From the
The
The replicative polymerase of Escherichia coli, DNA
polymerase III holoenzyme (holoenzyme), contains 10 nonidentical
subunits (reviewed in Refs. 1 and 2). The
Several subassemblies of the
holoenzyme can be purified from cell lysates. The smallest of these is
the core polymerase, a heterotrimer of
The core polymerase is neither
highly processive nor rapid in DNA synthesis; it polymerizes
nucleotides at a rate of
The
For several years, the
genes encoding five of the holoenzyme subunits have been known. The
This first report
of the series focuses on the
Gel filtration of mixtures containing
less than all five subunits of the
The recoveries of
Combining the Stokes radius and the s value in the mass equation of Siegel and Monty
(35) yields
a minimum estimated mass of 256 kDa for the reconstituted
For structural studies
to be described below, the
The molar
ratio of
The molar ratio of subunits
in the
In
Fig. 5
, the remaining combinations of subunits were analyzed by
gel filtration, specifically those containing
The mixture of
Does the
Perhaps a
The analysis of Fig. 5shows
that
In each of the panels in Fig. 7, a different
subunit of the
In Fig. 7A,
In experiments using
Analysis of
immobilized
Since
The apparent k
The schematic in Fig. 8depicts a subunit organization of the
A direct interaction between
The Stokes radii and s values
of the
Molar ratios of subunits in the constituted
complex of DNA polymerase III holoenzyme, the
replicase of Escherichia coli, couples ATP hydrolysis to the
loading of
sliding clamps onto primed DNA. The
sliding
clamp tethers the holoenzyme replicase to DNA for rapid and processive
synthesis. In this report, the
complex has been constituted from
its five different subunits. Size measurements and subunit
stoichiometry studies show a composition of
`
.
Strong intersubunit contacts have been identified by gel filtration,
and weaker contacts were identified by surface plasmon resonance
measurements. An analogous
complex has also been constituted and
characterized; it is nearly as active as the
complex in clamp
loading activity, but as shown in the fourth report of this series, it
is at a disadvantage in binding the
,
`,
, and
subunits when core is present (Xiao, H., Naktinis, V., and
O'Donnell, M. (1995) J. Biol. Chem. 270,
13378-13383). The single copy sub-units within the
complex
provide the basis for the structural asymmetry inherent within DNA
polymerase III holoenzyme.
subunit is the DNA
polymerase; the
subunit is the proofreading
3`-5`-exonuclease; and the
subunit is a dimer in the shape
of a ring with a central cavity for encircling duplex DNA. The function
of the
ring is to slide freely along duplex DNA while tethering
the polymerase machinery to the template for highly processive
synthesis
(3, 4) .
(5) . The
polymerase (Pol)
(
)
III` subassembly is composed
of four different subunits: a
dimer and two core polymerases
(core
-
)
(6) . The
complex,
composed of five different subunits (
`
), is
a molecular matchmaker that couples ATP hydrolysis to load
clamps
onto primed DNA. The largest subassembly of the holoenzyme is Pol III*,
which contains nine different subunits: two cores,
, and the
complex
(9) . Two DNA polymerases in one molecular particle fits
nicely with the hypothesis that replicative polymerases act in pairs
for coordinated synthesis of both leading and lagging strands of a
chromosome
(7, 8) .
10/s and dissociates from DNA after
incorporating
11 nucleotides
(10, 11) . The Pol III`
and Pol III* assemblies are not very processive either, but are
severalfold more efficient than core
(10, 12) . The
subunit sliding clamp is needed for rapid and highly processive DNA
synthesis. Once the
ring is assembled onto DNA, it confers
efficient synthesis on all these polymerase
subassemblies
(3, 13, 14) .
subunit
does not assemble onto DNA by itself, but requires the ATP-dependent
clamp loading activity of the
complex
(3, 13, 15, 16) . The individual
functions of
complex subunits are largely unknown. Past studies
have shown that
binds ATP
(17) and can function with
to place
onto DNA
(18, 19) , although the
`
subunit stimulates this reaction considerably
(19, 20) ,
and
and
are needed at physiological ionic
strength
(18) . Elucidating the exact function of each subunit
and the role(s) of ATP is an important goal.
subunit is encoded by dnaE,
by dnaQ (also
mutD),
by dnaN, and
and
are both
encoded by dnaX. The
subunit is approximately the
amino-terminal two-thirds of
and is generated by a -1
translational frameshift that results in only one unique amino acid in
, the C-terminal Glu residue
(21, 22, 23) .
Two years ago, the remaining five genes of the holoenzyme were
identified
(19, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) and used to produce large amounts of pure
subunits
(19, 24, 25, 26, 27) .
With all 10 subunits in hand, detailed questions of structure and
function can be addressed. This series of reports presents five related
topics in which the pure subunits are used to assemble the holoenzyme.
Studies of the assembly process have outlined the subunit contacts and
the overall structure of this replicative machine.
complex. The
complex has been
constituted in quantity from its five separate subunits, revealing
several subunit contacts and providing a framework for its overall
organization. Compositional studies show that four of the five subunits
are present in single copy, which defines the
complex as a
structure lacking a 2-fold axis of symmetry (i.e. it is
asymmetric). In the second report, the point of contact between the
clamp and the
complex is identified as residing nearly
completely, if not solely, in the
subunit
(41) .
Surprisingly, ATP is needed for the
complex to
``present'' the
subunit for interaction with
,
providing insight into the role for ATP and the mechanism of clamp
loading. The third report defines the assembly path of the nine-subunit
Pol III* assembly
(38) . Study of Pol III* and its subunit
composition reveals that contact between
and
is the central
touch point between the clamp loader (
complex) and the twin
polymerase (Pol III`) within the holoenzyme. Compositional study of Pol
III* shows that only one copy of some of the subunits is present, and
therefore, the holoenzyme must be structurally asymmetric. The first
report shows that the single copy subunits can assemble with either
or
to form a clamp loader. The fourth report utilizes
ATP-binding site mutants of
and
to show that in Pol III*,
the single copy subunits of the clamp loader reside on
(39). The
preference of
over
appears to be caused by association of
core with
(but not
), which puts the association of
with the single copy subunits at a kinetic disadvantage. The last
report shows that four different forms of Pol III can be assembled in
the presence of primed DNA
(42) . These distinct polymerase forms
may perform different tasks in DNA metabolism.
Materials
Radioactive nucleotides were obtained
from DuPont NEN, and unlabeled nucleotides were from Pharmacia Biotech
Inc. Proteins were purified as described: ,
,
, and
(33) ;
(4);
and
`
(24) ;
and
(25) ; and
(27) . Pol III* was purified from
cell lysates
(9) ; the
complex was purified from cell
lysates
(16) ; and the
complex was constituted and
purified as described
(33) . Protein concentrations were
determined from their extinction coefficients at 280 nm, except for Pol
III* and the
complex purified without overproduction, which were
quantitated using the protein assay from Bio-Rad and bovine serum
albumin as a standard. M13mp18 ssDNA was phenol-extracted from phage
that was purified by two consecutive bandings (first down and then up)
in cesium chloride gradients as described
(43) . M13mp18 ssDNA
was primed with a DNA 30-mer (map positions 6817-6846) as
described
(33) . DNA oligonucleotides were purchased from Oligos
Etc. Buffer A contained 20 mM Tris-HCl (pH 7.5), 0.1
mM EDTA (pH 7.5), 20% glycerol, and 5 mM DTT.
Replication buffer contained 20 mM Tris-HCl (pH 7.5), 4%
glycerol, 0.1 mM EDTA, 40 µg/ml bovine serum albumin, 5
mM DTT, 8 mM MgCl
, 0.5 mM ATP,
60 µM dCTP, 60 µM dGTP, 60 µM
dATP, and 20 µM [
-
P]TTP
(specific activity of 2000-4000 cpm/pmol). Column buffer
contained 20 mM Tris-HCl (pH 7.5), 10% glycerol, 2 mM
DTT, 0.1 mM EDTA, and 100 mM NaCl. SPR buffer
contained 10 mM Hepes-NaOH (pH 7.4), 150 mM NaCl, 3.4
mM EDTA, and 0.005% Tween 20. Sedimentation buffer contained
20 mM Tris-HCl (pH 7.5), 2 mM DTT, 0.1 mM
EDTA, and 100 mM NaCl.
Replication Assays
Replication assays contained 63
ng (32 fmol) of singly primed M13mp18 ssDNA, 0.82 µg of SSB, 8.8 ng
(56 fmol) of complex, and 20 ng (246 fmol as dimer) of
in a final volume of 25 µl of replication buffer (after the
addition of remaining proteins). All proteins were added to the assay
on ice and then shifted to 37 °C for 5 min. DNA synthesis was
quenched and quantitated using DE81 paper as described
(34) .
When needed, proteins were diluted in 20 mM Tris-HCl (pH 7.5),
2 mM DTT, 0.5 mM EDTA, 20% glycerol, and 50 µg/ml
bovine serum albumin.
Gel Filtration
Gel filtration of the reconstituted
and
complexes was performed at 4 °C (unless indicated
otherwise) using a Superose 6 HR 10/30 column (Pharmacia Biotech Inc.)
equilibrated with column buffer containing 5 mM MgCl
and 0.2 mM ATP. To constitute these complexes,
(78
µg, 0.88 nmol as dimer) or
(118 µg, 0.83 nmol as dimer)
was incubated with
(39 µg, 2.4 nmol as monomer) and
(30 µg, 2 nmol as monomer) for 30 min at 15 °C. To this protein
mixture was added
(77 µg, 2 nmol as monomer) and
` (74
µg, 2 nmol as monomer) for 30 min at 15 °C. The protein mixture
was then concentrated to 100 µl by spin dialysis using a Centricon
30 apparatus (Amicon, Inc.) and gel-filtered. After the first 7 ml,
fractions of 200 µl were collected and analyzed on 15%
SDS-polyacrylamide gels (100 µl/lane) stained with Coomassie Blue
R-250. Densitometry of stained gels was performed using a Pharmacia
Ultrascan XL laser densitometer. Replication assays of column fractions
were performed by first diluting a 2-µl aliquot 20-fold and then
adding 2 µl to the assay.
complex (i.e.Fig. 5
) were performed using a Superose 12 column (in one
case, Superdex 75 was used as indicated in the legend to Fig. 5).
Subunit mixtures were incubated for 30 min at 15 °C in 200 µl
of column buffer and included (when present) the following:
, 84
µg (0.9 nmol as dimer);
, 57 µg (1.5 nmol);
`, 56
µg (1.56 nmol);
, 41.8 µg (2.52 nmol); and
, 25
µg (1.67 nmol). Subunit mixtures were gel-filtered as described
above, except that after collecting the first 6.0 ml, fractions of 170
µl were collected. Additions of
in the absence of
required 0.5 M urea as described
(26) . To remove urea,
mixtures containing both
and
were first mixed using a
1.5-fold excess of
to
in a final concentration of 0.5
M urea in buffer A and then dialyzed against sedimentation
buffer.
Figure 5:
Search for subassemblies of the
complex. Gel filtration analysis of mixtures of
complex subunits
was performed as described under ``Experimental Procedures.''
The SDS-polyacrylamide gels of the column fractions were stained with
Coomassie Blue. Fraction numbers are shown at the top; positions of
molecular weight markers are noted on the left; and
complex
subunits are identified on the right. A-G were analyses
on Superose 12, and H was an analysis on Superdex
75.
Protein standards (Bio-Rad and Sigma) were a mixture of 50
µg each in 100 µl of column buffer. The K value was calculated using the following equation:
K
= (V
- V
)/(V
- V
), where V
is the observed elution volume, V
is the included volume, and V
is the
exclusion volume. The V
value for both
Superose 6 and 12 columns was 24 ml (manufacturer's
specifications), and the V
value, determined using
M13mp18 ssDNA saturated with SSB (total molecular mass of
25 MDa),
was 6.0 ml for Superose 12 and 7.0 ml for Superose 6.
Glycerol Gradient Sedimentation
The
`
and
`
complexes
were constituted as described above for gel filtration analysis and
then layered onto separate 11.6-ml 10-30% glycerol gradients in
sedimentation buffer. Protein standards (50 µg each in 100 µl
of sedimentation buffer) were layered onto a parallel gradient, and the
gradients were centrifuged at 270,000
g for 30 h at 4
°C. Fractions of 170 µl were collected from the bottom of the
tube and analyzed on a 13% SDS-polyacrylamide gel (100 µl/lane)
stained with Coomassie Blue and analyzed in replication assays as
described for the gel filtration column fractions. Stability of the
[
H]
Complex-The
and
subunits were tritiated by
reductive methylation as described
(3, 44) to specific
activities of 7.5
10
and 1.4
10
cpm/pmol, respectively. The
[
H]
complex was constituted upon
mixing 4 mg of
with 0.125 mg of [
H]
,
2.7 mg of unlabeled
, 0.25 mg of [
H]
,
and 1.7 mg of unlabeled
in a final volume of 3.0 ml of buffer A
(0.5 M urea final concentration due to the urea in
).
After 1 h at 15 °C, the mixture was applied to a 1-ml Mono Q column
and eluted with a 19-ml linear gradient of 0-0.4 M NaCl
in buffer A. The
[
H]
complex was
the last to elute (0.22 M NaCl). Fractions containing
[
H]
were pooled (3.1 mg;
specific activity of 1.8
10
cpm/pmol), dialyzed
against 4 liters of buffer A, and then aliquoted and stored at
-70 °C. Dissociation of the
[
H]
complex was assayed by gel
filtration on Superose 12, which resolves
[
H]
from dissociated
[
H]
. A total of 8.9 µg (71 pmol)
of
[
H]
complex was incubated for
5 min at the indicated temperature in either the absence or presence of
5.5 µg (141 pmol) of
and 5.25 µg (140 pmol) of
` in
100 µl of column buffer containing 0.3 M NaCl, 0.5
mM ATP, and 8 mM MgCl
. After incubation,
reactions were analyzed by gel filtration on Superose 12 essentially as
described above, except that the column buffer contained 300
mM NaCl, 8 mM MgCl
, and 0.5 mM
ATP. The amount of [
H]
in each column
fraction was determined by liquid scintillation counting. Different
temperatures of gel filtration were achieved by immersing the column
and the injection loop in a water bath containing ice or held at 28 or
37 °C.
Preparative Constitution of the
A
mixture of 11.1 mg of Complex
(118 nmol as dimer), 13.7 mg of
(355
nmol as monomer), 8.7 mg of
` (235 nmol as monomer), 5.9 mg of
(354 nmol as monomer), and 3.6 mg of
(237 nmol as monomer
in 4 M urea) was incubated in a final volume of 63.75 ml of
buffer A for 30 min at 15 °C (incubation was started with
and
proteins combined together in a final volume sufficient to
bring the urea concentration to 0.5 M, and then the other
subunits were added). This mixture was loaded onto an 8-ml Mono Q HR
10/10 column (Pharmacia Biotech Inc.) and eluted with a 180-ml linear
gradient of 0-0.4 M NaCl in buffer A at 0.3 ml/min.
Fractions of 2.5 ml each were collected. Fractions 46-52, which
contained
`
, were pooled (18.5 ml, 18 mg),
dialyzed against 4 liters of buffer A, and then aliquoted and stored at
-70 °C.
HPLC
Reversed-phase HPLC analysis was performed on
a Waters system. The reconstituted complex (30 µg) in 100
µl of buffer A was adjusted to 1% trifluoroacetic acid and injected
onto a Dynamax C
(butyl) reversed-phase HPLC column (250
4.6 mm) with a pore size of 300 Å equilibrated with 40%
HPLC-grade acetonitrile (J. T. Baker Inc.) in 0.125% trifluoroacetic
acid. Subunits were eluted with a 48-55% acetonitrile gradient in
0.125% trifluoroacetic acid over 3 min followed by a 55-100%
acetonitrile gradient in 0.125% trifluoroacetic acid over 12 min at a
flow rate of 1 ml/min. Elution of subunits was monitored at 280 nm and
recorded on chart paper. The areas under the peaks were measured
manually by cutting the peaks out and weighing them. The molar
extinction coefficients of the subunits calculated from their amino
acid sequences are as follows:
, 20,340 M
cm
;
, 46,130 M
cm
;
`, 60,136 M
cm
;
, 29,160 M
cm
; and
, 24,040 M
cm
).
complex
subunits from the HPLC column were determined by passing a mixture of
the subunits of known concentration (measured by their absorbance at
280 nm and known extinction coefficient) over the same HPLC column. The
total absorbance recovered for each subunit was as follows:
,
75.6%;
, 87.3%;
`, 100.4%;
, 90.6%; and
, 79.1%.
Hence, all the subunits were recovered from the HPLC column in a
reasonably high yield. These recoveries were not used to correct the
observed molar ratio of subunits in the
complex as the recovery
measurements have an error of their own.
SPR
Immobilization of subunits was performed on
the carboxymethyldextran matrix-coated sensor chip CM5 by carbodiimide
covalent linkage following the manufacturers' instructions
(Pharmacia Biosensor AB) using 30-µl solutions of subunits in SPR
buffer in 10 mM sodium acetate at the following subunit
concentrations and pH values: , 1.24 µM and pH 5.5;
`, 0.35 µM and pH 5.5; and
, 1.13
µM and pH 4.5. To immobilize the
complex, the
subunit was immobilized, and then a 35-µl solution of
(0.5 µM in SPR buffer containing 0.5 M urea) was
passed over the chip for 3 min, followed by washing with SPR buffer
(28% of immobilized
became complexed with
). The final
change in response units for each immobilization was as follows:
,
3542 response units;
`, 3562 response units;
, 2963 response
units; and
, 3683 response units (2936 response units of
plus 747 response units after adding
). SPR analysis was
performed by injecting 30 µl of a 1 µM solution of the
indicated protein (as monomer, except for
and
, which are
expressed as dimers) in SPR buffer for 3 min at 25 °C. All proteins
were dialyzed against SPR buffer to reduce buffer-related artifacts. To
obtain a value for the k
, injections were
followed for 3 min by SPR buffer lacking protein. After completing each
analysis, the surface of the chip was regenerated by injecting 10
µl of 0.1 M glycine (pH 9.5), which released remaining
bound protein without decreasing the capacity of the immobilized
protein to bind in future injections. When the
complex was
immobilized, regeneration was performed by consecutive injection of 30
µl of 6 M urea and 30 µl of 2 M urea in SPR
buffer, followed by de novo binding of
to
as
described above. Data are presented as the observed change in response
units divided by the molecular mass of the subunit in the mobile phase
(
,
`, and
as monomer;
and
as dimer; core
as
;
as
; and
` as
`
). All apparent k
and k
values were determined through
nonlinear curve fitting using the Pharmacia Biosensor kinetics software
(BIAevaluation 2.0) assuming the simplest case: A + B
AB.
Constitution of the
To determine
if the Complex
complex (
`
) can be
reconstituted from purified overproduced subunits,
was mixed with
a 2-fold molar excess each of
,
`,
, and
and
then gel-filtered (Fig. 1). A complex of
`
formed as indicated by comigration of all
five subunits (Fig. 1A, panel 1, fractions
21-25); excess proteins eluted later as the
` complex
(fractions 40-49) and the
complex (fractions
46-49). Column fractions containing the
`
complex were active in assembly of the
clamp onto primed DNA, leading to processive DNA synthesis by the
polymerase (Fig. 1A, panel3).
Figure 1:
Native mass of
constituted `
and
`
complexes. A, either
(panel 1) or
(panel 2) was incubated with
,
`,
, and
, followed by gel filtration on a
Superose 6 column as described under ``Experimental
Procedures.'' Column fractions (Frx) are identified above
and below the Coomassie Blue-stained SDS-polyacrylamide gels. The first
lane of each gel contains protein standards, and their molecular
weights (MW) are indicated to the left. The
,
,
,
`,
, and
subunits are identified to the right
of each gel. Panel3 shows the activity of the column
fractions of
`
(
) and
`
(
). Panel4 shows
the elution of
`
and
`
relative to protein standards of known
Stokes radii calculated from their diffusion coefficients (as described
in Ref. 36). B, shown is the glycerol gradient sedimentation
analysis of
`
(panel1)
and
`
(panel2). Either
or
was incubated with
,
`,
, and
,
followed by sedimentation in a 10-30% glycerol gradient as
described under ``Experimental Procedures.'' Panel3 shows the activity assays of glycerol gradient
fractions of
`
(
) and
`
(
). Panel4 compares the migration of protein standards of known s values with the migration of
`
and
`
. Tgb, thyroglobulin (670 kDa,
85.0 Å); Apf, horse apoferritin (440 kDa, 59.5 Å);
Amy,
-amylase (200 kDa, 8.9 S); IgG (158 kDa, 52.3
Å, 7.4 S); BSA, bovine serum albumin (67 kDa, 34.9
Å, 4.41 S); Ova, chicken ovalbumin (43.5 kDa, 27.5
Å, 3.6 S); Myo, horse myoglobin (17.5 kDa, 19.0 Å,
2.0 S).
Comparison with protein standards showed that the complex has
a Stokes radius of 67 Å (Fig. 1A, panel4), close to the 61-Å radius determined for the
complex purified from E. coli lysates
(25) . The
constituted
complex sedimented in a glycerol gradient with an
s value of 8.7 S (Fig. 1B, panels1, 3, and 4), similar to the value of
8.2 S for the
complex purified from E. coli lysates
(16) . Trailing of the
complex in the
sedimentation analysis indicates that some dissociation occurs during
the 30-h procedure. Hence, the s value should be taken as a
minimum estimate.
complex
(), similar to the value of 210 kDa for the
complex
purified from E. coli(16) .
complex was prepared in quantity and
purified away from excess subunits that were not bound in the complex.
Preparation of the
complex is shown in Fig. 2, in which the
subunits were mixed using limiting
, followed by chromatography on
a fast protein liquid chromatography Mono Q column eluted with an NaCl
gradient. The
complex was tightly retained on the column and was
the last to elute. This method is highly efficient as the 11 mg of
present initially was recovered as 18 mg of
complex for an
overall yield of 79%.
Figure 2:
Preparative constitution and purification
of the complex. A, the subunits of the complex were
incubated, and the
complex was separated from free components on
a fast protein liquid chromatography Mono Q column as described under
``Experimental Procedures.'' B, 7-µl aliquots of
the indicated column fractions were analyzed by 13% SDS-polyacrylamide
gel electrophoresis as described under ``Experimental
Procedures.''
Molar Ratio of Subunits in the
Two
techniques were applied to determine the molar ratio of subunits within
the Complex
complex. The simplest was to scan the Coomassie Blue-stained
SDS-polyacrylamide gels of the
complex that was either
constituted from pure subunits or purified as a complex from E.
coli lysates. The molar ratio of
complex subunits from this
analysis is presented in after correcting the areas under
the peaks for the relative molecular mass of each subunit and
normalizing to the
` subunit. The results suggest a molar ratio of
`
,
similar to the conclusions of an earlier study using the
complex
purified from cell lysates
(16) . Different proteins may take up
different amounts of Coomassie Blue dye, and therefore, we used as
standards individual subunits of known concentration determined by
absorbance from their extinction coefficients at 280 nm.
complex subunits was also determined by an HPLC
approach. The pure constituted
complex was treated with 1%
trifluoroacetic acid to disperse the subunits and then applied to an
HPLC column with a four-carbon chain as a ligand. Elution of the column
was monitored at 280 nm (Fig. 3), and the peaks were assigned by
collecting fractions and analyzing them on an SDS-polyacrylamide gel.
The 280 nm absorbance is due mainly to the Trp and Tyr residues in each
protein. From the subunit gene sequences, the number of Trp and Tyr
residues in each subunit allowed us to calculate their relative molar
ratio from the area under their respective 280 nm absorbance peaks. The
results () indicate a molar ratio of
`
.
An HPLC analysis of the
complex purified intact from E. coli cell lysates was also performed. Due to insufficient material,
only one analysis was performed at 280 nm, but the observed molar ratio
of subunits
(
`
)
was consistent with the subunit molar ratio measurements of the
complex constituted from pure subunits. The composition of the
complex most consistent with the measured molecular mass (256 kDa) and
the observed subunit molar ratios is as follows: one each of
,
`,
, and
and two or three of
(
`
)
(). A simple explanation of the value of two to three
subunits in the complex is that the
complex preparation is a
mixture of two complexes:
`
and
`
.
As discussed in the third report of this series,
and
migrate on gel filtration columns as homotetramers and a heterotetramer
of
(38) . Only one copy each
of
,
`,
, and
can associate with the tetramer
(e.g. to form
`
,
`
,
or
`
)
(38). The
and
subunits can also form homodimers as they
appear as such in glycerol gradient analysis
(17, 36) and are present as two copies each in Pol III*
(38) .
If the
,
`,
, and
subunits first associate with
a dimer (of
or
), then the formation of the
tetramer is prevented (i.e. yielding a complex of either
`
or
`
).
Hence, the value of two to three
subunits in the
complex
may reflect the proportions of
and
that the single copy subunits become associated with. However, a
trimer as the basis for the observed stoichiometry of
in
the
complex cannot be rigorously ruled out.
Figure 3:
Molar ratio of subunits in the
complex. Separation of the subunits of the pure
complex on a
C
reversed-phase HPLC column was performed as described
under ``Experimental Procedures.'' Elution of subunits was
monitored at 280 nm. The assignment of a peak with its respective
subunit, shown above each peak, was determined upon collecting the
peak, evaporating the sample to dryness, and analysis on a 15%
SDS-polyacrylamide gel.
The
The Complex
subunit (72 kDa) is
encoded by the same gene as
(47 kDa) and therefore contains the
amino acid sequence of
within it (except for the C-terminal amino
acid of
) and an additional extension of 213 amino acids at the C
terminus (21-23). Hence,
can probably bind
,
`,
, and
to form a ``
complex''
(
`
). Indeed, our previous studies showed
that the
subunit was capable of forming
and
` complexes
(19, 26) . Upon mixing
with
,
`,
, and
, a
complex was formed as
anticipated. Fig. 1(panel2 in A and
B) shows the gel filtration and sedimentation analyses of the
complex. The replication assays in panel3 show
that the activity corresponds to the position of the
complex.
Combining the Stokes radius (75 Å) and the s value (8.3
S) of the
complex in the equation of Siegel and Monty yields an
observed mass of 271 kDa ().
complex, determined by densitometry of the Coomassie
Blue-stained SDS-polyacrylamide gel, yielded a subunit stoichiometry
similar to that of the
complex (). A stoichiometry
of two
protomers and one monomer each of
,
`,
,
and
predicts a mass of 250 kDa for the
complex, consistent
with the observed mass of 271 kDa (). The value of two to
three
protomers in the
complex is possibly due to a mixture
of two complexes
(
`
and
`
)
due to the variable aggregation state of
as discussed above for
in the
complex.
Replicative Activity of Constituted Complexes
Are
these constituted clamp loader complexes as active as the naturally
purified complex in loading
onto primed DNA for processive
synthesis by the core polymerase? In Fig. 4, the complexes were
titrated into a replication assay containing the
subunit and core
polymerase in saturating amounts. The DNA template is a long (7.2
kilobases) M13mp18 circular ssDNA primed with a single synthetic DNA
oligonucleotide and coated with SSB. In this assay, there is no
detectable DNA synthesis unless the
clamp has been assembled onto
DNA for use by the core polymerase. The results show that the activity
of the constituted
complex is comparable to (in fact, slightly
higher than) the
complex purified intact from E. coli cell lysates. In contrast, the
complex appears to be
slightly less active and does not reach the same plateau level as the
complex.
Figure 4:
Activity of constituted and
complexes. Replication assays were performed as described under
``Experimental Procedures.'' Shown is the stimulation of DNA
synthesis upon the addition of increasing amounts of the following: the
complex purified from E. coli lysates (
), the
constituted
complex (
), or the constituted
complex
(
).
Strong Intersubunit Contacts within the
Gel filtration is a nonequilibrium technique, and thus,
only strong subunit contacts survive. To determine which subunits of
the
Complex
complex are in strong contact with one another, different
combinations of subunits were incubated together and analyzed by gel
filtration. Previously, we analyzed all combinations of
,
,
and
` with the finding that a
`
complex was stabile to gel filtration, as was a
`
complex, but the
+
and
+
` mixtures
did not result in gel-filterable complexes
(19) . Also, all
combinations of
,
, and
have been analyzed by gel
filtration with the result that
,
, and
complexes were observed, but the
+
mixture did not result in a stabile complex
(26) .
and either one or
both of
and
` plus one or both of
and
. In
Fig. 5A, the mixture of all five subunits (i.e. the entire
complex) was analyzed as a basis of comparison
for the other panels. The analysis shows a similar pattern as in
Fig. 1A in which the
complex (fractions
16-22) eluted ahead of the
` complex (fractions
34-44) and the
complex (fractions 40-48). In
Fig. 5B, a mixture of the
,
,
, and
subunits did not result in a four-subunit complex, but assorted
as a
complex (fractions 18-28) and a free
subunit (fractions 40-46).
,
`,
, and
in Fig. 5C resulted in a stabile
four-subunit
`
complex in fractions 18-28,
eluting earlier than the excess
` subunit (fractions 40-46)
and the
complex (fractions 40-48). This result was
somewhat surprising as
` does not coelute with either
or
during gel filtration. A mixture of
,
`, and
in Fig. 5D resulted in a stabile heterotrimer of
`
(fractions 20-28), but a mixture of
,
`, and
(Fig. 5E) showed no complex. Hence,
is not needed for
` to form a stabile complex with
.
` complex bind
? In
Fig. 5F, a mixture of
,
,
`, and
was analyzed, but
was not assimilated into the
`
complex (i.e.
migrated alone in fractions 50-58
and not with
` in fractions 20-26). Since both
` and
are stabile to gel
filtration
(19, 26) , a
`
complex
should also be stabile. Fig. 5G shows the anticipated
formation of a gel-filterable
`
complex (fractions
18-26).
`
complex can form in
the absence of
. This four-subunit mixture was tested in
Fig. 5H using a Superdex 75 column (hence its own set of
fraction numbers). The pattern on the SDS-polyacrylamide gel showed
formation of the
` and
complexes, but not a
`
complex.
strengthens the binding of
` to
. In
Fig. 6
, we examined this cooperative binding further. We used a
complex that was reconstituted using
[
H]
and [
H]
to
easily follow
(and
that dissociated from
it) in the column fractions. The
[
H]
complex was gel-filtered at
three different temperatures (Fig. 6, A-C). At 0
°C, the
[
H]
complex remained
nearly completely intact, but as the temperature was increased to 37
°C, most of the [
H]
dissociated
from
. The addition of
` provided stabile association of
[
H]
with
at 37 °C
(Fig. 6D), consistent with cooperative binding of
and
` to
. Analysis of column fractions by 13%
SDS-polyacrylamide gel electrophoresis followed by fluorography to
visualize
H-labeled subunits showed that the dissociated
peak was an equal mixture of [
H]
and
[
H]
, and therefore, both dissociated from
simultaneously (data not shown).
Figure 6:
Cooperativity between ` and
in binding to
. The
[
H]
complex was gel-filtered at
different temperatures as described under ``Experimental
Procedures.'' The earliest peak to elute was the
[
H]
complex, and the second peak
was the [
H]
complex. A,
analysis of
[
H]
at 0 °C;
B, analysis of
[
H]
at
28 °C; C, analysis of
[
H]
at 37 °C; D,
analysis of
[
H]
` at 37
°C.
Surface Plasmon Resonance Analysis
Next, the
weaker intersubunit interactions were analyzed using the SPR technique
in a Biosensor instrument. In SPR, one protein is immobilized on a
sensor chip, and a second protein is injected over the surface in a
mobile phase. If the two proteins interact, the resulting increase in
mass on the chip is detected and plotted as an increase in response
units over time, allowing calculation of the apparent k value. After the injection of protein in the mobile phase is
complete, buffer is passed over the chip, and the dissociation of the
proteins is observed as a loss in mass over time from which the
apparent k
value can be calculated. From these
data, the apparent dissociation constant
(K
) is obtained
(K
=
k
/k
). It is important to
note that these constants apply to the specific conditions used in
these experiments and could vary under other conditions. In this
report, similar levels of immobilized protein and 1 µM
concentrations of protein in the mobile phase were used. Hence, the
values should be approximately comparable for the different
experiments.
complex was attached to the sensor chip, and then
1 µM solutions of the other subunits were serially passed
over the chip. The
subunit was also included in the analysis, as
was the core polymerase. In Fig. 7, openarrowheads mark the start of protein injection, and closed arrowheads mark the end. Since
is insoluble by itself, the
complex was analyzed, and therefore, subunits that bind
specifically to
must be inferred upon comparison to results
using
alone. The immobilization of
appeared to inactivate
it, and therefore, SPR analysis could not be performed.
(
)
The data are plotted as response units divided by the
molecular mass of the protein in the mobile phase to normalize the
results for the different masses of subunits in the different
injections. In general, the stoichiometry of protein in the mobile
phase bound to immobilized protein was
15-30% of the known
stoichiometries. This level of ligand binding to immobilized protein
often occurs when using the SPR technique. Control experiments showed
no detectable binding of these subunits to a sensor chip that was
pretreated with the immobilization reagents.
Figure 7:
Identification of weak interactions within
the complex by SPR. The scheme for protein injection and buffer
(buf.) injection over an immobilized protein is shown in the
panel at the top. In each panel, the immobilized
complex subunit
on the chip is indicated at the top left corner, and the subunits in
the fluid phase are indicated in the panel. The closed arrowheads (above the data line) mark the start of protein injection, and the
openarrowheads (below the data line) mark the start
of buffer injection. A, immobilized
; B,
immobilized
`; C, immobilized
; D,
immobilized
complex. RU, response
units.
All the gel-filterable
complexes were also observed in the SPR analysis, but we hoped that the
sensitive SPR technique would detect new interactions that are too weak
to detect by gel filtration. For example, a mixture of and
will assemble
onto DNA, although the reaction is feeble,
indicating that
and
may weakly interact. Also, a mixture of
and
` results in DNA-dependent ATPase activity, suggesting
that they interact. Another possible weak interaction is one between
and
` as an explanation of how these subunits cooperate in
binding to
. In summary, SPR analysis detected the predicted weak
`-
interaction, but no interaction was observed between
and
or between
` and
. Hence, these latter two subunit
combinations probably cooperate by means other than direct contact
(discussed below). Furthermore, a putative
-
interaction was
observed for which there is no other supporting evidence. The detailed
experiments were as follows.
was
attached to the chip, and each subunit of the
complex was passed
over it. Included in the analysis were the
subunit and the core
polymerase. SPR analysis detected the previously identified strong
interaction of
with
`, but no other significant interaction
was observed, not even with the
subunit.
` immobilized on the chip (Fig. 7B), an interaction
with
was observed as anticipated from the ATPase assays. A
particularly strong interaction was observed for
, consistent with
the previously identified
` complex.
, in Fig. 7C, showed a slight, but
reproducible, interaction with
, but not with
` or
. To
determine if
` would bind
tighter, the
`
complex was passed over the
chip, but it showed only the same
interaction with
as
alone. These results indicate that
may bind to
, but it is important to note that experiments
with immobilized
failed to detect significant interaction with
or the
complex (see Fig. 7A).
Perhaps the surface on
that interacts with
may be
preferentially cross-linked to the sensor chip, preventing interaction
with
. The
subunit binds tightly to
, but this is
not shown in Fig. 7since
is insoluble. However, the
interaction of
with immobilized
was observed by injecting
in 0.5 M urea over the
chip (data not shown).
This resulted in a huge increase in response units due to the change in
refractive index caused by urea in the
preparation. Nonetheless,
upon finishing the injection and replacing urea with SPR buffer, the
final response units indicated that
had bound to
(28% of
immobilized
bound
).
is inactive and
insoluble by itself, but is soluble when bound to
, we
immobilized
(Fig. 7D) by first coupling
to
the chip and then adding
to it as described above. In the SPR
analysis of immobilized
(Fig. 7D), we
expected to observe a weak interaction between
and
`
or the
` complex as an explanation of their cooperativity in
binding
, but no interaction between
and
` was
observed. A slight interaction of
with
and
` was observed, presumably due to weak contact between
and
. Although this putative contact may lend cooperativity
between
` and
in binding
, it does not
explain the cooperative nature of
` and
in forming a
`
complex.
and
k
values for the interactions observed in
Fig. 7
were determined from the kinetic traces. It is important to
note that since one ligand is immobilized and thus not free to diffuse
in three dimensions, the apparent rates may differ from those measured
by other means. Since the experiments in Fig. 7were performed
using a uniform concentration of protein in the mobile phase and
similar amounts of immobilized protein, the apparent rates should be
comparable within the set of experiments. The association and
dissociation rates of
with immobilized
` and of
` with
immobilized
are similar (within 2-fold), and the apparent
K
value of 68-124 nM is
consistent with the ability to detect the
` complex in the
gel filtration approach. The kinetic parameters for binding of
`
to
and
are similar (within 3-fold), and the apparent
K
value for
` and
`
complexes is in the 100-300 nM range, apparently too
weak to detect by gel filtration. The putative k
value
for the interaction of
with
is 1-2 µM.
Likewise, the k
for the interaction of
with
` and of
with
and
` is also
1-2 µM. Hence, the putative interaction between
and
` appears to reside in the
-to-
contact. The
and
subunits bind tightly to
(38-150 nM), consistent with the ability to gel filter
and
complexes. These experiments
were performed in the absence of MgCl
and ATP. We have
performed a similar set of experiments in the presence of 0.5
mM ATP and 10 mM MgCl
, but no significant
differences were observed (data not shown).
Lack of Interaction between the
In a previous study, we showed that Complex and
Core
bound core
tightly, but
did not
(36) . Now that we have reconstituted
the
complex in quantity, we re-examined whether the
complex
binds to core by gel filtration analysis, but still did not detect an
interaction (data not shown). In the event that core binds a subunit of
the
complex weakly, we have used SPR to look for an interaction
of core with
,
`,
, or
, but no interaction
was detected (Fig. 7).
Structure of the
Individual
overproduced subunits have been used to constitute the Complex
complex in
quantity for structural studies. The constituted
complex is as
active as the
complex purified from E. coli lysates. The
hydrodynamic measurements and subunit molar ratios indicate a
stoichiometry of
`
,
in agreement with estimates of a preliminary study
(16) . As the
aggregation state appears to consist of tetramers (i.e. in gel filtration analysis) and dimers (i.e. observed in
glycerol gradients)
(17, 36) , the value of two to three
subunits in the
complex likely has its basis in a mixture
of two species,
`
and
`
.
complex consistent with the stoichiometry of subunits in the
complex and the observed interactions among them. The
subunit is
an oligomer, probably a tetramer, by itself
(17, 37) ,
but stoichiometry and native mass measurements indicate that
is a
dimer in the
complex. Only two subunits of the
complex
appear to have appreciable affinity for the
dimer. The
subunit has strong affinity for
and was detected in an earlier
study by gel filtration (26). Interaction of
` with
was
inferred by DNA-dependent ATPase activity that depends upon both
`
and
(19, 20) . Interaction of
` with
was
not detected by gel filtration, but was detected by the SPR technique.
Once
is bound to
, association of
` becomes strong
enough to survive gel filtration. One explanation for this is direct
contact between
and
`. However, no interaction between
and
` or between
and
` was
observed in this study. Hence, the
subunit may induce a
conformational change in
such that
binds
` tighter.
The
and
` subunits are shown as lacking direct contact in
Fig. 8
to reflect this possibility. The
and
subunits
are placed in contact with one another in Fig. 8to reflect the
weak interaction detected by SPR analysis.
Figure 8:
Putative subunit arrangement of the
complex. A dimer of
binds one monomer each of
and
`.
A
monomer forms a firm contact with
. A monomer of
associates with
` and
.
Because is a dimer,
it should have two binding sites for
` and two sites for
.
There are at least four obvious explanations of how the
dimer may
bind a single monomer of
` and
. 1) The two sites may
overlap. 2) Binding may be negatively cooperative, whereupon binding
one
` to a
protomer induces a conformational change in the
other
protomer, lowering its affinity for a second
`
(likewise for
). 3) The binding sites of
and
`
overlap such that once
` is bound to
, it
occludes one
site, and conversely, once
binds
, it occludes one
` site. 4) The
dimer may
not be isologous, and thus, one site may be formed by two protomers,
and this site would not be repeated elsewhere in the dimer. The third
possibility predicts that in the absence of
`, two
subunits
would bind a
dimer. Our earlier study observed a ratio of
subunits in the
complex of
, indicating
that the third explanation is not the basis for the binding of only one
to a
dimer
(26) .
and
was expected on the basis of the clamp loading activity
that depends upon both
and
(18, 19), but no interaction
between these subunits was detected in this study. Hence,
and
act separately to load
onto DNA, or their interaction is
very transient, or the
subunit and/or DNA present in clamp
loading assays is needed for direct interaction of
with
.
However, the
-
` contact is a strong interaction observed in
our earlier study
(19) . Hence, in Fig. 7C,
is shown to associate with
indirectly through association with
the
` subunit. The
subunit is known to bind
strongly
(26) , and the present report suggests that
may
also bind
.
The
The Complex
subunit contains the
amino acid sequence of
(and an extra 213 residues at the C
terminus) and therefore may be expected to bind the
,
`,
, and
subunits. Indeed, a
complex
(
`
)
can be constituted and is within 2-fold of the activity of the
complex. The native aggregation state of
, like
, appears to
consist of dimers and tetramers
(17, 36) , and thus, we
propose that the
complex (like the
complex) is a mixture
consisting predominantly of
`
with some
`
.
It is important to note that a
complex has yet to be purified
from E. coli cells. Furthermore, the
,
`,
,
and
subunits are not present in Pol III`
(core
-
)
(6) . The third report of
this series demonstrates that only one each of the
,
`,
, and
subunits assembles into Pol III*, and therefore,
these subunits must reside on either
or
, but not
both
(38) . The location of these single copy subunits in Pol
III* is the subject of the fourth report of this series
(39) .
Implication of
Single copy subunits
bound to a Complex Subunit Stoichiometry to the
Asymmetric Structure of Pol III Holoenzyme
dimer would eliminate the 2-fold axis of symmetry
relating the two protomers of the
dimer, thereby imposing a
structural asymmetry to the
complex. In other words, since
monomeric proteins are asymmetric structures, the
complex must
also be asymmetric. This has important implications for the putative
asymmetric holoenzyme as discussed more fully in the third report on
the constitution of Pol III*
(38) . The Pol III* assembly process
indicates that at the center of the structure, a
dimer forms a
heterotetramer with a
dimer. The
dimer also binds two
molecules of the core polymerase, presumably one core for each
protomer. Hence, in this arrangement, the two core polymerases are
likely to be related by a 2-fold rotational axis of symmetry to the two
protomers, and since
and
would be 2-fold symmetric,
the cores would have a 2-fold symmetry relative to the two
protomers also. Only one copy each of
,
`,
, and
is assimilated into Pol III*, and therefore,
,
`,
, and
must each be disposed asymmetrically relative to the two core
polymerases. Thus, it is these single copy subunits of the
complex that confer the structural asymmetry on the entire holoenzyme
structure. Whether they all confer special properties on the lagging
strand core or whether one or more function with the leading strand
core is a topic for future studies.
`
and
`
complexes were determined from the gel filtration and glycerol gradient
analysis in Fig. 1. Molecular masses and frictional coefficients were
calculated from the Stokes radii and s values as described
(35). These calculations require the partial specific volumes of
`
and
`
, which
were calculated by summation of the partial specific volumes of the
individual amino acids for
`
and
`
(assuming
and
as dimers) (40).
Molecular masses of the
`
and
`
complexes were calculated from the gene
sequences of
,
,
`,
, and
.
Table:
Molar ratio of subunits in the and
complexes
and
complexes purified by gel filtration as in Fig. 1 were
determined by densitometry of Coomassie Blue-stained 15%
SDS-polyacrylamide gels. The values are the average of three
determinations and were normalized to
`. In the HPLC analysis, the
molar ratio of subunits of the constituted
complex and of the
complex purified from E. coli lysates was determined by
monitoring their elution from the HPLC column at 280 nm. The areas
under the peaks of 280 nm absorbance (Fig. 3) were divided by their
respective
values (see ``Experimental
Procedures'') and normalized relative to the value for
`.
Each value is an average of three independent analyses.
subunit still bound the
complex, but showed no detectable interaction with
`, yet
` is a tight gel-filterable complex. In
addition, immobilized
did not show interaction with
`.
Hence,
appears to be largely inactive during the immobilization
procedure (probably due to the low pH needed for the carbodiimide
chemistry).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.