(Received for publication, June 2, 1995; and in revised form, August 23, 1995)
From the
A physical characterization of the and
subunits of
the Escherichia coli DNA polymerase III holoenzyme and their
complexes with the
,
`,
, and
subunits is
presented. The native molecular mass of the
and
subunits
was determined to be 255,000 and 189,000 Da, respectively, by
sedimentation equilibrium analytical ultracentrifugation. Both values
indicate a tetrameric quaternary structure. The
and
complexes were reconstituted and purified using two different methods.
Both complexes assembled readily and were reconstituted at subunit
concentrations approaching physiological levels. The stoichiometries of
the
and
complexes, as determined by quantitative
densitometry of SDS-polyacrylamide gels, were found to be
`
and
`
.
BIAcore analysis demonstrated that the formation of
large multiprotein complexes of holoenzyme subunits depends on the
presence of the
subunit;
could not substitute. We present a
model for a
-less form of DNA polymerase III holoenzyme that has
asymmetrical structural features that may be responsible for the
functional asymmetry observed in holoenzyme. The stoichiometry of the
reconstituted DNA polymerase III* component of holoenzyme in this model
is
(
)
DnaX
`
.
The DNA polymerase III holoenzyme, ()the replicative
polymerase of Escherichia coli, contains three essential
subassemblies: (i) a polymerase that is able to interact with other
specialized proteins at the replication fork, (ii) a
sliding
clamp that tethers the polymerase to the template, enabling high
processivity, and (iii) a DnaX complex that loads the sliding clamp
onto the primer-template (see the companion study in this series,
Dallmann et al.(1995), for a more thorough explanation and
references). The holoenzyme is dimeric and functions asymmetrically,
presumably because it contains distinct leading and lagging strand
polymerases. We have demonstrated that the asymmetric function of the
holoenzyme can be reconstituted using only the
subunit,
eliminating the possibility that an asymmetric placement of
relative to
is responsible for this asymmetry (Dallmann et
al., 1995).
In addition to the finding that alone permits
reconstitution of native holoenzyme, other evidence implicates
as
a key essential component and relegates
to nonessential roles
that can be replaced by
. Olson et al.(1995) demonstrated
that the salt resistance of native holoenzyme required a
-
-
complex;
-
-
would not substitute.
This suggests that
is found complexed with auxiliary clamp
loading subunits within holoenzyme. Blinkova et al.(1994)
showed that E. coli are viable when their dnaX gene
is mutated so that they cannot frameshift and only the
product is
produced. DNA polymerase III* isolated from these strains starts out in
the
-less form, but during purification,
is rapidly
proteolyzed to
. We (Dallmann et al., 1995) and others
(Tsuchihashi and Kornberg, 1989) have demonstrated that overproduction
of both
and
in vivo from the wild-type dnaX gene results only in assembly of
and
oligomers; no mixed
-
oligomers are detectable. We have also
shown that only
complexes function at low, physiologically
relevant concentrations of pol III core and that addition of free
, although it stimulates the reactions, does not efficiently
recruit
complexes into an efficient holoenzyme during the 5-min
time course of the reaction, a time adequate for replication of 300
Okazaki fragments in vivo.
As a step toward resolving these
issues, we analyzed formation of DnaX complexes under conditions that
allow accurate determination of their stoichiometry and analysis of
their interactions. We demonstrate that both and
can form
well defined complexes with
-
` and
-
, but that
only the
complex can efficiently enter holoenzyme. This work has
led to a model for the DNA polymerase III* component of holoenzyme:
(
-
-
)
(
)
(DnaX
-
-
`-
-
),
where DnaX can be
with preservation of all of the
known properties of native holoenzyme.
On-line formulae not verified for accuracy
where A(x) is absorbance at radial position x; A is absorbance at radial position x
; H =
(1-
)
/RT;
= protein
partial specific volume;
= solvent density;
= angular velocity; R = gas constant; T = temperature (K); M = native molecular
weight, and E is the base-line offset.
On-line formulae not verified for accuracy
where RU is the measured response (units) obtained at binding saturation and M is the molecular weight.
Figure 1:
Sedimentation equilibrium of and
. Sedimentation equilibrium studies of
and
were done
using the Beckman Optima XL-A analytical ultracentrifuge as described
under ``Experimental Procedures.'' Representative data, curve
fit, and residual plots are shown for
(14 µM)
sedimented at 15,000 rpm for 40 h (A) and for
(10
µM) sedimented at 15,000 rpm for 40 h (B). Data
were fit to the IDEAL1 model (). Residuals are expressed in
absorbance units (280 nm).
Figure 2:
Activity of reconstituted and
complexes are not stimulated by additional
,
`, and
. The
(
) and
(
) complexes
were assayed as described under ``Experimental Procedures.''
Both DnaX complexes were present at 100 fmol/assay (4 nM), and
core and
were present at 480
fmol/assay.
Preliminary studies on the reconstitution
of DnaX complexes indicated no preferential order of addition of
subunits and no requirement for prolonged stepwise preincubations or
for ATP or Mg in obtaining maximal activity yields.
The DnaX complexes could be quantitatively assembled at various
concentrations in buffer N at room temperature for 15 min before
purification (Fig. 3).
Figure 3:
Assembly of complexes at low protein
concentrations. The
(2.5 nmol of monomer),
(5 nmol) and
` (5 nmol) subunits, and the
complex (5 nmol) were
diluted into three different volumes of buffer N (0.5, 3.5, and 12.5
ml) at three different concentrations (1.25 µM, 180
nM, and 50 nM
complex equivalent) in order to
reconstitute the
complex. After mixing, samples were incubated at
room temperature for 15 min, then chromatographed over a Mono Q
ion-exchange column as described under ``Experimental
Procedures.'' Samples of
complexes prepared at each
concentration were denatured in SDS sample buffer and subjected to
electrophoresis on a 12% SDS-polyacrylamide gel. Following staining in
Coomassie Brilliant Blue G-250, subunits were quantitated by laser
densitometry to determine subunit stoichiometry. This was compared to
the subunit stoichiometry of
complex prepared by gel filtration
which was run on the same gel. Lane 1,
complex prepared
at 500 nM by Mono Q (8 µg); lane 2,
complex
prepared at 180 nM by Mono Q (8 µg); lane 3,
complex prepared at 50 nM by Mono Q (8 µg); lane
4,
complex prepared at 2.5 µM by Superose 6 gel
filtration (8 µg).
The stoichiometries of the DnaX complexes were
consistent with the subunit stoichiometries determined for the DnaX
subunits by analytical ultracentrifugation, but inconsistent with
previously reported stoichiometries, which assumed two copies of the
DnaX component in the DnaX complexes (Maki and Kornberg, 1988; Onrust
and O'Donnell, 1993). The reconstitutions of the and
complexes described above were carried out at fairly high protein
concentrations (2.5 µM DnaX tetramer equivalent), raising
the possibility that the observed stoichiometry reflected the use of
nonphysiological protein concentrations far exceeding the DnaX
dimer-tetramer equilibrium dissociation constant. To rule out this
possibility,
complexes were reconstituted and prepared by
ion-exchange chromatography at concentrations approaching the estimated
intracellular holoenzyme subunit concentration (
30 nM assuming 20 copies of holoenzyme/cell) (
)(McHenry and
Kornberg, 1981) to determine whether the stoichiometry of
decreased in these complexes. Visual inspection of an
SDS-polyacrylamide gel of samples of
complex prepared at 1.25
µM, 180 nM, and 50 nM
complex
equivalents, along with a standard of
complex prepared by gel
filtration (Fig. 3), indicated no obvious changes in the
stoichiometry of the
complexes. Laser densitometry of each of
these samples and comparison to defined standards indicated that
stoichiometry of all
complexes was the same as the values
reported in Table 2, with four to five copies of
for each
of
`,
, and
.
Figure 4:
Interaction of with
does not block interaction of
with core. Sensorgram overlays are
shown for
(200 nM) and core (200 nM)
sequentially injected over immobilized
(3800 RU immobilized). The
subunit was immobilized to a CM5 sensor chip as described under
``Experimental Procedures.'' Proteins were diluted in HKGM
buffer. To completely dissociate bound protein, the sensor chip was
regenerated with a 15-s pulse of 10 mM NaOH, which allowed for
>95% retention of the original binding activity of the immobilized
. Control injections over an underivatized flow cell were
performed but not subtracted from the data. Background contributions
due to these injections were equal to
50 RU for the
injection and
400 RU for the core injection. Sensorgram 1,
sequential injection of
(200 nM, 20 µl) and
core (200 nM, 40 µl). Sensorgram 2, sequential injection
of core (200 nM, 40 µl) and
(200 nM,
20 µl). The sensorgram at the bottom of the figure shows the
subtraction of sensorgram 1 from sensorgram 2 to extract a
representation of
binding to
without the background
of core simultaneously dissociating from
. The thick line
indicates binding of
to
in the presence of core
bound to
.
Figure 5:
Participation of in a DnaX complex
does not block the binding of core. Sensorgram overlays are shown for
mixtures of
,
,
`, or
,
,
`
(200 nM each subunit) sequentially injected before core (200
nM),
(200 nM), or
(200 nM
monomer) over immobilized
(660 RU immobilized), The
subunit
was immobilized to a CM5 sensor chip as described under
``Experimental Procedures.'' All proteins were diluted in
HKGM buffer. To completely dissociate bound protein, the sensor chip
was regenerated with a 15 s pulse of 10 mM NaOH, allowing for
>95% retention of the original binding activity of the immobilized
. Control injections over an underivatized flow cell were
performed but not subtracted from the data. Background contributions
due to these injections were equal to
200 RU for the
(
),
, and
` injections,
400 RU for the core
injection,
10 RU for the
injection, and
100 RU for the
injection. Similar sensorgrams were obtained when
` was
immobilized to the sensor chip and
was injected with the other
subunits (not shown).
The
DnaX complexes act to load the subunit onto primed DNA. To
determine whether the
and
complexes bind
, the
subunit was injected over the
and
complexes assembled on
the BIAcore
. As expected,
rapidly bound to both
complexes (
400 RU) and dissociated fairly rapidly (as indicated by
a more pronounced exponential decay of the signal seen after the end of
the
injection) (Fig. 5). The apparent binding
stoichiometry was calculated to be 1
monomer per
DnaX
`
complex, although this represents a minimal estimate since the
concentration was not varied to ensure that it was saturating. To test
the postulate that the
subunit functions to recruit the
complex into holoenzyme (O'Donnell, 1994; Stukenberg et al. 1994), we analyzed whether the
subunit could bind to either
or
complexes assembled on the BIAcore
(Fig. 5). Under the conditions used, no binding of either
or
complex to immobilized
subunit was detected.
Additional experiments showed no binding of
to
immobilized
on the BIAcore
, or
to
immobilized on the
BIAcore
(data not shown).
Figure 6:
Reconstitution of a -dependent
initiation complex on BIAcore
. A sensorgram is shown for
sequential injections of
,
, and
` (200 nM each subunit), core (200 nM),
(200 nM),
and primed, SSB-coated M13G
(30 fmol, 10 nM as
circles) over immobilized
(660 RU immobilized). All proteins were
diluted in HKGM buffer + 200 µM ATP. Immobilization
and regeneration conditions and control injections were done as in Fig. 5, and under ``Experimental Procedures.''
Background contribution due to the injection of primed, SSB-coated
M13G
was
1300 RU (
1000 RU were
bound).
We have used sedimentation equilibrium analytical
ultracentrifugation to determine the native molecular weights of
and
(Fig. 1, Table 1). Both proteins sedimented as
ideally behaved particles at molecular weights consistent with a
tetrameric quaternary structure. Numerous previous studies implied
stoichiometries from dimer to trimer for DnaX (Tsuchihashi and
Kornberg, 1989; Studwell-Vaughan and O'Donnell, 1991;
O'Donnell, 1994). These studies were conducted using gel
filtration and glycerol gradient centrifugation which are affected by
molecular properties other than molecular weight (such as particle
shape and frictional ratio), whereas sedimentation equilibrium is
dependent only on the weight of the sedimenting particle and not on
molecular shape factors (van Holde, 1971). Thus, our determination of
the native molecular weights of
and
must be considered the
most reliable estimate to date.
Sedimentation velocity analytical
ultracentrifugation of and
was also performed (Table 1). For
, the sedimentation coefficient, Stokes
radius and frictional ratio determined here were comparable to those
determined by Studwell-Vaughan and O'Donnell(1991). The
frictional ratios for both
and
indicate that they are
asymmetric.
The purified and
subunits were used to
reconstitute DnaX complexes along with the
and
` subunits
and the
complex. These complexes were readily assembled
with no requirement for stepwise preincubation of subunits or
requirement for ATP or Mg
before purification.
Previous reports (Blinkova et al., 1993; Stukenberg et
al., 1994) have indicated that a stepwise preincubation of
subunits is required for the reconstitution of
complex and
holoenzyme before purification. We attribute this requirement to the
use of
that had been purified in 6 M urea from
inclusion bodies (Xiao et al., 1993). Presumably this
procedure reflects the time required for the refolding of
and
its recruitment into a reconstituted holoenzyme. The
used in our
study was co-expressed with the
subunit as a soluble complex
which has been shown to readily interact with the DnaX proteins and is
required for cooperative assembly of
and
` into DnaX
complexes (Olson et al., 1995).
The and
complexes were purified by either gel filtration or anion exchange
chromatography. Both methods yielded complexes that were
indistinguishable by activity and subunit stoichiometry (Table 2). Stoichiometries of the
and
complexes were
found to be
`
and
`
,
respectively, inconsistent with a previous report of
complex
stoichiometry
(
`
)
(Maki and Kornberg, 1988), which was estimated by densitometry of
stained SDS-polyacrylamide gels using relative staining intensity for
quantitation. We believe that the stoichiometries determined here are
reliable and can be used to infer the stoichiometry of DnaX complexes in vivo for a number of reasons. (i) Defined amounts of each
subunit (determined from subunit extinction coefficients) were used as
standards in the quantitation, not relative staining intensity. (ii)
Reconstitutions of
complex at low protein concentrations (down to
50 nM
complex) approaching the estimated intracellular
holoenzyme subunit concentration (
30 nM
assuming 20 copies of holoenzyme/cell) (McHenry and Kornberg,
1981) produced complexes with stoichiometries identical to complexes
prepared at high concentrations (Fig. 3). (iii) Addition of
,
`, and
to
and
complex assays (Fig. 2) caused no stimulation, indicating that the complexes
were replete in these subunits. This last point implies that the
dissociation constant for the DnaX dimer-tetramer equilibrium is no
higher than 4 nM, the concentration of DnaX complex used in a
typical assay and below the estimated intracellular concentration. If
the
`
or
`
stoichiometry were an artifact due to high protein concentration
favoring DnaX tetramer formation, the observed stoichiometry might have
been caused by the use of protein concentrations far exceeding the
dimer-tetramer dissociation constant. In that case, addition of
,
`, and
to
and
complex assays (Fig. 2) should have caused a stimulation of DnaX complex
activity at the high dilutions used in the assay as the DnaX tetramer
reequilibrated to the dimer form. This was not observed.
The
participation of the and
subunits in higher order
multiprotein complexes with other holoenzyme subunits and subcomplexes
was qualitatively examined using the Pharmacia Biosensor BIAcore
instrument (Malmqvist and Granzow, 1994). Optimally, the
BIAcore
can be used to determine kinetic and equilibrium
constants for simple A + B
AB associating systems
(Fagerstam et al., 1992). For the interactions studied in this
report, where binding cooperativity (Olson et al., 1995) and
the assembly and dissociation of multisubunit complexes are involved,
kinetic and equilibrium constants could not be determined without more
extensive experimentation and mathematical modeling. The qualitative
results presented here can be considered analogous to protein-protein
cross-linking.
We have shown that the subunit, when
immobilized to the BIAcore
, can simultaneously interact
with core and the
complex (Fig. 4), as expected
based on the ability of
complex to reconstitute holoenzyme with
stoichiometric amounts of core and
(Dallmann et al.,
1995). We are confident that the final complexes assembled in this
experiment represent a core-
-
complex and not a mixed
population of
-
and
-core complexes since the
intermediate
-
and
-core complexes were formed to
completion, leaving no free
subunit present during the next
injection phase. The core-
-
complex also appeared to
assemble at the expected 2:4:1 core:
:
ratio.
The
and
complexes were reconstituted on BIAcore
via
or
` coupled to the sensor chip (Olson et
al., 1995). Not surprisingly, both
and
complexes bound
(Fig. 5). The
complex readily bound a stoichiometric
amount of core to form a complex with a dissociation half-life of 1.5
h; the
complex did not (Fig. 5), consistent with previous
results (Dallmann et al., 1995) showing that the
complex
requires a large excess of core for activity. These results can be
interpreted as a mass action effect. The activity of
complex
increases with increasing core since the interaction of core with
(required for high processivity) (Fay et al., 1982) on the
primed DNA will be diffusion-limited in the absence of any core-
complex interaction. With
complex, core-
interaction can be
established rapidly since the polymerase,
-clamp loader and
are all associated in one large complex ( Fig. 5and Fig. 6), effectively increasing the local concentration of core
relative to
.
We do not detect any interaction between the
complex and the
subunit on the BIAcore
(Fig. 5). The small effect of the
subunit on
complex activity assays (Dallmann et al., 1995) also supports
our present observation. All the components of a
-less initiation
complex can be assembled on the BIAcore
via the
subunit as a large, stable multiprotein-DNA complex dependent on the
presence of the
subunit (Fig. 6). Furthermore, the
apparent binding stoichiometry of core was consistent with the presence
of tetrameric DnaX complexes, as expected based on the DnaX subunit
stoichiometry determined by analytical ultracentrifugation and DnaX
complex stoichiometry determined by quantitative gel scanning.
The
results presented in this report and elsewhere (Olson et al.,
1995; Dallmann et al., 1995; Blinkova et al. 1993)
indicate is a dispensible subunit, both in vitro and in vivo. Asymmetry in the function of holoenzyme can be
conferred by the
subunit alone (Dallmann et al., 1995)
without apparent requirement for the
subunit/complex. In Fig. 7, we present a model for a
-less DNA polymerase III
holoenzyme that possesses all of the properties of native holoenzyme.
When
is present in this complex, it must replace two
s at a
position away from the two
s that dimerize pol III core. This
model is also consistent with the results of Blinkova et
al.(1993), who reconstituted and purified a functional
-less
holoenzyme from the
,
,
,
,
,
`,
,
and
subunits, and additionally found that expression of
,
but not
, was essential for cell viability. We have also observed
that holoenzyme reconstituted from all 10 subunits fractionates upon
gel filtration into a 9-subunit holoenzyme, lacking
(data not
shown), indicating that the mechanism of
entry is complex.
Figure 7:
Model of a -less DNA polymerase III
holoenzyme. A model for DNA polymerase III holoenzyme lacking the
subunit is shown. The model is based on observations in this report and
others (Blinkova et al., 1993; Olson et al., 1995;
Dallmann et al., 1995). In
-containing holoenzyme,
must replace 2
s distal to those that dimerize core pol III, since
is not a participant in this
interaction.
The
model shown in Fig. 7remains an asymmetric complex, considering
that the interaction of the single copy ,
`,
, and
subunits with the tetrameric
subunit is an inherently
asymmetric arrangement. An analogous situation can be observed in the
recently determined crystal structure of bovine heart mitochondrial
F
-ATPase (Abrahams et al. 1994) where the
interaction of the single copy
,
, and
subunits impose
asymmetry in the tertiary structures in each copy of the 3
and 3
subunits. The asymmetric placement of
,
`,
, and
on the tetrameric
subunit could then confer the asymmetric
properties of the leading and lagging strand polymerase halves of
holoenzyme.
While this manuscript was under review, a paper by
O'Donnell and colleagues (Onrust et al., 1995a)
concluded that and
exist principally as dimers and that the
stoichiometry of the
and
complexes are
`
and
`
,
respectively. Our data consistently indicate a tetrameric assembly for
both
and
, based on the molecular weight determinations for
and
obtained by the more rigorous method of sedimentation
equilibrium. Note that no discrepancy exists when the stoichiometry
assigned by O'Donnell and colleagues for the
complex (their Table 2) is divided by the yield obtained in the HPLC experiment
used to separate them (HPLC section of their ``Experimental
Procedures'').
In the same series of reports (Onrust et
al., 1995b), O'Donnell and co-workers described a method for
reconstituting into holoenzyme that depends on the presence of
limiting amounts of
and a molar excess of
in the
reconstitution mix. Furthermore, the presence of
-
` during
the early stages of reconstitution blocked the entry of
into
holoenzyme. Those results are also consistent with a tetrameric DnaX
assembly within holoenzyme. Our observations that DnaX assemblies are
tetrameric and that
-
` bind to (and presumably stabilize)
this tetrameric complex provide additional insight into holoenzyme
assembly. With
present in limiting concentrations and
present in excess, the formation of a mixed
-
complex (
)is the only way to accommodate the maximal quantity of
energetically favorable
-
contacts (K
<1 nM
) while preserving
a tetrameric DnaX arrangement. If
-
` binding to
stabilizes a tetrameric assembly, it may not be able to dissociate to
, a requisite for assembly with
. It seems unlikely to us that a
lengthy stepwise incubation in the absence of
-
` represents a
biologically relevant pathway for holoenzyme assembly. Given our
finding that
is the favored assembly state and that
pol III can bind
and form pol III` containing a
dimer
(McHenry, 1982; Studwell-Vaughan and O'Donnell, 1991), formation
of this complex may provide an assembly intermediate for the entry of
into holoenzyme. In any case,
appears to be dispensable,
since all of the known functional properties of DNA polymerase III
holoenzyme, including functional and structural asymmetry can be
reconstituted in
-less holoenzyme.