(Received for publication, June 2, 1995; and in revised form, August 23, 1995)
From the
An artificial operon that contains tandem holC-holD genes was used to overproduce a complex of
the and
subunits of the DNA polymerase III holoenzyme.
Normally insoluble by itself,
forms a tight soluble complex with
. A purification procedure that yields pure, active
complex in 100-mg quantities suitable for
biophysical studies is reported. Sedimentation equilibrium studies
demonstrate that
is a 1:1 heterodimer. The presence
of
dramatically lowers the level of
` required to reconstitute holoenzyme to levels
expected in vivo. That
accomplishes this by
binding to
or
and increasing their affinity for
` was demonstrated by surface plasmon resonance using a
Pharmacia BIAcore
instrument. In the absence of
`,
binds to either the
or
DnaX protein with K
= 2
nM.
The DNA polymerase III holoenzyme ()is the
replicative polymerase responsible for synthesis of the Escherichia
coli chromosome. Holoenzyme is composed of a DNA polymerase III
core (
-
-
) plus auxiliary subunits that confer the
special properties expected of a replicative polymerase (for reviews,
see McHenry(1991) and Kuriyan and O'Donnell(1993)). These include
high processivity and the ability to communicate with primosomal
proteins at the replication fork to permit coordinated replication (Wu et al., 1992a, 1992b ). The holoenzyme auxiliary subunits can
be divided into two subassemblies: 1)
forms a sliding clamp that
apparently encircles DNA (Kong et al., 1992) and tethers the
pol III core to the template by protein-protein interactions (LaDuca et al., 1986; Stukenberg et al., 1991); and 2) the
DnaX complex sets the sliding clamp onto the template-primer (Wickner,
1976).
The DnaX clamp-setting apparatus contains either the or
dnaX gene product complexed to
` and
(McHenry et al., 1986; Maki and Kornberg,
1988, Xiao et al., 1993b; Dallmann and McHenry, 1995). The
and
subunits are ATPases within the clamp-loading assembly
(Lee and Walker, 1987; Hawker and McHenry, 1987; O'Donnell et
al. 1993). Presumably, these subunits function to couple the
energy achieved from ATP hydrolysis to the assembly of the
sliding clamp. The
subunit also functions to dimerize pol III by
direct contact with the
subunit (McHenry, 1982; Studwell-Vaughan
and O'Donnell, 1991). The ATPase activities of
and
are stimulated by the presence of
` or
(Onrust and O'Donnell, 1993; Xiao et
al., 1993a), suggesting direct binding of one of the subunits. Gel
filtration of mixed subunits show that
and
` bind weakly to
and
to form a complex (Onrust and O'Donnell, 1993).
In a minimal holoenzyme assembly, a strong requirement is observed for
both the
and
` subunits (Onrust and O'Donnell, 1993).
The and
subunits have not been assigned a clear
function. They were initially identified by their association with
purified
complex (McHenry et al., 1986; Maki and
Kornberg, 1988), and it was not clear until they had been partially
sequenced and their structural genes cloned that they were distinct
proteins instead of proteolytic products of
or
` (Xiao et al., 1993a; Carter et al., 1993a, 1993b). No
requirement has been observed for
other than a
modest stimulation of holoenzyme reconstituted with the
DnaX
protein in the presence of elevated levels of salt (Xiao et
al., 1993b). It has been shown that
and
form a 1:1
complex (Xiao et al., 1993b). Gel filtration studies indicated
that
forms a complex with
or
in solution
and that
bridges the interaction of
with
(Xiao et al., 1993b).
The insolubility of the subunit and
its tendency to aggregate has limited its utility in physical and
functional studies (Xiao et al., 1993a).
required 6 M urea for all purification steps, and the resulting protein
was inactive and aggregated when urea was removed. The resulting
denatured purified
was useful only if rapidly gel-filtered to
remove urea immediately before conducting an experiment or if diluted
to 0.5 M urea, clearly a complication for kinetic and
biophysical experiments. Presumably as a result of the need to refold,
assembly reactions proceeded slowly, typically requiring 30 min (Xiao et al., 1993a).
The discovery, cloning, overexpression, and
purification of each subunit of the DnaX complexes has allowed us to
study their contributions to the holoenzyme replicative reaction. We
now report the purification and physical and functional
characterization of the complex. Exploiting an
artificial operon that overproduces both
and
, we show
that these subunits assemble in vivo to form a soluble 1:1
complex that is suitable for biophysical studies. We demonstrate that
the most striking contribution of
to the holoenzyme
reaction is its ability to bind
or
and increase their
affinity for
` so that they can form a functional
clamp-loading complex at physiological subunit concentrations.
In preliminary studies, we found that the subunit,
when overproduced by itself, formed aggregates that require denaturing
conditions for solubilization. O'Donnell and colleagues published
a similar observation and developed a purification for
that
started with denatured material (Xiao et al., 1993a). They
found that assays using
required extensive incubations,
presumably to permit proper folding and assembly of
into
complexes. We found that
, when overproduced from an artificial
operon with
, forms a soluble, monodisperse complex with
in vivo. We exploited the availability of this artificial
operon (Carter et al., 1993a) to generate a
complex that could be purified intact without a requirement for
denaturation and refolding.
Figure 1:
Purification of and
as a
complex. Fractions I-V were denatured and subjected to
electrophoresis on a 15% SDS-polyacrylamide slab gel. Protein was
detected by Coomassie Blue staining as described under
``Experimental Procedures.'' Lane 1, fraction I
(cell lysate, 250 µg protein). Lane 2, fraction II (45%
ammonium sulfate pellet, 200 µg protein). Lane 3, fraction
III (Q-Sepharose peak, 40 µg protein). Lane 4, fraction IV
(SP-Sepharose peak, 40 µg protein). Lane 5, fraction V
(Sephacryl S-100 peak, 40 µg protein). The migration of marker
proteins (phosphorylase B, bovine serum albumin, ovalbumin, carbonic
anhydrase, soybean trypsin inhibitor and
-lactalbumin) is
indicated on the left.
Figure 2:
Sedimentation Equilibrium of
. The
complex was sedimented as
described under ``Experimental Procedures.'' A,
sedimentation equilibrium boundary scans from the XLA analytical
ultracentrifugation runs at 84, 88, 92, and 96 h with
at a concentration of 4 µM. B,
data and residual plot of
(4 µM)
sedimented for 92 h at 35,000 rpm and fit to the IDEAL1 model.
Residuals are expressed as A
units. The curve
fit to the data assumes a heterodimeric form of
. C, same as in B, except the curve fit to the data
assumes
and
sediment independently as monomers. D, same as in B, except the curve fit to the data
assumes
sediments as a trimer
(
or
). E, same as in B, except the curve fit to the data assumes
sediments as a tetramer (
,
, or
).
We modeled
as single species sedimenting independently, and as
a dimer, trimer and a tetramer. Only the dimer (1:1
complex) fit the data (Fig. 2B), yielding very low
residuals (±0.02 A
units) distributed
around the theoretical curve. Sedimentation equilibrium data from all
three concentrations and each angular velocity were in close agreement.
They provided native molecular mass for
complex of
31,755 ± 178 daltons. The fit for the other models was
unacceptable, giving nonrandom residuals that deviated as much as 0.2 A
units from the theoretical curves (Fig. 2, C-E). Based on the amino acid
composition predicted from the DNA sequence and the protein sequence of
lacking its amino-terminal methionine,
and
are
16,599 and 15,043 Da, respectively (Xiao et al., 1993a; Carter et al., 1993a, 1993b). Thus, the species that best represents
is a heterodimer of 31,642 Da.
Figure 3:
confers salt resistance
to
-reconstituted DNA polymerase III holoenzyme but not
-reconstituted holoenzyme. DNA synthesis was measured as described
under ``Experimental Procedures'' using holoenzyme
reconstituted with
or
in the presence of the indicated
potassium glutamate concentrations. A, each assay contained
600 fmol of pol III core (
complex), 500 fmol of
,
500 fmol of
, 600 fmol of
, 600 fmol of
`, and 500 fmol
of
. B, same as in A, only
holoenzyme was reconstituted with 500 fmol of
instead of
.
Data represent DNA synthesis by reconstituted holoenzyme in the
presence (
) or absence (
) of
. Each data
point represents the average of a duplicate
determination.
Holoenzyme reconstituted with the product of the dnaX
gene instead of
, in the presence of
, is
extremely resistant to increasing potassium glutamate concentrations up
to 800 mM (Fig. 3B). The salt resistance is
similar to that observed for native purified holoenzyme (
)(Griep and McHenry, 1989). However, in the absence of
, the DNA polymerase activity of
-reconstituted
holoenzyme decreased dramatically as a function of increasing potassium
glutamate concentration. At 400 mM potassium glutamate, in the
presence or absence of
, the amount of dNTP
incorporation was 190 and 21 pmol, respectively, a 9-fold difference.
Thus the
complex is the key component
required for the salt resistance observed in native holoenzyme. This
9-fold dependence for
in the presence of 400 mM glutamate provided a convenient functional assay to monitor the
purification of
reported in Table 1. The assay
is linear over a broad range, from 10-50 fmol
per 25-µl assay (Fig. 4).
Figure 4:
Dependence of DNA polymerase activity of
-reconstituted holoenzyme on
in 400 mM potassium glutamate. DNA synthesis was measured with DNA
polymerase III holoenzyme reconstituted as described under
``Experimental Procedures'' and in the Fig. 3legend
using
and the indicated amount of
. Each data
point represents the average of a duplicate
determination.
Figure 5:
The presence of reduces
the level of
and
` required to reconstitute holoenzyme. DNA
synthesis was measured as described under ``Experimental
Procedures'' except that 75 fmol of the dnaX gene
products (
or
) were included, and the potassium glutamate
concentration was 100 mM. Holoenzyme was reconstituted with
(
) and
(
) Closed and open symbols represent holoenyme reconstituted with or without
, respectively. Each data point represents the
average of a duplicate determination.
Figure 6:
BIAcore analysis of
interaction with immobilized
. Sensorgram
overlays of various concentrations
(15-100
nM) injected over immobilized
are shown. The
and
subunits were immobilized to a CM5 sensor chip as described under
``Experimental Procedures.'' Solutions of
at the indicated concentrations in HKGM buffer were injected over
immobilized
. To completely dissociate bound protein, sensor chips
were regenerated with two 1-min pulses of 1 M urea, 0.1 M KNO
. These conditions allow for >95%
retention of the original binding activity to the immobilized
.
Control injections of each
concentration over an
underivatized flow cell were subtracted from the data to eliminate
contributions due to minor refractive index changes Data were analyzed
using the BIAEvaluation 1.0 and 2.0 software
packages.
In preliminary
BIAcore experiments, no high affinity interactions were
observed between the following pairs of proteins:
-
,
-
`,
-
,
-
`, or
`. With
or
coupled to the BIAcore chip, injection of either
or
` (up to 200 nM each) over the coupled DnaX subunit
resulted in a signal essentially equal to a control injection over a
blank chip. The same result was observed when either
or
`
was coupled to the BIAcore
chip and
or
(up to
400 nM each) was injected. Injection of
over immobilized
`, or
` over immobilized
also failed to show
significant interaction at analyte concentrations up to 2 µM (data not shown). These observations did not rule out possible
interactions between these subunit pairs at significantly higher
concentrations, but might indicate that the establishment of these
pairwise interactions is either kinetically slow or the resulting
equilibrium is sufficiently unstable that these interactions do not
represent central steps in the holoenzyme assembly pathway. Likewise,
(up to 400 nM) also did not appear to
interact with either
or
` when they were immobilized on
BIAcore
sensor chips (data not shown).
When equimolar
mixtures (200 nM) of +
or
+
were passed over a
`-bound chip, no binding was
observed (Fig. 7). The small signal observed was due to a
refractive index change caused by residual glycerol from the protein
storage buffer and is identical to the signal obtained from an
injection over a blank sensor chip. When a mixture of
and
was injected over the
` chip, complex formation occurred (Fig. 7). Since neither
or
alone binds, the binding
observed must represent a highly cooperative assembly of a
` complex. When
was
injected along with
and
, the rate and the extent of binding
was greater, indicating that
stimulated the rate of
binding of
` to
. Identical binding curves were
obtained when
was used in place of
or when
was
coupled to the BIAcore
chip and
` was used in the
mixture of analyte proteins (data not shown). Due to the complexity of
this associating system, no kinetic and equilibrium constants could be
obtained since the data could not be fit to the relatively simple
binding models available in the BIAcore
evaluation
software. Nevertheless, the qualitative conclusion that
functions to stabilize the interaction between
` and DnaX is consistent with the interpretation of the
functional experiments.
Figure 7:
increases the affinity
of
for
`. Sensorgram overlays of combinations of
,
, and
injected over immobilized
`
are shown.
` was immobilized to a CM5 sensor chip as described
under ``Experimental Procedures.'' All samples were injected
at equimolar concentrations (200 nM) in HKGM buffer. The
sensor chip was regenerated after each injection with a 15-s pulse of
10 mM NaOH. These conditions allow for >95% retention of
the original binding activity to the immobilized
`. Control
injections over an underivatized sensor chip were done but were not
subtracted from the data in order to illustrate the magnitude of the
refractive index changes due to the
injection.
and
were purified to homogeneity as a tightly
associated complex following overexpression of both subunits from a
vector containing an artificial holC-holD operon. We
pursued this strategy because of the insolubility of
when
overproduced individually. Having this material by itself permitted the
important demonstration that
binds to DnaX and bridges an
interaction with
(Xiao et al., 1993b), but more
detailed biophysical experiments required defined folded material so
that interactions could be studied without the complicating step of
protein folding in assembly reactions.
when
overexpressed as a complex constitutes 15% of the total cellular
protein and 11% of the total soluble protein. Together, ammonium
sulfate fractionation and Q-Sepharose chromatography yielded nearly
pure material. A trace 115-kDa contaminant and smaller molecular mass
polypeptides were removed upon SP-Sepharose chromatography. Sephacryl
S100 gel filtration chromatography provided only a marginal
purification, but ensured that the final material was free of
aggregates or unassembled subunits and permitted exchange into a
defined buffer. Purified fraction V
complex,
subjected to polyacrylamide gel electrophoresis, appeared as only two
bands of
15,100 and 16,600 Da even when 40 µg of protein was
loaded (Fig. 1, lane 5). Laser densitometry of a
Coomassie-stained gel containing 1-10 µg of
demonstrated 99% purity.
The molar extinction
coefficient of and
purified independently were calculated
based on the amino acid composition (Xiao et al., 1993a). The
sum is the calculated extinction coefficient for the
complex 53,200 M
. The actual native
extinction coefficient is 43,145 M
, a 20%
difference from the calculated molar extinction coefficient. Use of
this rigorously defined extinction coefficient will allow more
precision in future experiments.
Sedimentation velocity analysis
indicated an s of 2.6, a Stokes
radius of 28 Å, a native molecular mass of 31,400 daltons, and a
frictional coefficient of 1.3, data that are in reasonable agreement
with glycerol sedimentation of
(Xiao et
al., 1993b) and the sedimentation equilibrium data presented here.
The frictional coefficient of 1.3 suggests that
is
an asymmetric molecule.
Sedimentation equilibrium experiments were
conducted to examine the composition of in solution.
This technique is particularly powerful because at each position within
the boundary established, all components are at sedimentation and
chemical equilibrium. The shape of the curve at varying protein
concentrations permits a particularly sensitive way to detect multiple
molecular species in a mixture and to determine the equilibrium between
them.
sediments as a single ideal species with
native molecular mass of 31,755 Da, a difference of less than 1% from
the calculated molecular mass for a 1:1 heterodimer. Although it is
possible that
sediments independently as monomers,
or as a trimer or tetramer, these curve fits do not correlate with the
data generated by sedimentation equilibrium studies (Fig. 2).
Holoenzyme activity can be reconstituted without
under optimal levels of the other holoenzyme subunits. Thus, to develop
a functional assay, we needed to define a set of conditions that
provided a maximal stimulation by
. We reproduced the
findings of O'Donnell and colleagues that a modest
requirement exists at elevated salt concentrations
using reconstitution of
complex and holoenzyme as an assay (Xiao et al., 1993a), but we found that the resulting
-reconstituted holoenzyme did not exhibit the salt resistance
observed for native holoenzyme (Griep and McHenry, 1989). In a search
for conditions that permitted reconstitution of native holoenzyme at
high salt levels (400 mM glutamate), we found that both
and
are required. This result not only provided a
convenient linear assay for
, but also suggested that
a
interaction occurs within native
holoenzyme, providing additional evidence for our model that
plays a central role as a clamp loader in holoenzyme (Dallmann et
al., 1995; Dallmann and McHenry, 1995).
We examined the
influence of on the binding of
` to
DnaX in the BIAcore
. This instrument permits real-time
direct monitoring of the binding of protein in the flow phase to a
protein immobilized on a chip. By monitoring binding of
to
and
, we determined a K
of
2 nM. That the K
was
roughly equivalent for
and
suggests that the site for
interaction is entirely within the amino-terminal
domain of
. The estimated K
is
consistent with the requirement for
1 nM
in our functional assays.
Analyses using the
BIAcore to monitor DnaX complex formation supported our
model of
function.
` bound to DnaX
to a greater extent and more rapidly in the presence of
than in its absence. Binding on the BIAcore
is directly proportional to a change in the amount of mass bound
to the chip. Because
constitutes 8% of the mass of
the
complex (Dallmann and McHenry, 1995), the 2-fold increase in
binding is not due solely to the mass contribution by
.
The DnaX complex functions to load the
sliding clamp onto DNA. A defined position with the initiation complex
that results when pol III core assembles also suggests an elongation
role and
-DnaX contacts (Reems et al., 1995). The
function of
,
`,
, and
subunits within the
complex remains poorly understood. From the results of this study, we
propose that
, though not required for DNA synthesis per se, is an important component for proper holoenzyme
function in vivo. High concentrations of
` can
overcome most of the
requirement in vitro,
but concentrations of
200 nM are required to saturate
DnaX under our standard assay conditions (100 mM glutamate).
To date, only the
subunit is known to be present in excess. Other
components of the complex, including
and the DnaX subunits with
which
` interacts, are present at
20 copies per
cell, which corresponds to
28 nM.
This level
of
` would permit sequestration of DnaX in a complex in
the presence of
. In the absence of
, only a fraction of the maximal amount of complex
would be formed. The ability of
to alter the amount
of functional clamp loader present in the cell could also enable
to serve a regulatory role or as a modulator of the
holoenzyme assembly pathway.