From the
The
DNA polymerase III holoenzyme (holoenzyme) is tethered to DNA by
the ring-shaped
The holoenzyme has both
Whether the clamp loader within the holoenzyme is the
Wild-type
The effect of core on the binding kinetics of
Are these ATP-binding site mutants
inactive in loading
In the
assembly scheme shown in Fig. 2A, the
These four forms of Pol
III* were assayed in replication assays using
The ATP-binding site mutant
Consistent with this
notion, the addition of all the subunits of Pol III* except
If
Note Added in Proof-Neither the
The apparent kinetic constants of
complex (
`
) and
complex (
`
) clamp loaders require ATP
hydrolysis to load
sliding clamps onto DNA. The
sliding
clamp tethers the polymerase (Pol) III* replicase to DNA for processive
synthesis. Pol III* contains both
and
, but only one each of
the
,
`,
, and
subunits. Hence, there is
ambiguity with respect to which clamp loader, the
or
complex, exists in the Pol III* replicase structure. In this study,
ATP-binding site mutants of
and
have been prepared, and
these mutants, when assembled into either the
or
complex,
are inactive in clamp loading. These mutants have been used as a tool
to determine the identity of the clamp loader in Pol III*. The
nine-subunit Pol III* has been assembled using either mutant
or
in place of wild-type
or
. The results show that
mutation of
inactivates Pol III* activity, but mutation of
does not, indicating that the
complex (and not the
complex)
is the clamp loader of Pol III*. The
subunit carries the task of
dimerizing the core polymerase, and it is this association of
with core that appears to direct the single copy subunits away from
and onto
.
clamp for high processivity while replicating the
chromosome
(1, 2) . The
clamp is assembled onto DNA
by a clamp loading activity inherent in the holoenzyme
structure
(3, 4, 5) . The first report of this
series showed that a clamp loader can be assembled from the
,
`,
, and
subunits and from a dimer of either
or
to produce a
complex
(
`
)
or a
complex
(
`
),
respectively
(6) . Use of either
or
in forming a
clamp loader complex with the
,
`,
, and
subunits is not surprising as the
and
subunits are encoded
by the same gene
(7, 8, 9) . The
subunit is
produced by a translational frameshift such that
(47 kDa) is the
N-terminal 430 amino acids of
(71 kDa) plus one unique C-terminal
residue of glutamic acid. Hence, since both
and
bind the
,
`,
, and
subunits, their binding sites must be
located within the N-terminal two-thirds of
. Unique to the
subunit is the ability to bind tightly to the core
polymerase
(10, 11) . In the holoenzyme,
is a
dimer, and it dimerizes core, presumably for coordinated replication of
the leading and lagging strands of a duplex
chromosome
(12, 13) . The C-terminal sequence unique to
is essential for the strong interaction between
and core,
and thus, the core-binding site is probably located within the
C-terminal third of
(11) .
and
subunits and therefore may contain both clamp
loaders
(14) . However, structural studies in the third report of
this series showed that polymerase (Pol)
(
)
III*
contains only one copy of the
,
`,
, and
subunits, and therefore, it can have either a
or
complex,
but not both
(13) . Assembly studies showed that
` had to be
on either
or
for Pol III* assembly to occur, but if
`
was present on both
and
, Pol III* assembly was
prevented
(13) . These results further support the presence of
only one clamp loader in the holoenzyme. However, since both
and
are present in the holoenzyme, the exact location of the single
copy subunits, whether they are on
or
, remains ambiguous.
or
complex is the subject of this report. It has been presumed that the
subunit is associated with the
,
`,
, and
subunits in the holoenzyme because a
complex can be purified from
Escherichia coli cell lysates
(15) . Furthermore, a Pol
III` assembly, consisting of two core polymerases bound to a dimer of
, can also be purified from cell lysates, but it does not contain
the
,
`,
, or
subunit
(10) . These
observations suggest that the holoenzyme contains the
complex,
not the
complex. If the
,
`,
, and
subunits are located on either
or
, then there must exist a
mechanism of selectivity that targets them to one subunit rather than
another. In this study, we have used
and
subunits that
carry one amino acid replacement in the ATP-binding site that
inactivates their clamp loading activity. These mutants are used in
combination with wild-type
and
to assemble Pol III*
containing either mutant
or
(or both) as a means to
establishing the location of
,
`,
, and
within
Pol III*.
Materials and Methods
All sources and procedures
not described here are described in the first report of this
series
(6) .
Construction of the
The dnaX gene was amplified by the polymerase
chain reaction from pZT3
(7) using as upstream primer,
5`-CCCTCTAGAAGGAGATATAAATATGAGTTA-3`, hybridized over the initiating
ATG of dnaX, and as downstream primer,
5`-ACTGGTGGATCCTCAAATGGGGCGGATACT-3`, hybridized over the termination
codon of dnaX. Amplification was for 30 cycles using
TaqI polymerase in the following sequence: 1 min at 94 °C,
2 min at 60 °C, and 2 min at 72 °C, according to the
manufacturer's instructions (Perkin-Elmer). The amplified
1.9-kilobase product was purified by phenol extraction in 2% SDS
followed by digestion with XbaI and BamHI (cleavage
sites underlined in the upstream and downstream primers, respectively)
and ligated into pET-3c, cut with the same restriction enzymes, to
yield pET3dnaX. Sequence analysis of the entire dnaX gene confirmed that no errors had been introduced during
polymerase chain reaction amplification. The
XbaI/BamHI fragment containing dnaX was
excised from pET3dnaX and subcloned into M13mp18 (digested
with XbaI and BamHI), and oligonucleotide-directed
mutagenesis
(16) was performed at the ATP-binding site. The
mutagenic oligonucleotide (5`-CCCGTGGCGTCGGAGCGACCTCTATCGCCC-3`)
contained a 3-base pair mismatch (underlined) to produce a lysine to
alanine replacement at amino acid 51 of the and
ATP-binding Site
Mutants
and
proteins.
Mutation of the dnaX gene was confirmed by sequence analysis
and then excised from M13 by XbaI/BamHI digestion and
subcloned into pET-11a (digested with XbaI and BamHI)
to yield pET11dnaX(Ala).
Purification of
Eight liters of
BL21(DE3) pET11dnaX(Ala) cells were grown at 37 °C in LB
medium containing 100 µg/ml ampicillin. Upon growth to an
A and
of 0.6,
isopropyl-1-thio-
-D-galactopyranoside was added to 0.4
mM. After 3 h, the cells (32 g) were collected by
centrifugation; resuspended in 32 ml of ice-cold 50 mM
Tris-HCl (pH 7.5), 10% sucrose; and frozen at -70 °C. The
subsequent purification steps were performed at 4 °C, and the
mutant
and
proteins were followed on an SDS-polyacrylamide
gel. The cells were lysed; the cell debris was pelleted; and the
soluble lysate (2.2 g of protein) was fractionated using ammonium
sulfate as described
(17) . The ammonium sulfate pellet was
dissolved in buffer A (275 mg of protein in 50 ml), dialyzed against
buffer A to a conductivity equal to 40 mM NaCl, loaded onto a
60-ml Q-Sepharose Fast Flow column equilibrated with buffer A, and
eluted with a 600-ml linear gradient of 0-0.5 M NaCl in
buffer A. Seventy-two fractions were collected. Fractions 41-49
containing
and
(232 mg in 75 ml) were pooled, dialyzed
against buffer A to a conductivity equal to 20 mM NaCl, loaded
onto a 60-ml heparin-Affi-Gel column equilibrated with buffer A, and
then eluted with a 600-ml linear gradient of 0-0.5 M
NaCl in buffer A. Seventy-two fractions were collected, and fractions
18-30 containing
and fractions 40-50 containing
were pooled separately and dialyzed against buffer A to a conductivity
equal to 28 mM NaCl.
(180 mg in 108 ml) and
(50 mg
in 92 ml) were passed through separate 16-ml N-6-linked
ATP-agarose columns (Sigma) equilibrated with buffer A. Mutant
and
flow through this column, but wild-type
and
bind
tightly. The mutant
(162 mg) and
(48 mg) preparations were
aliquoted and stored at -70 °C.
and
were purified from 18 liters of BL21(DE3) pLysS pET3dnaX cell
culture as described for mutant
and
, except they bind the
ATP-agarose column. The ATP-agarose columns were eluted using a 160-ml
linear gradient of 0-2 M NaCl in buffer A; fractions
containing
and
were pooled and dialyzed against buffer A;
and then
(230 mg) and
(44 mg) were aliquoted and stored at
-70 °C.
ATPase Assays
The complex was constituted
from its five subunits (
,
`,
,
, and either
wild-type or mutant
) and then purified from excess proteins as
described in the first report of this series
(6) . ATPase assays
were performed in 20 µl of 20 mM Tris-HCl (pH 7.5), 8
mM MgCl
, 11 µg of M13mp18 ssDNA, 1 mM
[
-
P]ATP, and one of the following: 0.64
µg of wild-type
complex, 3.84 µg of mutant
complex,
0.65 µg of wild-type
, or 3.84 µg of mutant
. The
reaction was incubated at 37 °C, and aliquots of the reaction were
quenched with 25 mM EDTA (final concentration) at different
time points from 2 min to 3 h. The aliquots were analyzed by spotting
them (0.5 µl each) onto a TLC sheet coated with
polyethyleneimine-cellulose MN300 (Brinkmann Instruments). TLC sheets
were developed in 0.5 M lithium chloride, 1 M formic
acid. Autoradiograms of TLC sheets were used to visualize P
at the solvent front and ATP near the origin, which were then cut
from the TLC sheets and quantitated by liquid scintillation counting.
The extent of ATP hydrolyzed was plotted, and the initial rate of
hydrolysis was used to calculate the moles of P
released
per mole of protein/minute. One mole of
assumed the mass of a
dimer, and 1 mol of
complex assumed a stoichiometry of
`
.
Replication Assays
The complex was
constituted upon mixing 7.8 µg of
(83 pmol as dimer), 7.0
µg of
(181 pmol), 4.8 µg of
` (130 pmol), 3.0 µg
of
(181 pmol), and 2.5 µg of
(164 pmol) in 50 µl
of buffer A and incubated at 15 °C for 30 min. The
complex
was constituted as the
complex, except 11.0 µg of
(77
pmol as dimer) was added in place of
. The assay consisted of 66
ng of M13mp18 ssDNA (27.5 fmol as circles) uniquely primed with a DNA
30-mer
(1) and coated with 800 ng of SSB, 30 ng of
(0.37
pmol as dimer), 55 ng of core (0.35 pmol), and different amounts of
constituted
or
complex in a final volume of 25 µl of 20
mM Tris-HCl (pH 7.5), 8 mM MgCl
, 5
mM dithiothreitol, 4% glycerol, 40 µg/ml bovine serum
albumin, and 60 µM each dCTP and dGTP. The reaction was
incubated at 37 °C for 5 min, and then 60 µM dATP and
20 µM [
-
P]TTP were added to
initiate a 30-s pulse of replication before quenching the reaction by
spotting onto DE81 paper and quantitating the amount of DNA synthesis
as described
(6) . Replication assays of Pol III* were performed
as described above, except instead of adding core and the
complex, the constituted forms of Pol III* were titrated into the
assay, and 40 mM NaCl was included in the reaction.
Constitution of Pol III*
Pol III* containing 1)
wild-type and
, 2) wild-type
and mutant
, or 3)
mutant
and wild-type
was constituted as follows (all
incubations were in buffer A at 15 °C for 30 min). The
complex was formed upon incubating 174 µg of
and 107 µg
of
in 467 µl (urea from the
preparation was at a
final concentration of 0.1 M);
` was formed upon
incubating 610 µg of
and 260 µg of
` in 573 µl;
and core was formed upon incubating 455 µg of
, 145 µg of
, and 67 µg of
in 723 µl. Either wild-type or
mutant
(247 µg) and either wild-type or mutant
(124
µg) were added to core and incubated for an additional 60 min at 15
°C. In a separate tube, the
complex was mixed with
` and incubated for an additional 30 min. Then the mixtures
of
` and of
were
concentrated by spin dialysis using a Centricon-30 apparatus to a
combined volume of <200 µl. The two mixtures were then combined
and incubated at 15 °C for 5 min before injection onto a Superose 6
gel filtration column developed as described in the first report of
this series
(6) . Column fractions were analyzed by 15%
SDS-polyacrylamide gel electrophoresis, and fractions 16-22
containing Pol III* were pooled. Protein concentration was measured as
described in the first report of this series
(6) , and Pol III*
was aliquoted and stored at -70 °C. The Pol III* containing
ATP-binding site mutants of both
and
was constituted and
purified by Superose 6 gel filtration column using Method 1 of the
third report of this series
(13) ; the same amounts of protein
subunits were used as described above.
Surface Plasmon Resonance
Surface plasmon
resonance (SPR) was performed as described in the first report of this
series
(6) . The final levels of immobilized ` and
complex were 3325 and 2475 response units, respectively
(for
,
was 2095 response units, and the addition of
resulted in another 380 response units). A 30-µl solution of
1 µM
or
(both as dimer) in SPR buffer was
injected over the immobilized
` or
complex at a flow
rate of 15 µl/min. Then a 30-µl solution of SPR buffer was
injected at a flow rate of 15 µl/min. The surface of the
`
chip was regenerated by injection of 20 µl of 0.1 M
glycine (pH 9.5). The surface of the
chip was regenerated
by consecutive injection of 20 µl of 6 M urea and 10
µl of 2 M urea in SPR buffer. The
subunit was
reloaded onto immobilized
each time after regeneration; the
final response unit obtained was highly reproducible (±20
response units).
was determined by forming the core
-
complex (1 µM
(as dimer) and 3 µM
core (as
) were
incubated in 30 µl of SPR buffer for 30 min at 15 °C) prior to
injections. Controls for the effect of core on
were performed by
preincubating 3 µM core with 1 µM
(as
dimer) as described above for
. Response signals were normalized
to the molecular weight of the protein in the mobile phase:
, 94
10
;
, 142
10
; and core
-
, 472.2
10
. Apparent association and dissociation rates
were determined using the nonlinear curve fitting Pharmacia Biosensor
BIAevaluation 2.0 software. The sections of the kinetic traces used to
determine the rates were those that yielded a low
value (<2) and typically covered a range of 50-80 s.
ATP-binding Site Mutants of
Consistent with the known ability of and
and
to bind equally well to ATP, the sequence of the dnaX gene has a match with the consensus sequence of an ATP-binding
site
(18) in the region encoding both
and
(Fig. 1A). For several proteins with this consensus
sequence, the Lys residue has been shown to be important for function
with ATP (see Refs. 19-22). Hence, we replaced this Lys codon in
dnaX with an Ala codon, and then mutant
and
were
both expressed using the pET system and purified.
Figure 1:
ATP-binding site mutants of
and
are inactive. A, the lysine residue (position
51 in
and
) in the consensus sequence of the ATP-binding
site was mutated to alanine. The conserved residues of the site are
underlined. B, shown are ATPase assays of the
complex constituted using either mutant (mut) or wild-type
(wt)
. ATPase assays of mutant and wild-type
are
also shown. The DNA effector in the assay is M13mp18 ssDNA. C,
and
complexes were constituted using either wild-type or
mutant
and
subunits and then assayed for the ability to
assemble
onto DNA as determined by the ability to confer onto
core the ability to replicate a singly primed SSB-coated M13mp18 ssDNA
template in 30 s. Clamp loader complexes were constituted using
,
`,
,
, and
or
(either wild-type (
) or
mutant (
)).
In
Fig. 1B, the mutant proteins were tested for
DNA-dependent ATPase activity using M13mp18 ssDNA as effector. The
subunit is a known DNA-dependent ATPase (23-25), and as
expected, mutant
was inactive. The
subunit has very low
DNA-dependent ATPase activity alone; it requires the
and
`
subunits for significant activity and is most active as a complex of
all five subunits
(25) . The
complex was constituted from
its five subunits using either wild-type or mutant
, and then it
was assayed for DNA-dependent ATPase activity. The results show that
the
complex constituted using wild-type
is active, but the
complex constituted using mutant
is inactive. In the event
that mutant
and
bind ATP weakly but have the same maximal
velocity of hydrolysis, we have repeated these assays over a large
range of ATP concentrations, but little if any activity was
observed.
(
)
Hence, the replacement of Lys with
Ala was effective in blocking ATP hydrolysis of the
and
complexes. The
subunit stimulates the DNA-dependent ATPase
activity of the
complex
(25) , but the addition of the
subunit did not provide the mutant
or
complex with
ATPase activity (data not shown).
clamps onto DNA? We tested this by
investigating whether clamp loaders, constituted using
,
`,
,
, and either mutant
or mutant
, could clamp
the
subunit to DNA for processive synthesis with the core
polymerase on singly primed M13mp18 ssDNA coated with SSB
(Fig. 1C). In this assay, the core polymerase does not
give a detectable signal unless the
clamp is assembled onto
DNA
(1, 5) . The results show that use of wild-type
or
gives nearly equal and complete synthesis of the DNA template.
However, use of mutant
or
gives no detectable replication.
Hence, the mutant
and
complexes are inactive in assembling
clamps onto primed DNA, a result consistent with their loss of
ATPase activity. Furthermore, we have directly followed
clamp
assembly onto DNA using [
H]
and found that
the mutant
complex cannot assemble [
H]
onto DNA (data not shown), consistent with conclusions drawn from its
inactivity in replication assays. The absence of ATPase and replicative
activities is not due to a loss in native conformation of these mutant
proteins as they are fully capable of assembling into multiprotein
complexes (i.e. we have constituted the
complex
(e.g. used in Fig. 1B) and Pol III* from them
(e.g. as in Fig. 2)).
Figure 2:
Pol III* constituted using mutant is
active, but using mutant
is inactive. A, scheme for
assembly of Pol III* from individual subunits; B, Coomassie
Blue-stained 15% SDS-polyacrylamide gel analysis of the four forms of
Pol III* constituted using wild-type
and
(lane1), wild-type
and mutant
(lane2), mutant
and wild-type
(lane3), and mutant
and
(lane4); C, replication activity assays of four
different forms of Pol III*.
, wild-type
and
;
, wild-type
and mutant
;
, mutant
and
wild-type
;
, mutant
and
.
Constitution of Pol III* Using Mutant
The mutant and/or
and
subunits were used along
with wild-type proteins to constitute the nine-subunit Pol III*
containing mutant
, mutant
, or both mutant
and
.
If the single copy subunits (
,
`,
, and
)
associate randomly with either
or
, then two populations of
Pol III* may form, one with these subunits on
(i.e. a
complex) and one with these subunits on
(i.e. a
complex). In this event, use of one mutant subunit (either
or
) should inactivate Pol III* by 50%. If the
,
`,
, and
subunits associate preferentially with one subunit
(for example,
), then use of mutant
would result in complete
inactivation of Pol III*, but use of mutant
would not inactivate
Pol III*. In either case, double mutant Pol III* is expected to be
inactive since association of the single copy subunits with either
or
would not result in a productive clamp loader.
and
subunits were mixed with the subunits of core and incubated, and then
the
,
`,
, and
subunits were added. After a
further incubation, Pol III* was separated from excess proteins and
free subassemblies by gel filtration. Fig. 2B shows a
Coomassie Blue-stained 15% SDS-polyacrylamide gel analysis of four
forms of Pol III* prepared using wild-type
and
(lane1), wild-type
and mutant
(lane2), mutant
and wild-type
(lane3), and mutant
and
(lane4). Densitometry analysis shows that all four forms of
Pol III* have a similar subunit stoichiometry (within 15%) consistent
with the subunit stoichiometry of Pol III* shown in the first report of
this series
(
`
)
(6). Hence, mutant
and
are efficiently assembled into the
Pol III* structure, and use of ATP-binding site mutants of
and
does not affect the assembly process.
and singly primed
M13mp18 ssDNA coated with SSB (Fig. 2C). Double mutant
Pol III* (mutant
and
) was inactive in the assay as
expected. Of the two forms of single mutant Pol III*, one appeared as
active as Pol III* constituted using all wild-type subunits, and the
other was inactive, suggesting nonrandom assortment of the
,
`,
, and
subunits. The active single mutant Pol III*
was the one containing mutant
; the inactive Pol III* was the one
constituted using mutant
. Hence, it appears that the single copy
subunits prefer to associate with
over
.
Speed of Association of
Both ` and
with
and
and
associate with the
,
`,
, and
subunits to form active clamp loader
complexes
(26) . Then why in Pol III* do these subunits seem to
preferentially associate with
? Perhaps they are more stabile on
, and thus even if they associate randomly with either
or
, they eventually redistribute to their most stabile occupant.
Alternatively, they may associate with
more rapidly than with
. In Fig. 3, the surface plasmon resonance technique was
used to measure the association and dissociation kinetics of these
subunits with
and
. The first report of this series showed
that of the
,
`,
, and
subunits, only the
`
and
subunits show significant interaction with
and
(6) . Hence, we measured the rates of association of
and
with the
` subunit and with the
complex
(
was used since
by itself is insoluble). The
complex and
` were immobilized on separate sensor
chips, and then solutions of either
or
were passed across
the surface in the mobile phase. The results show that
and
have similar kinetics of association with the immobilized subunits.
Furthermore,
and
also have similar kinetics of dissociation
from the immobilized
complex and from the
` subunit.
The observed values of k
and k
and the K
values calculated from
them are shown in . Hence, the binding kinetics did not
reveal a significant difference in the interaction of
and
with these subunits.
Figure 3:
Effect
of the core polymerase on the binding kinetics of ` and
with
and
. The influence of core on the
kinetics of interaction between either
or
with
` and
was examined by SPR analysis. A, the
complex was immobilized on the sensor chip, and 1 µM
solutions (as dimer) of either
(rightpanel) or
(leftpanel) were passed over the immobilized
complex. B, the
` subunit was immobilized on
the sensor chip, and 1 µM solutions (as dimer) of either
(rightpanel) or
(leftpanel) were passed over the immobilized
` subunit.
In both A and B, the binding kinetics of core-
and of a mixture of
and core are also shown. Openarrowheads denote the start of protein injection, and
closedarrowheads mark the start of the buffer wash.
RU, response units
The subunit is known to bind tightly to
the
subunit of the core polymerase, whereas the
subunit
does not bind
(or core)
(11) . Perhaps the addition of a
bulky group, such as core, may affect the binding kinetics of
with
` and the
complex. This was tested using SPR by
forming the core
-
complex prior to passing
it over the immobilized
complex or
` subunit
(Fig. 3). The results show that the apparent dissociation
constant is elevated
5-fold for
and 10-fold for
`. As a control, core was mixed with
and passed over the
immobilized
complex and
` subunit. The results show
that core has little influence on the binding kinetics of
with
these subunits, as expected from the lack of interaction between
and core.
The
The Subunit Is the Site of ATP Action in the
Complex Matchmaker
complex couples ATP
hydrolysis to the assembly of a
clamp onto DNA. It has been
assumed in the past that
was the site of ATP action since
is known to bind ATP
(24) , and the amino acid sequence of
contains a consensus ATP-binding site sequence. However, the
subunit has also been reported to bind ATP (27), and within the
sequence, there is a close match with an ATP-binding
site
(28) .
(
)
The
complex,
constituted using the
mutant, was inactive in ATPase activity and
in replication assays, thus confirming that
is the subunit of the
complex that couples ATP hydrolysis to the clamp loading task. We
have also constructed a
mutant in which the Lys residue of the
putative ATP-binding site is replaced with Ala. The
complex,
constituted using the
mutant, was as active as the wild-type
complex in ATPase assays and in replication assays.
(
)
Hence, if
interacts with ATP, the interaction is not
needed in the
assembly reaction (or the putative site was not
correctly identified).
The
The Complex Versus
Complex in Pol
III*
subunit contains the amino acid sequence of
and an extra 213 residues at the C terminus. In fact, a five-subunit
``
complex''
(
`
)
can be constituted that is within 2-fold of the activity of the
complex
(6) . However, it is important to note that a
complex has yet to be purified from E. coli cells.
Furthermore, the
,
`,
, and
subunits are not
present in Pol III` (core
-
)
(10) .
The third report of this series demonstrates that only one each of the
,
`,
, and
subunits assembles into Pol III*, and
therefore, these subunits must reside on either
or
, but not both
(13) .
and
subunits were used to identify which subunit,
or
, is harnessed by the
,
`,
, and
subunits
to form the clamp loader in Pol III*. Only Pol III* constituted using
mutant
was active; mutation of
led to inactive enzyme.
Hence, the
complex appears to be the clamp loader in Pol III*.
Consistent with this result, SPR measurements show that the
and
subunits have similar binding kinetics with
` and
, but interaction of
with core significantly
decreases the efficiency of
binding to both
` and
. Presumably, core partially blocks the binding sites on
for
` and
(Fig. 4). Alternatively, core causes
a conformational change in
such that interaction with
` and
is weakened.
Figure 4:
Core hinders the binding of ` and
to
. The two core polymerases on
may sterically
prevent access of
` and
to their respective binding sites
on
(as shown in the figure). Alternatively, interaction of core
with
may result in a conformational change that reduces the
affinity of
` and
for
(negative
cooperativity).
What is the function of the ATP-binding
site of ? The
subunit is a DNA-dependent ATPase, but the
function of this ATPase is still not clear. The results of this study
suggest that its use will be downstream of
clamp assembly.
Perhaps it is needed during elongation or for interaction with other
replication proteins such as the helicase or priming apparatus.
Does
It seems strange that the holoenzyme contains
two proteins of similar structure and function in one macromolecular
particle. Furthermore, these proteins carry out very different
functions, one being recruited as a clamp loader and the other as a
glue protein that not only holds two core polymerases together, but
also holds the Function as a ``Glue
Protein''?
complex to the holoenzyme structure. One
hypothesis regarding the origin of this arrangement is that
was
the first to evolve and that a
tetramer carried out all these
tasks, one dimer to cross-link two cores and another dimer to function
with
,
`,
, and
as a clamp loader, with both
dimers being held to each other by an oligomerization domain
(still present on both
and
).
results in a ``
-less Pol III*'' that is as active as Pol
III* containing
(29) . In fact, E. coli survives
the genetic knockout of
(by mutating the frameshift site in
dnaX), suggesting the
-less Pol III* can replicate the
E. coli chromosome
(29) . Study of the
-less Pol
III* indicates that it has a tetramer of
and at least two core
polymerases and that the
,
`,
, and
subunits are
present in single copy
(13) .
can substitute for
in replication of the chromosome, then why has E. coli evolved a translational frameshift to produce
(or if
evolved before
, then why has not evolution removed
)? One
possibility is that the arrangement of both
and
in the same
structure carries an advantage that has not been detected yet.
Alternatively, the
complex may be recruited as a stabile entity
outside of the holoenzyme structure for use in other areas of DNA
metabolism such as repair or recombination. Production of only
would likely impose the use of the core polymerase with
since the
core and clamp loading activity would be physically linked. Production
of
would provide a means of separating the clamp loading activity
from the core polymerase, thus freeing it to load
clamps for use
with other enzymes.
complex nor
complex could load
onto primed DNA using
ATP
S in place of ATP. However, in replication assays containing
core, ATP
S supported the activity of the
and
complexes
to 24 and 28%, respectively, relative to the level of DNA synthesis
observed using ATP (dAMP-PNP was used in place of dATP).
Table:
Effect of core on interaction of with
` and
and
interaction with
` and
in the presence or
absence of the core polymerase are listed below. The values of
k
and k
were the average
of five independent experiments. The apparent K
was calculated from the apparent rates
(k
/k
). Standard errors are
shown in parentheses.
S, adenosine
5`-O-(thiotriphosphate); AMP-PNP, adenyl-5`-yl
imidodiphosphate.
and
for ATP, as measured using equilibrium gel filtration,
was
1 mM. We have also replaced the Lys residue in the
ATP-binding site with Arg and have purified and studied these Arg
mutants of
and
. They have nearly undetectable ATPase
activity, but in equilibrium gel filtration, they still bind ATP with a
K of
63 µM.
that most closely resembles an ATP-binding site
consensus sequence is AX
GKS at residues
220-226. The ATP-binding site consensus sequences are
G/AX
GKT/S, G/AXGKT/S, and
G/AX
GXGKT/S (18).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.