From the
The nine-subunit DNA polymerase (Pol) III* coupled to its
The Escherichia coli replicase, DNA polymerase III
holoenzyme (holoenzyme), has traditionally been difficult to obtain in
large quantity due to its low cellular concentration (10-20
molecules/cell)
(1, 2) . All 10 subunits of the
holoenzyme are now available in abundance by molecular cloning of their
genes and high level expression
techniques
(3, 4, 5, 6, 7, 8, 9, 10, 11, 12) .
In this report, we have used the pure subunits to determine how to
assemble the nine-subunit polymerase (Pol)
Previously, we described the
constitution of the four-subunit Pol III` subassembly
(
In this study, methods by which Pol
III* can be assembled in vitro are described. Assembly depends
upon staging the addition of subunits in a defined order. The resulting
Pol III* remains associated even when diluted to 30 nM. Thus,
once pol III* is assembled, it is tightly associated and does not
easily fall apart. The ability to assemble Pol III* from overproduced
subunits provided the material to analyze the stoichiometry of the
subunits in the particle. The assembly process, the stoichiometry of
subunits, and the implications of the results to the asymmetric
structure and function of Pol III holoenzyme are the subject of this
report.
In Method 2, 6.6 mg (52.5
nmol) of
In any assembly
scheme, the ``outer'' subunits such as
After all the subunit additions, Pol III* can be
purified from excess subunits and smaller subassemblies as shown in
Fig. 4
. Fig. 4A shows the profile of elution from
a heparin column, which partially resolves excess
The results for both constituted
and naturally purified Pol III* are most consistent with a dimer of
The activity of constituted Pol
III* in the
The approach was to mix combinations of two subunits, one
from Pol III` (
In
Fig. 10
, the interaction between
Panel A in
Fig. 11
is the mixture of Pol III` with a 3-fold molar excess of
the
In panel D in
Fig. 11
, the
A set of
The subunit arrangement of Pol III* based
on an earlier study
(16) differs from that proposed here. In the
earlier study, the core polymerase (
Although the
We have
constituted Pol III* using five variations of the two methods described
here. The three variations of Method 1 were as follows: 1a) placing
These Pol III* preparations have been
compared for differences in their DNA synthetic activity with
Coomassie
Blue-stained polyacrylamide gels were scanned as described under
``Experimental Procedures.'' The differences in subunit
staining by Coomassie Blue were corrected by comparison with subunits
of known concentration (determined by absorbance at
Molecular masses of the subunits were obtained from
the gene sequences.
sliding clamp is a rapid and highly processive replicating machine. The
multiple subunits are needed for the complicated task of duplicating
the Escherichia coli chromosome. In this report, Pol III* was
constituted from individual pure proteins, and its structure was
studied. Constitution of the Pol III* particle requires an ordered
addition of the subunits, and the final structure contains 14
polypeptides in the ratio
`
.
The structure can be summarized as being composed of two core
polymerases (
) held together by a dimer of
and
one
complex clamp loader
(
`
)
for loading
onto DNA. At the center of the structure, the related
and
subunits form a heterotetramer upon which the two core
polymerases and clamp loader proteins assemble. The single copy nature
of the
,
`,
, and
subunits confers a structural
asymmetry with respect to the two polymerases, presumably for the
different functions of replicating the leading and lagging strands.
(
)
III*
and to obtain it in large quantity.
),
which consists of a
dimer that serves, among other things, as a
scaffold to hold together two core polymerases
(13, 14) .
In the first report of this series, we described the constitution and
subunit composition of the five-subunit
complex clamp loader
(
`
),
the molecular matchmaker that assembles
clamps onto
DNA
(15) . Since the
complex and Pol III` together account
for the full complement of subunits of the nine-subunit Pol III*
assembly, we presumed that Pol III* would be constituted upon mixing
these two subassemblies. Hence, we were surprised to find that Pol III`
and the
complex did not associate with each other even when mixed
at high concentrations (25 µM each). The fact that Pol
III` and the
complex do not easily associate with one another
suggests that once they are formed in the cell, they stay separated.
Perhaps their inability to associate serves the purpose of isolating
their separate functions for specialized tasks with other proteins such
as in repair or recombination.
Materials and Methods
All methods, materials, and sources not described here were
described in the first report of this series
(15) . Gel
filtration was performed as described in the first report, except when
Superose 6 was used, 4.8 ml was collected before the start of
collecting fractions. Buffer B contained 20 mM Hepes-NaOH (pH
7.5), 2 mM dithiothreitol, 0.5 mM EDTA, and 10%
glycerol.
Replication Assays
Assays were performed as described for the complex in
the first report
(15) , except that in Pol III* assays, the
complex was omitted (only
was added to the assay).
Assays of column fractions from the analysis of 30 nM Pol III*
and
complex required the addition of 10 µl of undiluted
column fraction to each assay. Assays comparing the specific activities
of Pol III* forms were only allowed to proceed for 20 s, sufficient
time to finish one primed template.
Preparation of Reconstituted Complexes
Proteins were incubated in buffer A for 30 min at 15 °C
unless stated otherwise. The concentrations of all subunits except
and
are expressed as monomer. The concentrations of
and
are expressed as dimer since this is their final aggregation
state when assembled with the other subunits.
The Core Polymerase
A mixture of 10 mg (78 nmol) of
, 6.4 mg (229 nmol) of
, and 6 mg (698 nmol) of
was
incubated in a volume of 7.4 ml and then chromatographed on a Mono Q HR
5/5 column eluted with a 32-ml linear gradient of 0-0.4
M NaCl in buffer A. Fractions of 0.5 ml were collected and
analyzed on a Coomassie Blue-stained SDS-polyacrylamide gel. The core
polymerase eluted at
0.25 M NaCl (11 mg final
concentration).
The
The
`
Complex
`
complex was made in two steps. First, the
complex was constituted and purified as
described
(6, 19) and then
` was added, and the
resulting
`
complex was purified (however, the
`
complex can also be made by mixing all four
subunits, followed by purification on Mono Q). The
complex (1.5 mg, 52.5 nmol) was incubated with
` (3.89 mg, 105
nmol) in 3.06 ml, and then the mixture was chromatographed on a Mono Q
HR 5/5 column equilibrated with buffer A. The
`
complex was eluted from the column with a 32-ml linear gradient of
0-0.4 M NaCl in buffer A. The
`
complex eluted last at
0.28 M NaCl. Column fractions
containing
`
were pooled (4.2 mg in 3 ml) and
stored at -70 °C.
The
The Complex
complex was
constituted and purified by chromatography on a Mono Q column as
described in the first report of this series
(15) .
Pol III`
Pol III` was constituted by mixing ,
,
, and
at a molar ratio of 3:4.5:6.75:1 (molarity of
as dimer, the rest as monomer) in buffer A and incubating for 30
min at 15 °C (see figure legends for total protein concentrations).
Pol III` was concentrated to 100 µl by spin dialysis using a
Centricon 30 apparatus (Amicon, Inc.) and isolated by gel filtration on
a Superose 6 column to remove excess core,
complex, and
subunit.
Pol III* lacking -less Pol III*
was
constituted upon incubating
(239 µg, 0.84 nmol),
(40.5
µg, 2.44 nmol), and
(30 µg, 1.97 nmol, in 6 µl of 4
M urea) in a final volume of 98.1 µl, after which
(92 µg, 2.38 nmol) and
` (73.7 µg, 1.99 nmol) were added.
This mixture was further incubated for 30 min at 15 °C, at which
time
(239 µg, 1.99 nmol),
(67.6 µg, 2.46 nmol), and
(24.13 µg, 2.8 nmol) were added to a final volume of 260
µl containing 5 mM MgCl
and 0.2 mM
ATP. This mixture was further incubated for 30 min at 15 °C and
then concentrated to 100 µl at 4 °C with a Centricon 30
apparatus, followed by purification through gel filtration on a
Superose 6 column.
Pol III*
Two general methods were used to
constitute Pol III*. In Method 1, first the complex was
constituted and dialyzed to remove urea in the
preparation by
mixing 1.414 mg (85 nmol) of
with 0.864 mg (57 nmol) of
in a final volume of 4.2 ml (urea was present at 0.7 M) for 1
h and then dialyzing against buffer A. The
subunit (504 µg,
3.5 nmol) was incubated for 60 min with the
complex (224
µg, 7 nmol) in 488 µl. The
subunit (1.0 mg, 10.6 nmol)
was incubated with the
complex (676 µg, 22 nmol) for
60 min in 1.953 ml. The mixtures were then combined and incubated for 1
h, after which a 665-µl mixture of 787 µg of
` and 823
µg of
(21.3 nmol each) was added, followed by a further
incubation for 30 min. During this time, the core polymerase was
constituted in a separate tube containing
(2.77 mg, 21.3 nmol),
(878 µg, 31.9 nmol), and
(412 µg, 47.9 nmol) in
5.4 ml for 1 h. Then core was combined with the
`
mixture and incubated for 1 h. The
mixture was then loaded onto a 4-ml heparin-Sepharose column (Pharmacia
Biotech Inc.) equilibrated with buffer A and eluted with a 40-ml linear
gradient of 0-325 mM NaCl in buffer A. Eighty fractions
of 0.5 ml each were collected. Column fractions were analyzed on a 15%
SDS-polyacrylamide gel, and those containing Pol III* were pooled
(fractions 32-50), concentrated with the Centricon 30 apparatus
to 230 µl, and loaded onto a 24-ml Superose 6 column equilibrated
with buffer A containing 0.1 M NaCl. Fractions of 200 µl
were collected and analyzed on a 15% SDS-polyacrylamide gel. Fractions
containing Pol III* were pooled (total protein, 760 µg in 2 ml) and
stored at -70 °C. Assuming a mass of 670 kDa for Pol III*, if
all the
had been incorporated, there would have been 2.39 mg of
Pol III*. Hence, the recovery by this procedure is
32%. Two other
preparations of Pol III* were assembled by this same general method
with the following exceptions. The
complex was initially
placed exclusively on
by starting with the pure
complex (and
was not incubated with
). In the other
variation,
was initially placed only on
by using
pure
(prepared similarly to
), and
was not incubated with
.
complex (prepared as described
(6) )
was mixed with 3.89 mg (105 nmol) of
` in a volume of 3 ml,
followed by incubation for 30 min. The mixture was chromatographed on a
Mono Q HR 5/5 column equilibrated with buffer A and eluted with a 32-ml
linear gradient of 0-0.4 M NaCl in buffer A (see
Fig. 3
). Fractions 40-43 were pooled to yield 4.2 mg of
`
complex. The
subunit (0.19 mg, 1.34 nmol)
was mixed with 0.8 mg of
`
complex in 1.6 ml for
1 h followed by the addition of 0.36 mg (9.33 nmol) of
in a final
volume of 2.75 ml and incubated for an additional 30 min. During this
time, core was constituted upon incubating
(0.516 mg, 4 nmol),
(162 µg, 6 nmol), and
(77 µg, 8.95 nmol) in 183
µl for 30 min. Core was mixed with the
`
mixture and incubated for 60 min and
then was loaded onto a 2-ml heparin-Affi-Gel column (Bio-Rad)
equilibrated with buffer B and eluted with a 30-ml linear gradient of
0-0.325 M NaCl in buffer B. Fractions of 0.5 ml were
collected and analyzed on a 13% SDS-polyacrylamide gel for Pol III* and
the
complex. Fractions 38-54 were pooled and loaded onto a
Mono Q HR 5/5 column and then eluted as described for the
`
complex. Fractions were analyzed for Pol III*
on a 15% SDS-polyacrylamide gel, and fractions 45-50 were pooled,
concentrated to 200 µl using a Centricon 30 apparatus, and
gel-filtered on a 24-ml Superose 6 column in buffer A containing 100
mM NaCl. Fractions of 190 µl were collected and analyzed
by SDS-polyacrylamide gel electrophoresis, and fractions 25-30
were pooled (41 µg of Pol III*) and stored at -70 °C.
Recovery by this procedure is
5%. Another preparation of Pol III*
was assembled by this method using a
`
complex
instead of
`
and adding
later.
Figure 3:
Scheme of the two general methods used to
constitute Pol III* for the experiments of this report. Method 1
requires no purification of subassemblies until after all the subunits
are added. Method 2 requires the complex containing ` to be
purified from excess
` before the further addition of subunits.
See ``Discussion'' for the numerous variations of these
assembly schemes.
Chemical Cross-linking
Solutions of 32 fmol of (as dimer), 32 fmol of
(as dimer), or a mixture of 16 fmol each of
and
were
incubated at 15 °C for 30 min in 80 µl of 50 mM
Hepes-NaOH (pH 8.1), 1 mM EDTA, 10% glycerol, and 0.1, 0.3,
0.5, 1, or 2 M NaCl. The solutions were shifted to 25 °C,
and 8 µl of dimethyl suberimidate (DMS) at 11 mg/ml in the same
buffer (but at pH 10) was added. After 30 min at 25 °C, the
reactions were quenched upon adding 8 µl of 1 M glycine
and analyzed by electrophoresis on an 8% SDS-polyacrylamide gel.
Pol III` and the
To constitute Pol III*, we initially mixed Pol III`
( Complex Do Not Assemble into Pol
III*
)
and the
complex
(
`
) together under the assumption they would
associate into Pol III*
(
`
). In replication
assays, the mixture of Pol III` and the
complex is essentially as
effective in providing processive DNA synthesis with
as the Pol
III* assembly is. Hence, to assay formation of Pol III*, we could not
use simple replication assays, but needed to follow the assembly by
physical methods. In Fig. 1, we assayed Pol III* assembly by gel
filtration and analyzed the column fractions by Coomassie Blue staining
of an SDS-polyacrylamide gel. In Fig. 1(A and
B), the
complex and Pol III` assembly were analyzed
separately. The
complex elutes in fractions 26-34
(Fig. 1A), and Pol III` peaks in fractions 14-22
(Fig. 1B). If Pol III` and the
complex associate
into Pol III*, then all nine subunits should coelute in a position
earlier than either complex alone. Analysis of the mixture of Pol III`
and the
complex in Fig. 1C shows that they do not
associate to a significant extent; the subunits neither comigrate nor
elute substantially earlier. However, the slightly earlier elution of
the
complex in the presence of Pol III` may indicate a weak
interaction with Pol III` that is not stabile to gel filtration.
Figure 1:
Pol III` does not associate with the
complex. Pol III` and the
complex were analyzed by Superose
12 gel filtration either alone or after incubating them together as
described under ``Experimental Procedures.'' A, the
constituted and purified
complex (40.6 µg, 0.20 nmol) in 200
µl; B, Pol III` constituted upon incubating
(42.3
µg, 0.33 nmol) with
(13.4 µg, 0.50 nmol),
(6.4
µg, 0.75 nmol), and
(28.9 µg, 0.2 nmol) in 200 µl;
C, mixture of Pol III` and the
complex at the same
concentrations as in A and B in a 200-µl final
volume and further incubated for 60 min at 15 °C before gel
filtration. The firstlane contains molecular mass
standards, and their masses are shown to the left. The
,
,
,
,
`,
,
,
, and
subunits are
identified to the right. Frxn, fraction
number.
One
explanation for this result is that the association of Pol III` with
the complex is too weak to isolate the Pol III* complex under the
nonequilibrium conditions of gel filtration. An earlier report showed
that Pol III* isolated naturally from E. coli was isolable as
a complex on the same gel filtration matrix; however, it appeared to
partially dissociate as its concentration was decreased (16). The
experiment of Fig. 1was performed at a concentration of 1
µM each Pol III` and
complex and thus may be less
concentrated than needed. However, we have performed gel filtration of
these complexes at a concentration of 28 µM with the same
result as in Fig. 1(17) . In the experiments below, we
show that Pol III* can indeed be assembled by the appropriate order of
subunit addition and that once formed, Pol III* is stabile to gel
filtration even at a concentration of 30 nM.
Constitution of Pol III*
After numerous studies using different orders of subunit
addition, methods to constitute Pol III* were discovered. In overview,
formation of Pol III* requires that the and
subunits
associate with each other, and this
-
association does not
occur when using intact Pol III` and the
complex. The underlying
reason that
(in Pol III`) could not bind
(in the
complex) rested with the
subunit, which prevented their
interaction;
needed to be added after
and
were
preincubated.
(
)
In fact, the
-
contact
could be made upon incubating Pol III` with a complex of
`
(
complex lacking
), and subsequent
addition of
resulted in formation of Pol III*. In the course of
these studies, we identified an additional reaction that inhibited
interaction of
with
, thus preventing Pol III* formation.
The
` subunit can bind both
and
as shown previously
(
must also be present on
(
) for
` to stably
associate with
(
)
(15) ), but if
` is allowed to
bind both
and
, it prevents the essential association of
with
. The results of adding
and
` at different
points in the assembly scheme are summarized in Fig. 2. Study of
these necessary orders of addition is presented below (see
Fig. 10
and Fig. 11). Mixtures of complexes that do not
result in productive assembly are shown in Fig. 2with an
X. Although the addition of
and
is shown near
the beginning of the assembly scheme and core (
) is
added near the end,
,
, and core can be added at any stage
without preventing the formation of a Pol III* complex.
Figure 2:
Assembly schemes showing possible paths
leading to Pol III* and paths leading to dead-end complexes. Openwhitecircles indicate pure subunits. Shadedellipses indicate mixtures of subunits that result in the
indicated complex. These mixtures contain excess free subunits and/or
subcomplexes. The twohatchedellipses indicate the `
and
`
complexes, which must first be purified to remove excess unbound
`
subunit before continuing the assembly process. The productive paths
are shown as solidlines that end in another complex.
The unproductive paths converge to an X. Core
(
) and the
and
subunits can be added at
any point in the assembly scheme. See ``Results'' for
details.
Figure 10:
Chemical cross-linking analysis of the
-
interaction. Solutions of
,
, and a mixture of
and
were treated with DMS and then analyzed on an 8%
SDS-polyacrylamide gel stained with Coomassie Blue. The firstlane contains molecular mass markers; the nextfivelanes show treatment of
with DMS at
increasing concentrations of NaCl (0.1, 0.3, 0.5, 1, and 2 M).
The middlefivelanes show treatment of
with DMS at 0.1, 0.3, 0.5, 1, and 2 M NaCl. The lastfivelanes show treatment of the
and
mixture with DMS at 0.1, 0.3, 0.5, 1, and 2 M NaCl. The
positions of the
and
monomers and dimers are shown to the
right, as is the cross-linked
form (
heterodimer).
Figure 11:
The subunit inhibits interaction of
with
. A, in one tube, Pol III` was constituted by
incubating core (54.8 µg, 0.33 nmol) and
(28.4 µg, 0.20
nmol) for 30 min at 15 °C in 77 µl and then adding the
`
complex (32.6 µg, 163 nmol) in a 98-µl
final volume, followed by gel filtration on a Superose 12 column.
Column fractions were analyzed on a Coomassie Blue-stained 15%
SDS-polyacrylamide gel. B, the
`
complex
(32.6 µg, 163 nmol) was gel-filtered as described for A.
C, Pol III` was constituted and gel-filtered as described for
A. D, Pol III`, constituted as described for
A, was incubated 30 min at 15 °C with the
complex
(40.3 µg, 0.2 nmol) in 117 µl of buffer A and then
gel-filtered. E, Pol III* was constituted upon mixing Pol III`
(constituted as described for A) with the
`
complex (32.6 µg, 163 nmol) in 97.8 µl
of buffer A for 30 min at 15 °C followed by the addition of 19
µl of
(11.6 µg, 0.3 nmol) and incubated for an additional
30 min at 15 °C before gel filtration. Column fractions are shown
at the top; positions of molecular mass markers are indicated to the
left; and subunits are identified to the right. Frxn, fraction
number.
The schemes
in Fig. 3outline the two methods by which Pol III* was assembled
for the studies in this report. In Method 1, the key feature is that
and
are premixed before the addition of
and
`.
The determining feature of Method 2 is that
` is first assembled
onto
(or
); then excess
` is removed by ion-exchange
chromatography before adding
(or
) so that the
-
interaction is productive, and then
is added subsequently.
Provided the requirements described above are satisfied in the assembly
scheme, the rest of the subunits can be added at any step (or
together), and a nine-subunit Pol III* will assemble. For example, in
Method 1, the
subunits and the core polymerase can be
added to
and/or
before or after mixing
with
. In
Method 2,
(or
) must be added initially to stabilize
the association of
` with
(or
), but
and core
can be added at any point in the scheme. Staging the addition of
subunits in various ways may, in principle, result in different subunit
orientations within Pol III* (see ``Discussion''). We have
compared five preparations of Pol III* assembled using three variations
of Method 1 and two variations of Method 2 (see
``Discussion''). The Pol III* used in most of these studies
was assembled by Method 1 (unless stated otherwise).
and
must
be added in excess over subunits more ``central'' to the
structure such that all complexes are driven to completion. The
subunit is the most central since it binds both the
complex and
core, and therefore,
must be limiting in the assembly scheme. The
-
interaction appears to be an equilibrium. We typically push
the
-
association by adding a 3-fold excess of
over
; however, the excess
complex that forms is difficult to
separate from Pol III* (explained below). If insufficient
is used
to push this equilibrium, then an eight-subunit form of Pol III
assembles that has all the subunits except
(referred to below as
``
-less Pol III*''). It is desirable to reduce the
amount of
-less Pol III*, even at the expense of forming excess
complex, since it is most difficult to separate
-less Pol
III* from Pol III*.
complex
(fractions 23-38) from Pol III* (fractions 32-54).
Fractions 38-54 were pooled, resulting in some loss of Pol III*
at the expense of removing the bulk of excess
complex. Excess
and
` coelute with Pol III* on the heparin column, but are
cleanly separated from Pol III* by chromatography on a Mono Q column
(Fig. 4B). Fractions 45-50 were pooled,
concentrated, and passed over a Superose 6 gel filtration column to
remove the remaining
complex (Fig. 4C). The
persistent
complex contaminant (fractions 32-38) now
resolves from Pol III* (fractions 22-28), and the
-less Pol
III* contaminant becomes apparent (fractions 16-20).
-less
Pol III* has a higher molecular mass and elutes earlier than Pol
III*.
(
)
To remove
-less Pol III* and the
complex from the preparation, we pooled a narrow range of
fractions (fractions 25-30).
Figure 4:
Purification of constituted Pol III*. Pol
III* was constituted by Method 2 as described under ``Experimental
Procedures.'' Purification of the resulting Pol III* from
contaminating single subunits and subassemblies was achieved by
chromatography on a heparin-Affi-Gel column (A) and on a Mono
Q column (B) and by gel filtration on a Superose 6 column
(C) as described under ``Experimental Procedures.''
Column elution was monitored at 280 nm (leftpanels)
and by Coomassie Blue-stained 13% SDS-polyacrylamide gel
electrophoresis (rightpanels).
Characterization of Constituted Pol III*
The size of constituted Pol III* was estimated by gel
filtration analysis and comparison to size standards in Fig. 5.
Pol III* peaks at fractions 23-27, followed by remaining excess
complex in fractions 29-35 (Fig. 5A). Pol
III* activity assays (Fig. 5B) show that the active
fractions correspond to Pol III* with a Stokes radius of 85 Å for
a mass of
670-680 kDa (see ). The mass of Pol
III* purified from E. coli cell lysates (without protein
overproduction) was also analyzed by gel filtration
(Fig. 5C). It eluted in the same position as constituted
Pol III*, reasonably consistent with a previous study estimating the
mass of naturally purified Pol III* at 800 kDa
(16) .
Figure 5:
Size analysis of Pol III*. Pol III* (600
µg, 0.9 nmol), constituted by Method 1, was gel-filtered on
Superose 6 as described under ``Experimental Procedures.''
A, analysis of column fractions by Coomassie Blue-stained 13%
SDS-polyacrylamide gel. The firstlane contains
molecular mass standards. The positions of the ,
,
,
,
`,
,
,
, and
subunits are
identified to the right. The positions and masses of gel filtration
standards analyzed separately are shown above the gel. B,
activity assays of the column fractions. C, elution of
constituted Pol III* and Pol III* purified from E. coli relative to protein standards of known Stokes radii. The Stokes
radius was calculated from the diffusion coefficient using the
following equation: Stokes radius =
kT/6
D, where k is the
Boltzmann's constant, T is absolute temperature,
is viscosity, and D is the diffusion coefficient.
Tgb, thyroglobulin (670 kDa, 85 Å); Apf, horse
apoferritin (440 kDa, 59.5 Å); IgG (158 kDa, 52.3 Å);
BSA, bovine serum albumin (67 kDa, 34.9 Å);
Ova, chicken ovalbumin (43.5 kDa, 27.5 Å); Myo,
horse myoglobin (17.5 kDa, 19.0 Å).
We tried
to determine the molar ratio of subunits in Pol III* by the HPLC
technique
(14) , but could not identify a gradient that resolved
all the subunits.(
)
Hence, we estimated the molar
ratio of subunits in constituted Pol III* and naturally purified Pol
III* by laser densitometry of a Coomassie Blue-stained
SDS-polyacrylamide gel in which the differences in staining for each
subunit were corrected by comparison to individual subunits of known
concentration analyzed on the same gel (Fig. 6). The scan of
constituted Pol III* (Fig. 6A) is similar to that of
naturally purified Pol III* (Fig. 6B). The
concentrations of the individual Pol III* subunits used as standards
were determined from their extinction coefficients at 280 nm. The
quantitation of the analysis is shown in . All values are
normalized to the
subunit.
Figure 6:
Subunit molar ratio in Pol III*. Pure Pol
III* was gel-filtered on a Superose 6 column, and the fractions were
analyzed on a Coomassie Blue-stained SDS-polyacrylamide gel, followed
by densitometric scanning. Densitograms are presented. A, 600
µg of Pol III* constituted and purified by Method 1; B,
330 µg of Pol III* purified from E.
coli.
The ratio of the ,
, and
subunits was approximately equimolar for both constituted Pol
III* and Pol III* purified from cell lysates. The intensity of the
subunit was too weak to be determined accurately in this study;
but previous studies showed that the stoichiometry of the
complex is 1:1, and therefore, it is assumed here that
is
equimolar to
in Pol III*
(7) . The
subunit in both
reconstituted Pol III* and Pol III* purified from E. coli lysates is also approximately equimolar to
,
, and
. However, even though the
,
`,
, and
subunits were added in excess during the assembly of Pol III*, these
subunits are present at approximately half the amount of the
,
,
, and
subunits in both constituted Pol III* and
E. coli purified Pol III*.
and
, two each of the core subunits (
), and
only one each of the
,
`,
, and
subunits. The
single copy nature of
and
` can be seen by simple inspection
of the scans in Fig. 6by comparing the greater peak height of
the double copy
subunit (27.5 kDa) relative to the heights of the
larger molecular mass but single copy
(38.7 kDa) and
` (37.0
kDa) subunits. This result is also consistent with previous studies on
the composition of Pol III`
(
)
that showed it to be composed of two of each
subunit
(13, 14) . Although an earlier study of Pol III*
purified from cell lysates suggested there were two of each subunit,
analysis of the holoenzyme in that same study gave the following
composition (after setting
to two):
`
(16), quite consistent with the subunit ratios obtained in this
report. The subunit stoichiometry predicts the molecular mass of Pol
III* to be 673 kDa as summarized in , consistent with the
size estimated by gel filtration.
-dependent replication of singly primed M13mp18
single-stranded DNA coated with SSB was compared with that of Pol III*
purified from cell lysates. The results (Fig. 7) show that
constituted Pol III* is slightly more active than Pol III* purified
from cell lysates; their specific activities were 3.4
10
and 2.3
10
pmol of nucleotide incorporated
per mg/min, respectively. Constituted Pol III* is similar to Pol III*
purified from lysates with respect to size, subunit stoichiometry, and
replication activity, thus indicating that constituted Pol III* is
authentic.
Figure 7:
Activity of constituted Pol III* and of
Pol III* purified from cell lysates. Shown are -dependent
replication activity assays of Pol III* on singly primed and SSB-coated
M13mp18 single-stranded DNA of Pol III* purified from E. coli lysates (
) and Pol III* constituted by Method 1
(
).
How tightly associated is Pol III*? In the cell, there
are 10-20 molecules of Pol III* for a concentration of
17-34 nM(1) . In Fig. 8, we tested the
ability of the Pol III* particle to remain intact at a concentration
similar to that in the cell. At 30 nM Pol III*, the subunits
are far below the limit of detection in column fractions by Coomassie
Blue staining (or silver staining) of an SDS-polyacrylamide gel. Hence,
Pol III* was identified in column fractions by assaying for
-dependent replication of singly primed M13mp18 SSB-coated
single-stranded DNA. If Pol III* falls apart, the
complex, upon
dissociating from within Pol III*, should elute much later. Hence, the
column fractions were also assayed for the
complex. The results
show that Pol III* and the intrinsic
complex activities comigrate
in fractions 16-28, consistent with the size of Pol III* and with
the particle remaining intact during gel filtration at these dilute
conditions. As a control, a second gel filtration analysis using 30
nM
complex showed the expected elution position of
complex activity had Pol III* dissociated (Fig. 8, dashedline).
Figure 8:
Pol
III* remains intact at cellular concentrations. Pol III* was
constituted and purified by Method 1 and then diluted to a
concentration of 30 nM (assuming a mass of 670 kDa) in a final
volume of 200 µl of column buffer and gel-filtered on Superose 6.
The replication activities of Pol III* () and the
complex
(
) were followed in the column fractions. Also shown is a
separate experiment in which a solution of 30 nM constituted
complex was gel-filtered and followed in the column fractions by
activity assays (
).
Structural Insights from the Assembly Process
The
The
initial observation that Pol III` and the and
Subunits Interact
complex did not
assemble into Pol III* implied that extra subunits might be needed. For
example, perhaps the
complex was meant to assemble with
-less Pol III* to form a dimeric polymerase with a
complex
and a
complex. However, these assemblies did not interact either.
After that, we pursued a line of investigation based on the assumption
that one of the subunits of either Pol III` or the
complex was
preventing assembly of Pol III*, an assumption that turned out to be
correct.
,
,
, or
) and one from the
complex (
,
,
`,
, or
), and to assay for an
interaction by gel filtration (except for the previously established
interactions of
with
). Using this approach, a contact of
with
was identified (Fig. 9). The
complex
is somewhat difficult to observe by gel filtration because each subunit
migrates in gel filtration columns as a higher order oligomer,
consistent with a tetrameric state (14, 17), but upon mixing them, they
migrate as a
heterotetramer. The
heterotetramer elutes earlier than
alone, but later than
alone. A study of the
-
interaction is presented in Fig. 9. Panel A shows the
subunit alone, and panelD shows
alone.
PanelB shows the result of mixing
with a
4-fold excess of
. Comparison of the
position in panelB with
alone in panel A shows that
causes
to elute earlier (bigger) than
alone (compare
fractions 27-33 in panelB with fractions
33-37 in panel A). When
is in a 4-fold excess over
(panel C), it causes
to elute later (smaller) than
alone (fractions 29-33 in panel C relative to
fractions 27-31 in panel D). The mixed heterotetramer
does not resolve from the homotetramers, and therefore, the
stoichiometry of
and
in the heterooligomer cannot be judged
from the polyacrylamide gels.
Figure 9:
The and
subunits interact. The
and
subunits were incubated for 60 min at 15 °C and
then gel-filtered on a Superose 6 column. A, the
subunit
(94 µg, 1.0 nmol as dimer); B, mixture of the
subunit (32.9 µg, 0.35 nmol as dimer) and
(198.8 µg, 1.4
nmol as dimer); C, mixture of
(132 µg, 1.4 nmol as
dimer) and
(50 µg, 0.35 nmol as dimer); D, the
subunit (142 µg, 1.0 nmol as dimer); E, mixture of
(94 µg, 1.0 nmol as dimer) and
(142 µg, 1.0 nmol as
dimer). Column fractions are indicated at the top. In the firstlane are molecular mass standards, and their masses are
indicated to the left. The
and
subunits are identified to
the right. Frxn, fraction number.
That and
form a mixed
oligomer is not surprising as
and
are related structures;
contains the
sequence plus an additional 24 kDa of
polypeptide at the C terminus. Since they both form homooligomers, it
is reasonable to expect them to form a heterooligomer. The
complex does not appear to be
disproportionately favored relative to homotetramer formation as an
equimolar mixture of
and
does not yield a heterotetramer as
the sole product (Fig. 9E). The positions of
and
are both affected, but they do not fully comigrate, indicating
the presence of all three tetramers:
,
, and
.
and
is shown by
chemical cross-linking. The second through sixthlanes show cross-linking of
at increasing
concentrations of NaCl. The
monomer and dimer are clearly
visible; the trimeric and tetrameric forms are present, but on the gel
of Fig. 10, they are unresolved at the top. The seventh through eleventhlanes show cross-linking of
, and again at all salt concentrations, the
monomer and
dimer are evident, while the higher states are unresolved at the top.
The lastfivelanes show cross-linking of an
equimolar mixture of
and
. One band labeled
heterodimer is unique to the
mixture, and it migrates
between the
and
positions. The
presence of both
and
in this cross-linked band has been
confirmed using either [
H]
or
[
H]
(
H label is present in this
position regardless of which subunit is labeled (data not shown)).
Attempts to resolve the higher molecular mass cross-linked products in
lower percentage gels resulted in smeared bands and did not appreciably
resolve the higher order cross-linked products. Presumably, these
proteins are too large and the various inter- and intramolecular
cross-links are too heterogeneous to give sharp and clearly resolved
higher molecular mass products.
The
The assumption that a subunit of the Subunit Inhibits the Interaction of
with
complex or Pol
III` prevents their association into Pol III* led to the identification
of the
-
interaction (described above). Armed with the
knowledge that
and
interact, we turned the argument around
and asked which subunit, when added to
or
, prevents their
interaction? The binding of
, the
complex, or the
entire core (
) to
before (or after) the addition
of
did not inhibit formation of the
complex (Ref. 17
and data not shown). Likewise, prior formation of
,
, and
`
did not prevent
interaction with
or Pol III` (summarized in
Fig. 2
).
(
)
Hence, by exclusion, it would
appear that
is the culprit. The
subunit does not stabily
interact with
in the absence of
`, and thus, the smallest
complex that can be used to test the prediction is the
`
complex. As expected, the
` complex did not bind to Pol
III`, implying that
indeed prevents interaction between Pol III`
and the
complex. Fig. 11illustrates this finding. Pol III`
and
`
(
complex) do not interact, but
if Pol III` and
`
are premixed and
is added
last, Pol III* is formed. The effect of
on Pol III* assembly is
also summarized in Fig. 2.
`
complex; some of the
`
complex binds to and comigrates with Pol III`
(in fractions 16-20).
(
)
The
`
complex alone migrates much later (fractions
26-32; panel B) and is well resolved from Pol III`
(fractions 16-20; panel C).
complex is mixed with Pol III`, and the
filtration analysis shows that they do not associate to a significant
extent, as described more fully in Fig. 1. In panel E,
the addition of
has been delayed until
`
and Pol III` have been given time to associate. Then
is added,
and the filtration analysis shows that Pol III* has formed. Thus, the
binding of
to
`
prevents the formation of
Pol III* presumably by interfering with the
-
contact.
The
Both ` Subunit Bound to Both
and
Prevents
the
-
Interaction
and
can bind the
,
`,
, and
subunits, and both the
and
complexes are active as clamp loaders in replication
assays
(15) . Do both
and
complex clamp loaders exist
within Pol III*? To study this, we tried to construct a Pol III*
assembly containing both a
complex and a
complex.
Obviously, the
subunit must be excluded from the reaction until
after the
-
contact has been formed. Hence, we assembled a
Pol III`
`
complex and a
`
complex and analyzed the mixture by gel filtration (Fig. 12). The
results show that they did not interact (Fig. 12A), as
is most simply observed by examining the different peak positions of
(fractions 16-20) and
(fractions 26-32).
However, a Pol III`
complex was able to bind the
`
complex (Fig. 12B), as evidenced
by some comigration of
with
in fractions 16-22.
Hence, even though both
and
can bind
`, if
` is
on both
and
, they cannot establish the
-
contact
as though there is only enough room in the heterotetramer for one
molecule of
`. We have also confirmed the
` inhibition of the
-
contact using the minimal assemblies possible:
`
does not bind
`
, but
binds
`
, and
binds
`
.
(
)
The effect of
` on the
assembly of Pol III* is also summarized in Fig. 2.
Figure 12:
The
` subunit bound to both
and
inhibits the interaction
of
with
. A, the Pol III`
`
complex was formed upon incubating constituted Pol III` (formed as
described in the legend to Fig. 11), and then
` (11.1 µg, 0.3
nmol) and
(9.5 µg, 0.3 nmol) were added (final volume
of 90 µl) and incubated for 30 min. Then the
`
complex (32.6 µg, 163 fmol) was added, and
the mixture was incubated for another 30 min at 15 °C (111 µl)
and gel-filtered as described in the legend to Fig. 11. B, the
Pol III`
complex was formed as described for
A, except that
` was omitted from the incubation (final
volume of 108 µl) followed by the addition of the
`
complex as described for A, and then
gel-filtered. Column fractions are shown at the top; positions of
molecular mass markers are indicated to the left; and subunits are
identified to the right of the gels.
This study
of Pol III* assembly showing ` can be on
or
,
but not both, is consistent with the observed stoichiometry of one
` (and
,
, and
) in Pol III*. Furthermore, the
single copy of
in Pol III* follows from the fact that the
stoichiometry of the
` complex is 1:1, and the point of
attachment of
to the
complex is through interaction with
` (3). The fact that there is only one
` in Pol III*, coupled
with the fact that
` stabilizes the
complex
(15) , provides a ready explanation for why Pol III*
retains only one copy of
and
even though their presence
on both
and
does not prevent Pol III* assembly. Hence, only
the subunit (
or
) that receives
` will retain the
complex due to the stabilization incurred by
`.
The Pol III* Structure
The nine-subunit Pol III*
assembly has been constituted from individual pure proteins; it is
similar in size, subunit composition, and replication activity to Pol
III* purified from E. coli cell lysates. The particle contains
14 polypeptides in an arrangement consisting of two core polymerases
and a complex clamp loader all connected to each other through
as illustrated in Fig. 13. The two core polymerases bound
to the
dimer are most likely arranged with one core on each
protomer of
. Assuming the subunits of the
dimer are related
by a 2-fold axis of rotation, then each polymerase is also related to
the other polymerase by a 2-fold rotational axis. The
dimer forms
a heterotetramer with
, and assuming the heterotetramer is also
symmetric, each (core-
protomer-
protomer) unit will be
related to the other unit by a 2-fold rotational axis. However, the
single copy each of
,
`,
, and
imposes a
structural asymmetry onto the structure of Pol III* such that there can
be no overall 2-fold rotational axis. Presumably, the structural
asymmetry created by these single copy subunits imposes a functional
asymmetry onto the two polymerases for the different actions needed on
the leading and lagging strands. Whether these subunit are all on
or whether some or all are on
is discussed below. The
subunit associates with the
subunit of the core polymerase
(14) and with the
subunit of the
complex
(19) , and therefore, in principle, a total of three
dimers may associate with Pol III*. A previous study indicated
that two
dimers were present in the holoenzyme, and in
Fig. 13
, they are placed on the two core polymerases.
Figure 13:
Subunit arrangement of Pol III
holoenzyme. The and
dimers are each shown in an isologous
arrangement, and the
heterotetramer is also shown as
isologous. The two core polymerases are attached to the
dimer,
presumably one on each protomer. Depending on which face of the
dimer they bind, they could point in the same direction (as shown) or
in opposite directions. If
and
form a heterologous closed
tetramer, the two cores could be oriented 90° with respect to each
other. The single copy subunits,
,
`,
, and
, are
drawn on
, although they may be shared physically or in time with
(see ``Discussion'' for details).
` is positioned
between the
and
interface to explain the observation that
only one
` is accommodated in the heterotetramer, yet one
`
is present in the
complex (contains a
dimer) and in the
complex (contains a
dimer) (15). Two
dimers are shown
bound to the two cores.
It is
quite interesting to see how the subunit stoichiometry and native
aggregation state of the various Pol III subunits have been utilized to
form this machine. All the subunits of Pol III*, except and
, are monomers in isolation. The oligomeric structure of
is
needed to bring two copies of the core polymerase into the Pol III*
structure. Furthermore, the holoenzyme architecture has been sculpted
such that it contains only one each of the
,
`,
, and
subunits. Since either
or
can bind these single copy
subunits, it seems plausible that the holoenzyme would obtain at least
two copies of each of them: a set on
and a set on
. However,
the holoenzyme architecture precludes this by preventing the
complex from binding more than one
` monomer, perhaps by allotting only enough space inside the
junction of the heterotetramer to fit one monomer of
` (as
suggested in Fig. 13). The restriction of assembling only one
` monomer into Pol III* is consistent with the observed
stoichiometry of one
` in Pol III*. It is also in keeping with the
single copy of
in Pol III* as it has been shown previously that
` is a 1:1 complex
(3) .
and
subunits can be bound to both
and
without inhibiting the
-
interaction, and thus, it seems possible that Pol III*
could, in principle, have two copies of
and
.
Nevertheless, the stoichiometry studies suggest that only one of each
is present in Pol III*. The first report in this series showed that the
complex dissociated easily from
(and
) at 37
°C, but that
` greatly stabilized the
-
interaction
(15) . Hence, it may be presumed that whichever
dimer,
or
, that the
` complex is associated with
in Pol III* will also be the dimer that the
complex
stabily associates with.
) was proposed to
be dimerized by the
subunit of core. It is now known that the
subunit binds
tightly and dimerizes the core
polymerase
(14) . Furthermore,
is a monomer, and core does
not dimerize even at a concentration of 100 µM(7) .
Also in the earlier study, the
and
subunits were proposed
as the basis for asymmetry within Pol III*, in which one core
polymerase was on
and the other core was on
. This
asymmetric arrangement of subunits was proposed to endow each
polymerase with different properties, one suited for continuous
synthesis of the leading stand and the other for discontinuous
synthesis of the lagging strand. The arrangement in
Fig. 13
differs from the earlier study in that a
dimer
bridges both core polymerases and
binds the polymerase
indirectly, through the
dimer. In support of this, we detected no
interaction of core with
(14) or with any other subunit of the
complex
(15) or with the entire
complex (data not
shown). Both models propose that the
,
`,
, and
subunits are associated with the
dimer, and this report shows
that the asymmetric structure of Pol III* is based in the single copy
nature of these subunits. The earlier study of Pol III* structure
proposed two copies of each subunit in Pol III*. However, this
stoichiometry does not match the observed 2:1:1:1:1 stoichiometry of
the
complex
(
`
)
(15, 20) . Perhaps some
complex was present in the Pol III* prepared from cell lysates,
which would have increased the apparent stoichiometry of the
,
`,
, and
subunits. The stoichiometry of subunits in
the holoenzyme determined in the earlier study is consistent with the
results of this report, in which the holoenzyme consists of one subunit
each of
,
`,
, and
and two subunits each of
,
,
,
, and
(16).
Location of the Single Copy Subunits
Since the
,
`,
, and
subunits can be added after mixing
with
, there is an ambiguity in the exact position of these
single copy subunits (i.e. see Fig. 2). Are they on
or
, split between them, or shared? The
` subunit can
be placed on either
or
, but not on both at the same time or
else assembly of Pol III* is halted (i.e. as in Fig. 2).
Furthermore, the
subunit must be added after
and
are
mixed, and thus, it too may assemble with either
or
(presumably the one that has
`). In the fourth report of this
series, we found that the
complex forms in preference to the
complex in Pol III*
(21) . Apparently, the presence of core
on
decreases the efficiency of
` and
association with
(21) . Consistent with
as the
preferred target of association of these single copy subunits, Pol III`
purified from E. coli lysates does not contain the
,
,
, or
` subunit, nor has a
complex been purified
from E. coli lysates. Despite these arguments, it is still
conceivable that
and
share these subunits either in time
(e.g. by dissociation and reassociation of
` between
and
) or physically, in which the
,
`,
, and
subunits bridge the
/
boundary with some present on
and some on
.
complex has not been
purified from wild-type E. coli, a
-less form of Pol III*
can be reconstituted from individual subunits, and these assemblies are
active in replication assays
(15, 23) . Whether such
complexes exist in vivo is unknown; however, it has recently
been reported that E. coli cells are viable, even when the
signal for the -1 translational frameshift in dnaX that
produces
is removed
(23) . These cells contain only the
subunit, and
-less Pol III* appears to be present in them,
implying that
-less Pol III* can function at a replication
fork
(23) .
-less Pol III* appears to contain two
dimers, and we propose that one
dimer replaces
both
functionally and structurally.
Why both
and
are
present in the holoenzyme when
alone can do the job of both is
unknown; speculation on this issue is included in the fourth report of
this series
(21) .
Forms of Pol III* Resulting from Different Methods of
Assembly
By staging the assembly of Pol III* in vitro,
it should be possible to selectively place the ,
`,
,
and
subunits on
in a preincubation with
in the
absence of
(see Fig. 2as an aid in understanding the
assembly paths discussed below). For example, we have constituted Pol
III* starting from the
`
complex, followed by
adding
and then
and core. Provided the subunits do not
exchange from
to
during the assembly time (a real
possibility), the resulting Pol III* should at least have
`
on
rather than on
.
on both
and
before mixing them (as in
Fig. 2
), 1b) mixing the pure
complex with
(this may direct the assembly of
` to
), and 1c)
mixing the pure
complex with
(this may direct
the assembly of
` to
). The two variations of
Method 2 that we have performed include the following: 2a) starting
with
`
(as in Fig. 3; this may direct
to
) and 2b) starting with
`
(this may
direct
to
). Methods 1b and 2a may be expected to direct
,
`,
, and
onto
, and Methods 1c and 2b
should direct
,
`,
, and
onto
. Method 1a
is the least assuming of the five constitution reactions (even less
assuming is to premix
and
and then add a mixture of all the
rest of the subunits).
on
singly primed single-stranded DNA. However, all five forms were within
2-fold of the activity of Pol III* purified from cell lysates, and they
all had the same subunit composition as determined by scanning of
Coomassie Blue-stained SDS-polyacrylamide gels (data not shown).
Perhaps these forms would show different behavior in more complete
assays including initiation at an origin and function with the helicase
and primase during propagation of the replication fork. It is also
possible that all these forms reverted to a single species due to
subunit rearrangement over the time scale used to assemble them.
Comparison of the Pol III* Structure with Replicases of
T4 Bacteriophage and Eukaryotes
The T4 replicase has a
two-subunit complex (gene 44/62 protein complex) that is thought to act
as a clamp loader of the gene 45 protein clamp onto primed DNA, but
unlike the holoenzyme, it does not appear to have a homolog to
bring the polymerases and clamp loader into one macromolecular assembly
in the absence of DNA
(24, 25, 26) . The
structure of eukaryotic DNA Pol
is even more similar to the
E. coli holoenzyme
(25, 26, 27) . Pol
is composed of two subunits containing the polymerase and
3`-5`-exonuclease activity and may be compared with the E.
coli core. Pol
becomes highly processive upon assembly with
the PCNA clamp encircling DNA. The PCNA clamp is assembled onto DNA by
activator-1 (or RF-C), a five-subunit clamp loader that couples ATP to
load PCNA clamps onto DNA. Hence, the human replicase machinery is
quite similar to the E. coli holoenzyme. At the current state
of knowledge, Pol
is not organized into a twin polymerase, and
the clamp loader is not physically connected to Pol
in solution.
Hence, the human system lacks the equivalent of the E. coli
subunit for organizing its polymerases and clamp loader into
one particle.
Table:
Subunit stoichiometry of constituted Pol III*
and Pol III* purified from E. coli cell lysates
) on a Coomassie Blue-stained gel. Values for
constituted Pol III* are the average of four different preparations,
two by Method 1 and two by Method 2. Values for Pol III* purified from
cell lysates are from three adjacent lanes of one preparation. All
values are normalized to a value of 2 for
. The error represents
one standard deviation.
Table:
Average subunit stoichiometry and calculated
mass of Pol III*
assembly containing
(i.e.
`,
`
,
`
)
will not bind
. The converse is also true: a
assembly
containing
(i.e.
`,
`
,
`
) will not bind
(17) (R. Onrust and M. O'Donnell, unpublished data).
-less Pol III* has a Stokes radius of 97
Å for a mass of
1 MDa and a specific activity of 4.9
pmol/min/mg. We propose that
-less Pol III* is composed of a
tetramer of
in which a
dimer replaces the
dimer in
associating with
,
`,
, and
and the other
dimer has two core polymerases.
and
` subunits did not resolve on the HPLC column by any gradient or
elution program that we tried. For those subunits that did resolve, the
molar ratio was
(where ND is not determined).
, Pol III`
, or Pol
III`
`
did not prevent interaction of
with
(in Pol III`).
`
is
that Superose 12 was used in these experiments. Pol III` already elutes
in the excluded volume of this column.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.