatory
sequences and used to transform Escherichia coli
AR120 (25). In toto, three different RB69 SSB expression constructs were engineered for this work: the intact protein (gp32), a
truncation mutant without the N-terminal domain (gp32-B; residues 22-299), and a mutant without the C-terminal domain (gp32-A; residues 1-253). The clone for expression of gp32-A was generated by changing the codon for Val-254 to a stop codon on intact gene 32 using the
QuikChange site-directed mutagenesis kit (Stratagene).
Expression and Purification of RB69 gp43 exo Design of Primer-template DNA for Complex Formation--
The
design of a primer-template DNA to form a complex with RB69 DNA
polymerase and SSB was based on the crystal structure of RB69 DNA
polymerase bound to a primer-template DNA (13) and biochemical studies
on the site size of T4 SSB binding to ssDNA (27) (Table
I). Oligonucleotides were purified by
ion-pairing reverse phase chromatography (C4 resin; Vydac, Hesperia,
CA). Primer-template DNAs were annealed by mixing equal molar amounts of primer and template strands in 10 mM Tris-Cl, pH 7.5, 0.1 mM EDTA (TE) heated to 80 °C and allowed to
slow cool to 20 °C prior to use.
Light Scattering--
A miniDAWN three-angle light-scattering
photometer (Wyatt Technology, Santa Barbara, CA) with a 30-milliwatt
semiconductor diode laser of wavelength 690 nm was used to determine
the molecular mass of individual proteins and complexes.
200-300-µg samples were injected onto a Shodex KW803 gel filtration
column at a flow rate of 0.5 ml/min, and their elution was monitored by
Shimadzu SPD-10Avp UV-visible and Waters R401 refractive index
detectors. All measurements were made in the in-line flow mode. The
miniDAWN was calibrated according to the manufacturer's instructions
(Wyatt Technology). Bovine serum albumin was used as a standard to
evaluate the accuracy of the system. A dn/dc (change in refractive
index as a function of concentration) value of 0.184 was used
for bovine serum albumin and single proteins (28). dn/dc values for
complexes were determined by using both UV-visible detector and
differential refractometer. The Zimm method was used to determine the
molecular mass for all samples (29).
Equilibrium Fluorescence Titrations--
The interaction between
RB69 DNA polymerase and SSB to primer-template DNA was quantitated by
quenching of intrinsic tryptophan fluorescence. All measurements were
made in an SLM 8100 spectrometer (SLM Instruments, Urbana, IL), using
an excitation wavelength of 295 nm and an emission wavelength of 347 nm. All experiments were carried out in a 1-cm path length quartz
fluorimetric cuvette containing an active magnetic stirrer at 20 °C
and a 4-nm band-pass for both excitation and emission monochromators.
Initial protein concentrations were 1 or 2 µM in 10 mM Tris-Cl, pH 7.5, 150 mM NaCl in the cuvette.
Small aliquots (2 to 10 µl) of the corresponding DNA solution in TE
buffer were added to the sample cuvette, and TE buffer was added to the
reference cuvette. After mixing for 4 min, the spectrometer made ten
measurements, and the average was taken as one data point. The effects
of dilution and photobleaching were corrected by data from the
reference cuvette. Nucleic acids absorb excitation light at 295 nm, and
it was necessary to correct for this inner filter effect using an
n-acetyltryptophan amide (NATA) calibration titration (30).
Because there is no appreciable interaction between NATA and nucleic
acids, decrease in NATA fluorescence is a consequence of inner filter
effect. After correcting for photobleaching, dilution, and inner filter
effects, the titration curves were fitted using the program Dynafit
(BioKin Ltd., Pullman, WA) (31). In Dynafit, users give the reaction
mechanisms, which are translated by the program into the underlying
systems of mathematical equations by using the theory of matrices (32).
The data are then fitted using least squares regression.
RB69 DNA Polymerase·SSB·Primer-template Complex
Formation--
To analyze the formation of the RB69 DNA
polymerase·SSB·primer-template complex, it was necessary to find
conditions that support a discrete 1:1:1 stoichiometry. Light
scattering was used to assess complex stoichiometry by an accurate
determination of their molecular mass and by analysis of their contents
by SDS-PAGE and UV-visible spectrophotometry. As shown in Table
II, RB69 DNA polymerase
exo Determination of Binding Affinity of RB69 DNA Polymerase to
Primer-template DNA--
To assay whether the interactions between
RB69 DNA polymerase and SSB change the affinity of the polymerase to
primer-template DNA, we determined the binding affinity of RB69 DNA
polymerase to primer-template DNA by titration of 2 µM
RB69 DNA polymerase exo Determination of Binding Affinity of RB69 SSB to T6
DNA--
We also measured the affinity of RB69 SSB to a short ssDNA.
By measuring the individual affinities of RB69 DNA polymerase and SSB
to their substrates, we were then able to determine their cooperativity
when bound together on the same DNA. Bacteriophage T4 gp32 has been a
prototype for protein-nucleic acid interactions, and the RB69 homolog
also proved amenable to physicochemical analysis. The site size of
RB69 SSB to ssDNA and maximum fluorescence quenching were
determined by titrating 2 µM RB69 SSB intact protein with poly(dT) DNA. The site size was determined under stoichiometric binding
conditions to be n = 6 ± 1 nucleotides (Data not
shown), in good agreement with the previous studies (27) on T4 SSB. The
dissociation constant for gp32-B to T6 DNA at 10 mM Tris-Cl, pH 7.5, 150 mM NaCl was very weak,
10.9 ± 1.7 µM (data not shown), consistent with
previous studies using T4 gp32 (19).
The cis and trans DNA Binding Experiments--
We designed
experiments to determine the cooperativity between RB69 DNA polymerase
and SSB when bound to the same (cis) or different
(trans) DNA strands (Fig. 3).
In the cis experiment, equimolar ratios of RB69 DNA
polymerase and SSB bind to a single contiguous primer template. If the
presence of RB69 DNA polymerase at the primer-template junction
increases the affinity of SSB for the template strand, the increase in
binding through protein-protein interactions can be readily measured.
In the trans experiment, the template strand is broken into
a shorter primer-template junction, which only RB69 DNA polymerase will
bind, and a six-nucleotide DNA, which RB69 SSB will bind. Because no
significant association has been found between RB69 DNA polymerase and
SSB in solution in the absence of DNA, the experiment serves as a
control for the proteins when they are bound in trans to
separate DNA strands. To simplify our analysis of the fluorescence
data, we used a truncation fragment of gp32 (gp32-B) that binds with
the same intrinsic affinity to ssDNA as intact gp32 but without the
gp32:gp32 cooperativity that complicates analysis. At 10 mM
Tris-Cl, pH 7.5, 150 mM NaCl, RB69 DNA polymerase binds to
primer template with ~100 nM affinity whereas gp32-B
binds to ssDNA about 100 times more weakly (10.9 µM) and
as proposed in our model, suggests DNA polymerase would bind
preferentially at the primer-template junction whereas gp32-B would
associate with available single-stranded template DNA (Fig. 3). Under
identical solution conditions, 1 µM RB69 DNA polymerase and gp32-B were titrated with p/t+12 DNA or p/t+T6 DNA (see
Table I and Fig. 3). The saturation binding curves are shown in Fig. 4A. As expected, in the
cis situation, the interaction between RB69 DNA polymerase
and SSB increased the affinity of SSB to ssDNA, which appeared as a
tighter binding regime, whereas in the trans experiment, two
independent binding processes were displayed as a two-phase curve. The
mechanism and their associated fits to our data are shown in Fig.
4B. The overall calculated dissociation constant for RB69
DNA polymerase/gp32-B to primer-template DNA at 10 mM
Tris-Cl, pH 7.5, 150 mM NaCl was 353 ± 25 nM, similar to the affinity of DNA polymerase alone to
primer-template DNA (124 ± 16 nM). In the
trans experiment, we were readily able to fit the data
assuming there were two independent binding processes in the cuvette,
and the calculated dissociation constant for RB69 DNA polymerase to
primer-template DNA was 139 ± 14 nM and for RB69
gp32-B to T6 DNA was 20.0 ± 0.4 µM.
These data are in good agreement with the dissociation constant for
individual binding processes, 124 ± 16 nM and
10.9 ± 1.7 µM, respectively, and support the
proposed mechanism for the trans experiment. The strength of
the RB69 DNA polymerase-SSB interaction can be estimated from the
difference in apparent association of SSB to available single-stranded template DNA. At 10 mM Tris-Cl, pH 7.5, 150 mM
NaCl, the presence of RB69 DNA polymerase results in a 57-fold increase
in SSB affinity for ssDNA as a result of DNA polymerase:SSB
cooperativity.
The A-domain of RB69 SSB Is Not Essential for Interaction between
DNA Polymerase and gp32--
It has been postulated that the A-domain
of gp32 was the binding site for RB69 DNA polymerase (18). If this is
true, the cooperative RB69 DNA polymerase-SSB interactions should be
abolished by the removal of the A-domain. To test this hypothesis, we
performed our DNA polymerase:SSB cooperativity studies using core gp32
(without the A-domain). To our surprise, we obtained a curve that
demonstrated tighter DNA polymerase-SSB interaction than we observed
with gp32-B protein (Fig. 4A). The calculated dissociation
constant for RB69 DNA polymerase:gp32 core to primer-template DNA at 10 mM Tris-Cl, pH 7.5, 150 mM NaCl was 48.6 ± 7.7 nM. This is about a 350-fold increase in affinity
from protein-protein interactions and is 7-fold higher than observed
for RB69 DNA polymerase:gp32-B. The dissociation constants are
summarized in Tables IV and
V. We also obtained a saturation curve
for RB69 DNA polymerase:gp32 intact protein binding to primer-template
DNA and for RB69 DNA polymerase:gp32-A binding to primer-template DNA.
Interestingly, the gp32 and gp32-B proteins showed similar
cooperativity (~60-fold), whereas gp32 core and gp32-A consistently
showed stronger polymerase:SSB cooperativity (~350-fold).
The specificity and strength of protein-DNA and protein-protein
contacts within the replisome are critical for its correct assembly and
function. Even a minimal replisome in T4/RB69 consists of at least five
proteins: DNA polymerase, sliding clamp, single-stranded DNA-binding
protein, the primase, and the helicase. Each organism has proteins
fulfilling analogous conserved roles throughout DNA replication,
although eukaryotes typically have more subunits (10, 34). Although
there have been several very good qualitative studies of
protein-protein interactions within replication, quantitative analysis
has proven to be more difficult (3, 35). Typically, this is because the
assembly of the replisomal proteins is quite weak and dissociates
readily in the absence of DNA. Based on the crystal structure of RB69
DNA polymerase·primer-template DNA complex and the biochemical
studies on the site size of T4 SSB core protein, we have found solution
conditions that support formation of a homogeneous complex of RB69 DNA
polymerase, SSB, and primer-template DNA. Our results show that the
cooperativity between RB69 DNA polymerase and SSB increases the
affinity of SSB to the single-stranded region on the template strand
about 60-fold.
Studies on the SSB family of proteins suggest their function in DNA
replication is conserved across all organisms. In addition to being
essential to DNA replication, recombination, and repair, they are among
the first proteins that assemble onto the DNA for these diverse
processes and recruit other proteins to the site of action. A direct
interaction between SSB and replicative DNA polymerase has also been
proposed for eukaryotes. In eukaryotes, the SSB protein is replication
protein A (RPA), a heterotrimeric protein shown to act as a
common "touchpoint" (36) for the assembly of the pol More specifically for the RB69/T4 system, it has been hypothesized that
the C-terminal A-domain of RB69 SSB recruits proteins essential for
replication, recombination, and repair (18, 21) via protein-protein
interactions. Current models suggest that in the DNA-free state of RB69
SSB, the A-domain is at least partially buried within the ssDNA-binding
site (21, 38, 39). Upon binding to ssDNA, the A-domain is exposed and
can make interactions more readily with other proteins (38, 40, 41).
Hurley et al. (18) purified a C-terminal region of the T4
SSB (residues 213-301) and immobilized it on an agarose matrix. They
found that this affinity column was able to retain DNA polymerase and
several other replication and recombination proteins from T4 whole cell extract. If the A-domain is the domain responsible for interaction with
DNA polymerase, the DNA polymerase·primer-template DNA·SSB core
complex should not display any cooperativity between DNA polymerase and
SSB core in our studies. Surprisingly, we observed about 7-fold higher
cooperativity for this complex than with gp32-B protein (which has the
A-domain). This implies that the A-domain on RB69 SSB is not the site
for binding to DNA polymerase. On the contrary, it slightly hinders the
interaction between RB69 DNA polymerase and SSB. Our studies support a
model for DNA polymerase-SSB interaction in the RB69/T4 system, in
which the A-domain partially folds back into the DNA-binding pocket,
blocking the binding site for DNA polymerase (Fig.
5). Upon binding to ssDNA, the A-domain is displaced, and the binding site for DNA polymerase is exposed to
make interactions with DNA polymerase. The experiments performed by
Hurley et al. (18) used a peptide that includes not only the
A-domain of T4 SSB but also 42 extra amino acids (residues 213-255) on
the core domain, and this may account for differences between our
studies. Because the crystal structure of T4 gp32 core bound to DNA
shows that these 42 amino acids form a large surface on the back of the
DNA-binding pocket (16), the binding site for DNA polymerase may be
composed, in part, by residues 213-255. If the A-domain is not
present, the interaction surface with DNA polymerase may already be
accessible and could explain why we see a 7-fold higher cooperativity
for the DNA polymerase·gp32-A·primer-template complex. Our data
suggest the binding site for DNA polymerase in gp32-A is exposed,
sending a "false signal" that gp32 is bound to DNA and is
available for DNA replication. In the intact gp32, the binding site for
DNA polymerase is protected by the A-domain and is exposed only when it
is bound to ssDNA.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
family
of replicative DNA polymerases (11). Structural studies on the T4/RB69
system have been successful in determining high resolution x-ray
structures for the DNA polymerase (gp43) (12-14), sliding clamp (gp45)
(13, 15), and SSB (gp32) (16) and provide an excellent starting point
for the study of replisome assembly.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and
gp32 Proteins--
Clones for the overexpression of RB69 DNA
polymerase and an exo
mutant were the generous gift of
Dr. J. Karam (Tulane University School of Medicine). RB69 DNA
polymerase exo
was purified in a similar manner as
described previously (26). Intact RB69 gp32, gp32-A, and gp32-B were
purified in a manner similar to intact T4 SSB (25) using ssDNA
cellulose affinity column. RB69 gp32 core protein was produced from
trypsin digestion of gp32-A protein (19) and purified using ssDNA
cellulose affinity column (26). The proteins were flash-frozen in
liquid nitrogen and stored in 10 mM Tris-Cl, pH 7.5, 10 mM NaCl, 1 mM EDTA, 3 mM
dithiothreitol, and 5% (v/v) glycerol.
DNA constructs for cis and trans experiments
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, gp32-B, and gp32 core protein were well behaved
monomeric species whereas intact gp32 and gp32-A were highly
polydisperse, consistent with their known ability to self-aggregate
(33). We estimated the molecular mass of the RB69 DNA polymerase
exo
·primer-template DNA complex to be 122.2 kDa, which
was 2.2% away from the theoretical molecular mass of 119.6 kDa.
Finally, we determined the molecular mass for the complexes in the
presence of different RB69 SSB proteins (Table
III). Because some aggregation was always
seen in the front shoulder (less than 5% of the total material), the
middle of the peak was selected for molecular mass calculations. Under
the conditions used (10 mM Tris-Cl, pH 7.5, 150 mM NaCl), the RB69 DNA polymerase·SSB·primer-template
complex was monodisperse (Fig. 1) and
displayed a molecular mass of about 130 kDa. Molecular mass estimates
for complexes made up of both DNA and protein are somewhat more
difficult as the dn/dc value of the sample must be estimated from
refractive index changes and led to slightly larger residual
errors for all complexes tested. Although the molecular mass of the
1:1:1 stoichiometry was about 10% lower than the theoretical value, we
used SDS-PAGE and UV absorbance at 280 and 260 nm to ensure that DNA
polymerase, SSB, and primer-template DNA were all present (data not
shown). Alternative stoichiometries lead to much greater errors and
allow us to conclude that there was one molecule of DNA polymerase
(104.6 kDa), one molecule of primer-template DNA (15.0 kDa), and one
molecule of RB69 SSB (26.2-31.1 kDa) in the complex. Interestingly,
whereas intact gp32 and gp32-A were polydisperse alone, the DNA
polymerase·primer-template DNA complexes with these proteins were
monodisperse (Table III) and further confirm that only one RB69 SSB was
accommodated onto the template DNA. We also performed the same
experiments using 10 mM Tris-Cl, pH 7.5, 10 mM
MgCl2 as a buffer but found that the complexes were more
polydisperse, presumably because of nonspecific aggregation at low
ionic strength (data not shown).
Monodispersity and accuracy of molecular mass of individual proteins
determined by light scattering
Monodispersity and accuracy of molecular mass of DNA
polymerase: p/t + 12 complexes determined by light
scattering in the presence or absence of RB69 SSB
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Fig. 1.
The molecular weight distribution of DNA
polymerase exo (a), gp32-B
(b), DNA polymerase exo
and p/t+12
complex (c), and DNA polymerase exo
,
p/t+12, and gp32-B complex (d) across the gel
filtration elution peak showing that they are monodisperse in 10 mM Tris-Cl, pH 7.5, 150 mM
NaCl. The chromatographic peaks are values from
the refractive index detector, and the points are the
weight-averaged molecular mass across the chromatographic peak for each
species tested.
with p/t+12 primer-template DNA.
Fig. 2 shows the saturation binding
curves of DNA polymerase to primer-template DNA under different salt
concentrations generated by measuring the percentage change in protein
fluorescence as a function of DNA concentration. Under low salt
conditions (50-100 mM NaCl), the binding is too tight to
allow an accurate estimate of Ka; however, we can
estimate the stoichiometry of binding to be 1:1 using the break point
estimated from the initial and final slopes of the binding curve (19).
The dissociation constant for RB69 DNA polymerase to primer-template
DNA at 10 mM Tris-Cl, pH 7.5, 100 mM NaCl was estimated to be greater than 2 nM, in good agreement with
previous studies (4, 5) on other polymerases. To obtain an accurate measurement of binding, it was necessary to increase the concentration of NaCl to 150 mM. Data were fitted in Dynafit, and the
calculated dissociation constant for RB69 DNA polymerase to
primer-template DNA at 10 mM Tris-Cl, pH 7.5, 150 mM NaCl was 124 ± 16 nM. In addition, we
noted that the maximum change in fluorescence quenching decreased as a
function of salt concentration. As a consequence all our studies were
performed at 150 mM NaCl.
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Fig. 2.
Fluorescence titration curves for the binding
of p/t+12 to RB69 DNA polymerase exo under different salt
concentrations.
F is the fluorescence quenched and
F0 is the starting fluorescence. All
measurements were at 20 °C at a protein concentration of 2 µM. The maximum percentage fluorescence quenching
decreases as the salt concentration increases, and all fluorescence
experiments were performed at 150 mM NaCl.
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Fig. 3.
Experimental design used for the
determination of cooperativity of RB69 DNA polymerase and SSB by
equilibrium fluorescence. A 1:1 mixture of RB69 DNA polymerase
exo and SSB were titrated under identical solution
conditions with either a primer-template DNA complex with an
11-nucleotide overhang on the template strand (cis) or a 1:1
mixture of a primer-template DNA complex with a five-nucleotide
overhang on the template strand and T6 (trans).
The cooperativity of DNA polymerase and SSB is determined from the
cis experiment whereas the trans experiment
serves as a control for binding to separate DNA strands. In the
cis experiment, the primer-template junction constrains the
two proteins in two dimensions, increasing local concentrations of the
proteins relative to each other, and makes the measurement of weak
interactions between RB69 DNA polymerase and SSB possible.
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Fig. 4.
a, fluorescence titration curves for the
cis experiments using different RB69 SSB proteins and for
the trans experiments using gp32-B. F is the
fluorescence quenched, and F0 is the starting
fluorescence. All measurements were at room temperature at a protein
concentration of 1 µM each and 10 mM Tris-Cl,
pH 7.5, 150 mM NaCl. b, global least squares
fits of fluorescence titrations in Dynafit (31). Left,
cis titration of p/t+12 to 1 µM DNA polymerase
exo
and 1 µM gp32-B. Right,
trans titration of p/t+6/T6 to 1 µM DNA polymerase exo
and 1 µM gp32-B. The buffer conditions are 10 mM
Tris-Cl, pH7.5, 150 mM NaCl. The mechanisms used to fit the
data are shown under each panel.
Dissociation constants for RB69 DNA polymerase exo and SSBs
to different DNA ligands at 10 mM Tris-Cl, pH 7.5, 150mM NaCl
Dissociation constants for RB69 SSBs to different DNA ligands at 10 mM Tris-Cl, pH 7.5; 150mM NaCl
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
replication complex. Our studies show that binding in cis to
the same DNA strand is facilitated by protein-protein interactions and
thus promotes proper assembly of proteins to the primer-template
junction. Immunoaffinity chromatography has shown that DNA pol
from
calf thymus and the p70 subunit of RPA associate with several other
proteins involved in DNA replication (37), and RPA is also directly
responsible for the loading of replication factor C and pol
onto primer-template DNA in a coordinated manner (36). These data are
consistent with the scenario we found in our studies in which RB69 DNA
polymerase and SSB directly and coordinately assemble at the
primer-template junction.
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Fig. 5.
Model for RB69 DNA polymerase-SSB interaction
at the primer-template junction. Prior to binding to the
primer-template junction, the A- and B-domains on RB69 SSB are buried.
Upon binding to the primer-template junction with DNA polymerase in a
coordinate manner, the A-domain of SSB is displaced by ssDNA and
exposes the binding site for DNA polymerase, which may overlap with the
site for the B-domain. The B-domain of SSB contacts the adjacent SSB
for cooperative binding to ssDNA.
We have found that specific protein-protein cooperativity between RB69
DNA polymerase and its SSB (gp32) has the overall effect of increasing
the affinity of SSB for ssDNA proximal to the DNA polymerase by about
60-fold. This modest protein-protein association predicts that, in the
absence of DNA, the DNA polymerase and SSB will not associate to form
unproductive "pseudo-replisomes" at their in vivo
concentrations. Conversely, the cooperativity assures that when both
proteins are bound to the same DNA strand, they will productively
associate to facilitate accurate and processive DNA synthesis. Previous
studies have suggested that SSB from bacteriophage T4 and E. coli are able to diffuse along ssDNA, and therefore the
"search" for either another SSB or DNA polymerase is
confined to two dimensions (42). The protein-protein cooperativity
exhibited by DNA polymerase and SSB furthers the view that the dynamic
character of the replisome is the product of subtle and finely tuned
interactions at the molecular level. It is our expectation that these
organizing principles will be seen throughout DNA replication and in
all organisms.
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ACKNOWLEDGEMENTS |
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We thank Dr. Jim Karam and Dr. J. Borjac for clones and results prior to publication.
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FOOTNOTES |
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* This work was supported in part by American Cancer Society Grant RSG-03-051-01-GMC and an Oak Ridge Associated Universities Powe Junior Faculty Enhancement Award (to Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry and Cell Biology, Rice University, 6100 S. Main St.,
MS140, Houston, TX 77005. Tel.: 713-348-5493; Fax:
713-348-5154; E-mail: Shamoo@rice.edu.
Published, JBC Papers in Press, November 27, 2002, DOI 10.1074/jbc.M210497200
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ABBREVIATIONS |
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The abbreviations used are: SSB, single-stranded DNA-binding protein; ssDNA, single-stranded DNA.
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