From the
Several different subassemblies of DNA polymerase III holoenzyme
can be purified from Escherichia coli. Toward the goal of
understanding the functional significance of these subassemblies, we
have used the
DNA polymerase III holoenzyme (holoenzyme) is the replicative
polymerase of the Escherichia coli chromosome (reviewed in
Refs. 1 and 2). Besides the holoenzyme, three different polymerase
subassemblies can be purified from cells. 1) The three-subunit core
polymerase is a 1:1:1 complex of
Besides its
role as a replicase, Pol III also functions in other areas of DNA
metabolism
(1) . Perhaps the different polymerase subassemblies
serve distinct functions in DNA metabolism. Consistent with this is the
curious observation that Pol III* appears to be composed of one Pol
III` and one
Toward the goal of understanding the functional
significance of these subassemblies to replication and chromosome
maintenance, these subassemblies have been used to assemble processive
polymerases onto DNA using the
The results show that use of
different polymerase subassemblies results in different processive
polymerase structures attached to a
A derivative of
[
[
[
The [
Three preparations of Pol III* were constituted differing only in
which subunit was labeled. Pol III* was assembled in stages essentially
as described in the third report of this series
(8) . The
subunits were added in the following molar ratios before purification
(relative to
[
[
[
The
Pol III* and the
Prior to applying the reactions of
Fig. 2
onto the gel filtration column, a sample of each reaction
was analyzed for ability of these polymerases to fill the ssDNA gap
upon initiating DNA synthesis. The agarose gel analysis
(Fig. 2D) shows conversion of the gapped DNA to the
slower migrating replicative form II. Core
In the gel
filtration experiments of Fig. 2, the polymerase is in molar
excess over DNA, and therefore, even with the
core
In these experiments, Pol
III* contains only one molecule of
Multiple
Prior to this work, it had been shown that the
The
minimal processive polymerase, shown in Fig. 8A,
contains one core polymerase attached to a
Our observations of core-
An earlier study showed that at least two distinct
polymerases, either lacking or containing
There
are
During discontinuous synthesis on the lagging strand, the holoenzyme
must be capable of rapidly cycling from the end of a completed Okazaki
fragment to a new primed site to extend the next fragment. We have
shown previously that Pol III* remains tightly associated with its
We are grateful to Dr. Vytautas Naktinis for
construction and purification of the
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
complex clamp loader and the
ring to assemble
each different polymerase onto DNA. Through use of radioactive labeled
proteins, the subunit structure of each resulting processive polymerase
has been determined. Use of DNA polymerase III core, the
complex,
and
results in a core-
complex on DNA; the
complex is
not incorporated into the structure. The addition of
to the
assembly reaction to form either core
-
or
core
-
results in a more efficient
polymerase and more stabile association of core-
on DNA,
although the
complex still does not remain on DNA. The
complex clamp loader was retained on DNA with the other subunits only
if it was first assembled into the polymerase (Pol) III* structure. The
clamp loader within Pol III* appeared to be capable of loading two
clamps onto DNA for both core polymerases within Pol III*,
consistent with the hypothesis that one replicase can simultaneously
replicate both strands of a duplex chromosome. These findings extend
those of an earlier study showing that distinctive polymerases can be
assembled depending on the presence or absence of
(Maki, S., and
Kornberg, A.(1988) J. Biol. Chem. 263, 6561-6569). The
significance of these distinct polymerases in separate paths of DNA
metabolism is discussed.
(DNA polymerase),
(proofreading 3`-5`-exonuclease), and
. 2) The four-subunit
polymerase (Pol)
(
)
III` contains two molecules of
core polymerase attached to a dimer of the
subunit
(core
-
). 3) The Pol III* assembly consists
of nine different subunits
(
`
)
and lacks only the
sliding clamp. These polymerase subassemblies
do not have the rapid and processive character of the holoenzyme.
Generally, they incorporate residues at a speed of 10-20
nucleotides/s, and their processivity ranges from 10 to 50
residues/binding event
(3, 4) . However, when combined
with the
sliding clamp, each of these polymerases becomes rapid
(750 nucleotides/s) and processive
(1, 2) . Assembly of
the ring-shaped
clamp
(5, 6) onto DNA requires the
complex
(
`
),
which couples ATP hydrolysis to load
clamps onto
DNA
(1, 2) . The
sliding clamp confers a high
degree of processivity onto the holoenzyme by tethering it to the
primed template for continuous DNA synthesis
(5) .
complex
(7) , yet Pol III` and the
complex do not associate to form Pol III* unless a specific order of
addition is followed
(8) . If these subassemblies are formed
separately within the cell, then this order of addition requirement may
serve the purpose of providing the cell with a stabile quantity of each
of these assemblies for action in other areas of DNA metabolism such as
repair, recombination, and mutagenesis. Alternatively, Pol III` and the
complex may associate with each other in the presence of primed
DNA and
. For example, proximity at a primed site may lead to
their association. Yet another possibility is that the association of
Pol III` with the
complex may be facilitated in vivo by
a chaperonin.
complex clamp loader and
clamp, and each resulting polymerase on DNA has been analyzed for its
structural composition and activity. These subassemblies were studied
earlier using small amounts of isolated core, the
complex, and
overproduced
and
subunits with the finding that distinctive
polymerases (+
) could be assembled onto a primed
template
(9) . Now that each subunit is available in quantity and
the subassemblies can be constituted from them, we have tagged each
subassembly with a subunit of known specific radioactivity, providing
accurate stoichiometry measurements of each subassembly on DNA.
Depending on the relative amounts of core and
present in the
cell, one may expect either one core (limiting core) or two cores
(excess core) to associate with the
dimer
(10) . Hence, we
have constituted and studied both of these polymerase forms
(core
-
and
core
-
).
clamp on DNA. Furthermore,
comparison of the polymerase formed using Pol III* plus
to that
formed using Pol III` plus the
complex and
showed
polymerases of different compositions on DNA even though all 10
holoenzyme subunits were present in both reactions. If these several
different forms of DNA polymerase III also assemble onto primed DNA
sites in the cell, then it seems likely that they could be used in
different processes of DNA metabolism.
Materials and Methods
Materials, methods, and
sources not described here were described in the first report of this
series
(11) . A gapped circular DNA template was prepared by
nicking M13mp18 plasmid DNA (376 µg) at a specific site using 35
units of M13 gene protein II (a gift of Dr. Peter Model (The
Rockefeller University) and purified as described
(12) ) in a
20-min incubation at 37 °C in 3 ml of 40% sorbitol, 25 mM
Tris-HCl (pH 8.1), 6.7 mM MgCl, 6.7 mM
dithiothreitol, and 5 µg/ml BSA
(13) . This treatment results
in a single nick at the M13 origin in
60% of the DNA templates. A
gap was produced at the nick upon treatment with 1000 units of
exonuclease III for 1 min at 37 °C followed by phenol/chloroform
extraction and ethanol precipitation. The average size of the gap was
estimated to be 500 nucleotides from the amount of radioactive
nucleotide incorporated by the holoenzyme using
[
-
P]dTTP. Reaction buffer contained 5%
glycerol, 20 mM Tris-HCl (pH 7.5), 8 mM
MgCl
, 40 µg/ml BSA, 0.5 mM ATP, 50
µM dCTP, 50 µM dGTP, 0.1 mM EDTA,
and 5 mM dithiothreitol (ATP was omitted where indicated).
Radioactive Labeled Subunits
The ,
,
,
`, and
subunits were
H-labeled by
reductive methylation as described
(5, 15, 27) .
Briefly, 1 ml of protein at a concentration of 1-3 mg/ml was
incubated with 10 mM formaldehyde and 2 mM
NaB
H
(74 Ci/mmol) on ice for 15 min. The
H-labeled protein was separated from unspent reagents by
gel filtration in the fume hood on a 10-ml column of Sephadex G-25. The
excluded fractions were counted, pooled, and stored at -70
°C. The specific activities were as follows:
[
H]
, 15 cpm/fmol;
[
H]
, 29 cpm/fmol as dimer;
[
H]
, 29 cpm/fmol as dimer;
[
H]
`, 20 cpm/fmol as monomer; and
[
H]
, 67 cpm/fmol as dimer. The replication
activity of labeled protein relative to unlabeled protein was assessed
by comparative titrations in replication assays as follows
(14) .
[
H]
, assayed for replication of singly
primed SSB-coated M13mp18 ssDNA with Pol III*, was indistinguishable in
activity from unlabeled
. The activity of
[
H]
was determined by reconstituting
core
-
using either unlabeled
or
[
H]
and then comparing their replication
activity with
and the
complex on singly primed SSB-coated
ssDNA. Core
-
constituted using
[
H]
was within 2% of the activity of that
constituted using unlabeled
. The replication activity of
[
H]
was determined by constituting Pol III*
using either [
H]
or unlabeled
and then
testing their activity with
on singly primed SSB-coated circular
ssDNA. Pol III* constituted using [
H]
was
within 80% of the activity of that constituted using unlabeled
.
The activity of [
H]
` was within 10% of that
of unlabeled
` as determined by constituting the
complex
using either labeled or unlabeled
` and then comparing their
replication activity with
and core
-
on singly primed SSB-coated ssDNA. We initially tried labeling
core using [
H]
, but
[
H]
was only 40% as active as core
reconstituted using unlabeled
. Therefore, core was
H-labeled by reconstituting it using the
[
H]
subunit. The presence or absence of
has no effect on holoenzyme activity; however,
[
H]
was as effective as unlabeled
in
forming a tight nondissociable complex with
as determined by
comigration of
[
H]
subunits
during gel filtration analysis.
containing an
additional six amino acids at the C terminus
(NH
-RRASVP-COOH) serves as a substrate for phosphorylation
by cAMP-dependent protein kinase
(15, 27) . This
derivative was labeled using [
-
P]ATP and
protein kinase as described in the second report of this
series
(16) . Although this
derivative could be labeled to
very high specific activity, the typical specific activity of
[
P]
used in this report was low
(20-65 cpm/fmol) such that both
P and
H
could be assayed in the same sample by liquid scintillation.
Assembly of Multiprotein Complexes Using
Subassemblies containing
H-Labeled Subunits
H-labeled subunits were constituted by incubating purified
unlabeled subunits with one labeled subunit in buffer A at 15 °C
for 1 h, and then the resulting complex was purified from free subunits
by chromatography. Fractions from the chromatography columns were
analyzed on an SDS-polyacrylamide gel stained with Coomassie Blue.
Fractions containing the desired complex of proteins were pooled,
dialyzed against 1 liter of buffer A, aliquoted, and stored frozen at
-70 °C. All complexes used here retain full activity for over
1 year when stored in this manner. Specific activities of complexes
were determined by scintillation counting, and protein concentration
was measured using the protein assay from Bio-Rad with BSA as a
standard.
H]
was assembled
upon incubating 180 µg (2.5 nmol) of
[
H]
, 960 µg (7.5 nmol) of
, 510
µg (18 nmol) of
, and 294 µg (33 nmol) of
in 1.44
ml and then purified from free subunits by chromatography on a 1-ml
Mono Q column eluted with a 19-ml gradient of 0-0.5 M
NaCl in buffer A. The fractions containing
core
-[
H]
were
pooled, concentrated to 200 µl using a Centricon 30 apparatus, and
then gel-filtered on a 24-ml Superose 6 column (Pharmacia Biotech Inc.)
developed in buffer A containing 300 mM NaCl to remove any
remaining excess core (43.2 µg of final complex recovered).
Core
-[
H]
had a
specific activity of 30 cpm/fmol (474.2 kDa), consistent with the
specific activity of [
H]
.
H]Core
-
was
constituted by the same method as
core
-[
H]
(except
[
H]
and unlabeled
were used), and it
was purified on a Superose 6 column only. Its specific activity was 24
cpm/fmol, approximately half the specific activity of
, since
is only a monomer and two
subunits are present in the
[
H]core
-
complex.
H]Core
-
was
assembled by incubating 522 µg (3.75 nmol) of
, 132 µg (5
nmol) of
, 58.8 µg (7.5 nmol) of
[
H]
, and 801 µg (11.25 nmol) of
in 1.6 ml and then was concentrated using a Centricon 30 apparatus to
7.6 mg/ml and purified by Superose 6 chromatography as described above
(final amount of 67 µg).
[
H]Core
-
had a
specific activity of 18 cpm/fmol (304 kDa), consistent with the
presence of only one molecule of
/core
-
complex.
H]
complex
(
[
H]
`
) was assembled
by mixing 710 µg (9 nmol) of
complex
(reconstituted as described
(11, 17) ), 392 µg (11
nmol) of
, and 340 µg (9 nmol) of
` in 2.045 ml and then
was purified on a 1-ml Mono Q column using a 19-ml gradient of
0-0.5 M NaCl in buffer A. The fractions containing
`
were pooled (302 µg), dialyzed against
buffer A, and stored at -70 °C. The specific radioactivity of
the [
H]
complex was 20 cpm/fmol, consistent
with the presence of only one
` subunit within the complex.
):
, 3.0;
, 4.5;
, 7.0;
, 1.0;
, 3.0;
, 7.0;
`,
6.0;
, 7.0; and
, 6.0. Detailed procedures were as follows.
H]Pol III* labeled with
[
H]
was assembled upon incubating 352 µg
(3.7 nmol) of [
H]
with 178 µg (1.25
nmol) of
, 145 µg (8.73 nmol) of
, and 114 µg (7.5
nmol) of
in 789 µl. After 30 min, 328 µg (8.48 nmol) of
and 278 µg (7.53 nmol) of
` were added, bringing the
volume to 1394 µl, and then incubated for an additional 30 min.
During this time, a separate tube containing 200 µg (1.55 nmol) of
, 154 µg (5.6 nmol) of
, and 76 µg (8.84 nmol) of
was incubated for 30 min in 283 µl. Then the two tubes were
mixed and incubated for an additional 30 min. Pol III* was separated
from free subunits and subassemblies by chromatography on a 1-ml
heparin-agarose column eluted with a 15-ml gradient of 0-325
mM NaCl in buffer A. Fractions containing Pol III* were
pooled, dialyzed against buffer A, and then chromatographed on a 1-ml
Mono Q column eluted with a 30-ml gradient of 0-0.4 M
NaCl in buffer A. Fractions containing Pol III* were pooled, dialyzed
against buffer A, and stored at -70 °C. The specific activity
of [
H]Pol III* ([
H]
)
was 29 cpm/fmol, consistent with one dimer of
within Pol III*.
H]Pol III* labeled with
[
H]
was assembled as described above for
Pol III* labeled with [
H]
, and its specific
activity was 18 cpm/fmol, consistent with two molecules of
in
the Pol III* structure (final yield of 142 µg).
H]Pol III* labeled with
[
H]
was assembled as described for Pol III*
labeled with [
H]
, except that a pre-purified
complex was incubated with
[
H]
. A further difference was the use of
Superose 6 gel filtration in place of the Mono Q column, which removes
the small amount of
core
-([
H]
)
side
product of the assembly. Use of Superose 6 required concentration of
the sample after heparin-agarose chromatography to 70 µl using a
Centricon 30 apparatus (final yield of 23.8 µg).
Assembly of Subunits on DNA and Analysis by Gel
Filtration
In general, subunits and subassemblies were added to
DNA templates in 75 µl of reaction buffer on ice and then incubated
at 37 °C for 4 min to allow time for assembly onto primed DNA.
Reactions were then gel-filtered on 5-ml columns developed in column
buffer, and fractions of 200 µl were collected over a period of 30
min at 4 °C. Tritium and P were quantitated in the
same fraction by analyzing aliquots of 150 µl in 5 ml of Aquasol
(DuPont NEN) by liquid scintillation counting (
H window was
0-350;
P window was 400-1000). The specific
activities of [
P]
and of the
H-labeled subunits were comparable in all the experiments,
and therefore, bleed-over of one isotope into the window of another was
<1%. Reaction buffer contained 50 µM each dCTP and dGTP
in all experiments to prevent the 3`-5`-exonuclease activity of
the
subunit from digesting primed templates during assembly
reactions and on gel filtration columns. The individual volumes of
reactions and incubation times varied and are detailed in the figure
legends. The recovery of [
P]
was typically
75-90% using either Sephacryl S-400 or Bio-Gel A-15m. The
recovery of the [
H]
complex and complexes
containing [
H]
was only 50-70% on
Bio-Gel A-15m, but was 56-80% on Sephacryl. Therefore, after the
experiment of Fig. 2, subsequent gel filtrations were performed
with Sephacryl columns.
Figure 2:
stimulates the binding of core to a
sliding clamp.
[
P]
was assembled onto gapped M13mp18 DNA
using the
complex and ATP as described under ``Experimental
Procedures'' (see scheme at top) with the exception that 2.7 pmol
of DNA was present in the 382-µl reaction (instead of 1.8 pmol)
before dividing it into 87-µl aliquots. Then 1.77 pmol of
[
H]core (A),
[
H]core
-
(B), or
[
H]core
-
(C) was added and analyzed by gel filtration on agarose
A-15m as described under ``Experimental Procedures.'' Panels
to the right are controls that were performed just as those to the left
except ATP was omitted.
, [
P]
;
, the [
H]polymerase complex. In D,
aliquots of reactions (containing ATP) were replicated and analyzed on
a 0.6% native agarose gel as described under ``Experimental
Procedures.''
In experiments using
[P]
assembled onto gapped DNA by the
complex, the
complex (0.76 µg, 3.78 pmol) was preincubated
with [
P]
(0.76 µg, 3.78 pmol) and
gapped DNA (4.1 µg, 1.8 pmol) saturated with SSB (28.8 µg) in
382 µl of reaction buffer for 4 min at 37 °C. Then the reaction
was split into 85-µl aliquots in 1.5-ml Eppendorf tubes that
contained 15 µl of core (290 ng, 1.8 pmol),
core
-
(540 ng, 1.8 pmol), or
core
-
(840 ng, 1.8 pmol) in buffer A with
40 µg/ml BSA and incubated for an additional 2 min at 37 °C
prior to analysis of 75 µl by gel filtration.
Replication Reactions
Replication reactions
contained 130 ng (30 fmol) of gapped M13mp18 DNA, 0.32 of µg SSB,
22 ng (272 fmol) of , 6 ng (30 fmol) of
complex (omitted
when Pol III* was used), and one of the following: core,
core
-
, core
-
,
or Pol III* (amounts are indicated in the legend to Fig. 1) in
23.5 µl of reaction buffer. Reactions were shifted to 37 °C for
5 min to allow time for Polymerase assembly onto DNA, and then a 15-s
pulse of DNA synthesis was initiated upon the addition of 1.5 µl of
dATP and [
-
P]dTTP (final concentrations of
60 and 20 µM, respectively). Synthesis was quenched after
15 s upon spotting onto DE81 filter paper and quantitated as
described
(11) .
Figure 1:
Replication activity of four different
polymerases. Polymerase subassemblies were titrated into replication
reactions containing a clamp on gapped M13mp18 DNA as described
under ``Experimental Procedures.''
, Pol III*;
,
core
-
;
,
core
-
;
,
core.
In gel filtration experiments, polymerase-DNA
complexes were quantitated for DNA synthesis as follows. An 11-µl
aliquot of the reaction was removed, and a 1.5-µl aliquot of dATP
and [-
P]dTTP (final concentrations of 60
and 20 µM, respectively) was added to initiate DNA
synthesis. The reaction was shifted to 37 °C, and then after 15 s,
the reaction was quenched with 12.5 µl of 1% SDS and 40 mM
EDTA. The extent of replication in the reaction was analyzed by two
methods. First, a 10-µl aliquot was spotted onto DE81 paper, and
the amount of incorporated nucleotides was quantitated as
described
(11) . Second, a 10-µl aliquot was analyzed on a
0.6% native agarose gel, which separates unreplicated template from
replicative form II product. The DNA was visualized by UV-induced
fluorescence of ethidium bromide.
Experimental Strategy
Throughout this report,
the physical presence of protein on DNA was identified and quantitated
through use of radioactive subunits of known specific radioactivity.
H-Labeled protein and primed DNA were mixed, and then the
protein-DNA complexes were gel-filtered on a large pore molecular
sieving column. Protein bound to DNA comigrates with the large DNA
template in the excluded volume and resolves from unbound proteins in
the included volume. The molar amount of
H-labeled protein
bound to DNA was calculated from the known specific radioactivity.
There are 10 different proteins in the holoenzyme, and we have not
labeled each of them. Instead, we have labeled the
clamp, the
subunit, the
subunit of core, the
` subunit of the
complex, either the
or
subunit of Pol III`, and the
,
, or
subunit of Pol III*. Thus, some conclusions
drawn in this study rest on the assumption that these subassemblies
remain tightly associated through gel filtration. This assumption is
supported by the tight association between the subunits of these
H-labeled complexes through liquid chromatography resins
and gel filtration columns
(15, 18) and during their
preparation (see ``Experimental Procedures'').
dimer binds two core polymerases provided core is supplied in excess.
If core is limiting, then only one core is present on the
dimer
(10) . Therefore, we have prepared both forms for these
studies (core
-
and
core
-
). To follow the
clamp and
H-labeled proteins in the same experiment,
was
P-labeled by engineering a six-residue kinase recognition
motif onto the C terminus, followed by
P radiolabeling
with a protein kinase. This derivative of
is as active as
unmodified
(15, 27) .
complex interact nonspecifically with SSB-coated ssDNA (i.e. the interaction is independent of ATP,
, and a primed
site)
(5, 19) . The basis for this interaction is weak
binding between the
subunit and SSB that depends on
ssDNA.
(
)
This particular interaction must be
reduced for quantitative studies of other specific protein-DNA
complexes. Thus, the template used in these studies is an M13mp18
plasmid with a ssDNA gap of 500 nucleotides. This template minimizes
the amount of SSB-coated ssDNA in the assay and reduces to a low
background level this ``nonspecific'' interaction of the
complex and Pol III* with SSB-coated
ssDNA
(15, 18) .
Activities of Four Different Polymerases
In
Fig. 1
, the replication activities of core,
core-
, core
-
,
and Pol III* were compared on the SSB-coated gapped DNA template
containing a
clamp (assembled onto DNA by the
complex prior
to initiating replication). The results show that Pol III* is the most
active of these polymerases, core is least active, and the core-
polymerases are intermediate in activity. Similar results are observed
if the polymerase subassemblies are included during the assembly of the
clamp onto DNA and onto primed ssDNA (data not shown). This
hierarchy is consistent with earlier studies showing that
stimulates the activity of core with a
clamp
(9) and is
also consistent with studies in the absence of
, in which
polymerase subassemblies of increasing subunit complexity are also
increasingly active in DNA synthesis
(3, 4) . The
differences may lie in different processivity or speed of chain
elongation or in differential efficiencies of polymerase binding to a
clamp on primed DNA. Previous work has already shown that the
complex is similar to holoenzyme in its rapid and
processive chain elongation
(5) . Therefore, we have examined the
relative binding stability of different polymerase subassemblies to a
clamp on DNA.
The
In Fig. 2, a
[ Subunit Provides Stoichiometric Interaction of
Core with a
Clamp
P]
clamp was assembled onto circular
gapped DNA using the
complex and then incubated for 2 min with
[
H]core,
[
H]core
-
, or
[
H]core
-
prior to
analysis by gel filtration (see scheme at the top of Fig. 2).
Because only two of the four dNTPs were included, the polymerase was
unable to replicate the template and was stalled in an elongation mode.
Proteins bound to the large DNA template elute early (fractions
9-14) and resolve from free proteins that elute later (fractions
15-30). The number of
clamps assembled onto DNA is
approximately equimolar to the input DNA in all three experiments
(Fig. 2, A-C, left panels). The amount of
core
-
and core
-
bound to DNA in the excluded fractions was nearly equimolar to
the
clamp (Fig. 2, B and C,
respectively). However, in the absence of
, only half as much core
was retained with
on DNA (Fig. 2A, left
panel). Hence,
provides stoichiometric interaction between
core and
on DNA. In the absence of ATP, the
clamp is not
assembled onto the DNA, and none of the polymerases comigrate with the
DNA in appreciable amounts (Fig. 2, A-C, rightpanels). This result implies that these polymerases
require a
clamp on DNA for productive coupling to the template,
consistent with their need for a
clamp to become highly
processive in DNA synthesis.
-
and core
-
fill the gap in all the
circular DNA molecules, whereas core fills the gap in some DNA
molecules, but leaves others untouched (Fig. 2D, compare
second through fourth lanes). This action is
consistent with core being processive with a
clamp
(5) ,
but unable to bind to all the DNA molecules as indicated by gel
filtration (Fig. 2A), and is consistent with the lower
replication activity of core with
in Fig. 1.
-
dimer, which may be capable of
binding two
clamps/molecule, the limited availability of
clamps provides only one
clamp/molecule of
core
-
. We address the question of whether
a dimeric polymerase can bind two
clamps in experiments below
using Pol III*.
The
How does Subunit Is Present on DNA with Core and
increase the amount of core bound to
?
Is
action stoichiometric, or does it act as a molecular
matchmaker by transferring core to a
clamp and then dissociating
from the DNA to transfer other cores to
clamps? To distinguish
between these possibilities, we assembled a
core
-[
H]
complex onto DNA containing the [
P]
clamp, followed by gel filtration analysis. The results show
approximately equimolar association of
core
-[
H]
with
[
P]
clamps (Fig. 3A). The
interaction of core
-[
H]
with DNA requires a
clamp on the DNA as there is little
binding in a reaction where ATP is omitted (Fig. 3B).
Hence,
acts in a stoichiometric fashion and is present with core
and
on DNA rather than dissociating from DNA and acting as a
catalytic matchmaker.
Figure 3:
acts in stoichiometric fashion to
increase the efficiency of core binding to
on DNA. A,
[
P]
was assembled onto gapped DNA by the
complex in the presence of
core
-[
H]
as
described under ``Experimental Procedures,'' followed by gel
filtration. B, the analysis of A was repeated, but
ATP was omitted from the reaction.
,
core
-[
H]
;
,
[
P]
.
A simple mechanism by which may increase
the amount of core bound to
on DNA is that it may act as a brace
between core and
and/or the DNA. To test whether
can bind
to
on DNA without core, we mixed [
H]
with a [
P]
clamp on primed DNA and analyzed
the mixture by gel filtration (Fig. 4). The results show that
very little [
H]
binds to the
clamp.
This low level of [
H]
does not require the
clamp (data not shown) and therefore can be ascribed to the
previously documented weak association of
with DNA
(18) .
Presumably, the weak binding of
to DNA is sufficient to provide
the extra stabilization to the core-
contact needed to observe
stoichiometric comigration of the core-
complex to
clamps on
DNA. Alternatively,
may change the conformation of core such that
it binds
tighter.
Figure 4:
does not bind a
clamp in the
absence of core. Shown is the analysis of
[
H]
interaction with
[
P]
sliding clamps in the absence of core.
[
P]
was placed onto gapped DNA by the
complex as described under ``Experimental Procedures'' and
then treated with 1.77 pmol of [
H]
before gel filtration analysis.
,
[
P]
;
,
[
H]
.
Does the
Previous experiments have shown that after the
Complex Assemble with the Core Polymerase
and
on DNA?
complex assembles
onto DNA, the
complex can be
removed from the reaction by gel filtration
(5) . After gel
filtration, core can be added to the
clamp on DNA, resulting in
highly efficient synthesis similar to that of the entire
holoenzyme
(5) . But would the
complex have stably
associated with the
clamp on DNA if the core polymerase were
present before gel filtration? In Fig. 5, we used the
[
H]
complex and
[
P]
to determine whether the
complex
remains on DNA with
when core is added to the reaction prior to gel filtration. The gel filtration analysis showed that very
little
complex remained on DNA with
even in the presence of
these polymerase subassemblies (Fig. 5A).
Figure 5:
The complex does not remain
associated with the core-
or core-
complex on DNA.
[
P]
was assembled onto gapped DNA using the
complex as described under ``Experimental Procedures''
with the exception that the [
H]
complex
(labeled in
`) was used in place of unlabeled
complex.
A-C are gel filtration analyses of reactions containing
unlabeled core, core
-
, and
core
-
, respectively.
,
[
P]
;
, the
[
H]
complex.
The
subunit is known to interact directly with
(8) . The
complex interaction is essential for the
complex to be
assimilated into the Pol III* structure
(8) . However, the
complex does not associate with
(or Pol III`) unless a particular
order of subunit addition is followed, such as adding the
and
` subunits after the
-
contact has been
established
(8) . However, it remains possible that Pol III` and
the
complex will associate in the presence of a
clamp and a
primed template. This experiment is shown in Fig. 5C.
The results show that the [
H]
complex does
not remain with the [
P]
clamp on DNA
through the gel filtration column and thus did not assemble into Pol
III*. In Fig. 5B, core
-
was
incubated with the
complex,
, and DNA. Perhaps the
unoccupied protomer of
will associate with the
complex and hold it to DNA with the polymerase and
clamp.
However, the results show that the [
H]
complex does not assemble with the other proteins on the primed DNA.
Does the
A previous study indicated the physical presence of all
the subunits of the holoenzyme on DNA through gel
filtration
(7) . However, the large amounts of SSB-coated ssDNA
present in that study may have led to the nonspecific binding of Pol
III* to DNA (e.g. through the Complex within Pol III* Remain on DNA with
?
-SSB contact).
This early study also required use of silver-stained
polyacrylamide gels to identify subunits and thus was qualitative. Here
we have re-examined the composition of Pol III* on DNA with
using
radioactive labeled proteins and the gapped plasmid to minimize
nonspecific binding of Pol III* to DNA (Fig. 6). To perform these
experiments, Pol III* was reconstituted using
[
H]
(to follow the
complex),
[
H]
(to follow core), or
[
H]
. In Fig. 6(A-C), a
3-fold molar excess of [
H]Pol III* over gapped
template was incubated with
. Approximately equimolar amounts of
[
H]core
(there are two cores in one
Pol III*), [
H]
complex, and
[
H]
were bound to
[
P]
on gapped DNA in the excluded
fractions. Therefore, when the
complex clamp loader is already
part of the polymerase structure, it remains associated with the
polymerase and
on DNA.
Figure 6:
The complex within Pol III* is
retained with core,
, and the
clamp on DNA. Pol III*,
labeled in different subunits, was incubated with
[
P]
and gapped DNA, followed by gel
filtration. Proteins were incubated for 3 min at 37 °C in 100
µl of reaction buffer with 30 mM NaCl. Experiments in
A-C contained 1.2 µg (260 fmol) of gapped M13mp18
DNA, 3.2 µg of SSB, 81 ng (1 pmol) of
[
P]
, and 1 µg (1.4 pmol) of
[
H]Pol III* constituted with the indicated
tritiated subunit. Experiments in D-F contained 0.57
µg (400 fmol) of gapped DNA, 3.2 µg of SSB, 67 ng (830 fmol) of
[
P]
, and 277 ng (400 fmol) of
[
H]Pol III* reconstituted with the indicated
tritiated subunit. A and D, Pol III* and
[
H]core; B and E, Pol III* and
[
H]
; C and F, Pol III* and
[
H]
.
, [
H]Pol
III*;
,
[
P]
.
Up until now, the polymerase has been
used in molar excess over clamps on gapped DNA, and therefore,
the assemblies containing two core polymerases (i.e. core
-
and Pol III*) could bind only
one of the
clamps, even though these polymerases may be capable
of binding two
clamps/polymerase molecule. In the experiments
presented in Fig. 6(D-F), less Pol III* was
added, and the concentration of the gapped plasmid was increased,
resulting in an excess of
clamps over dimeric polymerases. The
results show that each molecule of [
H]Pol III*
(labeled in core,
, or
) binds approximately two
[
P]
clamps.
complex, yet two
clamps
are formed per Pol III*. Hence, these results further indicate that the
single
complex within Pol III* can assemble two
clamps on
DNA, one for each core within Pol III*.
clamps may be
assembled onto one primed template
(5) , and if this occurs, in
the experiments of Fig. 6, the amount of
each polymerase
binds would be an overestimate. The
clamp freely slides off
linear DNA unless it is held to DNA through stoichiometric association
with the polymerase
(5, 18) . Fig. 7shows an
experiment in which an excess of DNA was used to assemble
[
H]Pol III* with [
P]
clamps. The circular DNA was then linearized in a 1-min incubation with
BamHI prior to gel filtration in order to remove
clamps
not associated with a polymerase. The results show a stoichiometry of
one [
H]Pol III* bound to 1.7
[
P]
clamps, consistent with Pol III*
cross-linking two
clamps on separate DNA templates. These
experiments are technically difficult, and thus, these intriguing
conclusions remain to be rigorously tested in future studies. The
possibility that each core within Pol III* binds a
clamp on DNA
would be consistent with the hypothesis that both strands of the
chromosome are replicated concurrently by the twin polymerase within
the holoenzyme
(20, 21) .
Figure 7:
Pol III* binds two sliding clamps.
Initiation complexes were assembled onto 600 fmol of gapped M13mp18
template using 200 fmol (135 ng) of [H]Pol III*
(labeled in
) and 250 fmol (20 ng) of
[
P]
and incubated for 3 min at 37 °C in
100 µl of reaction buffer with 30 mM NaCl. After a 1-min
treatment with 100 units of BamHI to linearize the DNA,
reactions were gel-filtered, and the amount of
[
H]Pol III* and [
P]
in the column fractions was quantitated.
,
[
H]Pol III*;
,
[
P]
.
complex
is a clamp loader that assembles a
clamp on DNA, but then
dissociates and is not needed for
to tether core to DNA for
processive elongation of a 7-kilobase pair
template
(1, 2) . This may have led to the conception
that the
complex is not present in the holoenzyme structure on
DNA and that processive synthesis is performed only by a complex of the
clamp with the core polymerase (Fig. 8A). This
study has shown that the
complex is retained with the other
subunits (core,
, and the
clamp) on primed DNA
(Fig. 8D) provided that Pol III* is used instead of
separate subassemblies of the
complex and Pol III`. Hence, even
though the
complex is not essential to the polymerase-
contact, the clamp loader is present with the other proteins on DNA
when Pol III* is used (via contact with
). Presumably, Pol III* is
the form that replicates the chromosome since the function of the
complex in assembling
onto DNA is needed repeatedly during
discontinuous synthesis of the lagging
strand
(15, 22, 23) .
Figure 8:
Models of the four different initiation
complexes that DNA polymerase III can assemble onto DNA templates.
A, core- clamp; B,
core
-
clamp; C,
core
-
clamp; D, Pol
III*-
clamp. In C and D, both cores are presumed
to bind a
clamp, but for clarity, only one
clamp is shown.
See ``Results'' for details. 3`, the 3` termini of
the DNA being extended. The model of core is based on the crystal
structure of DNA polymerase I and duplex DNA
(26).
An interesting aspect of
this study is that the subunit structure of the holoenzyme on DNA can
be very different depending on how the subunits are added, even though
all the same subunits are present. Hence, the addition of
core-
(Pol III`) to the
complex and
results in a structure containing the two polymerases and
bound to the
clamp, but without the
complex
(Fig. 8C). However, if these subunits are first
assembled into Pol III* before mixing with
and primed DNA, the
complex is retained on DNA with the other proteins
(Fig. 8D). Consistent with this result, we showed in the
third report of this series that the constitution of Pol III* from pure
subunits requires a strict order of addition and that if the
four-subunit Pol III` is added to the five-subunit
complex, the
nine-subunit Pol III* does not form
(8) . In this study, we show
that these assemblies still do not associate with each other even in
the presence of primed DNA and the
clamp. Hence,
, either on
or off DNA, does not circumvent the order of addition needed to
incorporate the
complex into the holoenzyme structure.
clamp. Whether the
complex is removed from solution by gel filtration or is present
in free solution, this minimal polymerase is highly processive and is
nearly as efficient in DNA synthesis as the holoenzyme
(5) . The
subunit lends extra stability to the interaction of core with a
clamp on primed DNA, consistent with a previous observation that
in the presence of
, duplex structures in the path of replication
are more easily traversed by the polymerase
(9) . Use of the
subunit in molar excess over the core polymerase results in a
core
-
complex instead of
core
-
(Pol III`)
(10) . Study of the
core
-
complex showed that it too binds a
clamp, but like the core
-
complex,
it fails to retain the
complex, leading to the structures shown
in Fig. 8(B and C, respectively). Hence, the
availability of one
protomer lacking core does not lead to
assimilation of the
complex into the holoenzyme structure. For
simplicity, the dimeric polymerases in Fig. 8are shown with only
one
clamp on DNA, even though both polymerases can bind
clamps. Whether both polymerases within Pol III* are equally active in
DNA synthesis is under investigation.
and core-
complexes lacking the
complex appear to
contrast with results of an earlier study that showed the
complex
to be bound along with core and
on singly primed ssDNA coated
with SSB
(7) . However, in that study, the authors also formed a
processive polymerase that was presumed to consist of only
and
core, as in Fig. 8A (the
complex appeared
catalytic in their reactions). When a large excess of
complex was
added, two molecules of
complex were observed bound to DNA along
with one core and a
dimer, suggesting that the
complex may
bind core
(7) . However, the
complex binds nonspecifically
to SSB-coated ssDNA
(5, 19) ,
and therefore,
the
complex may have been bound nonspecifically to SSB-coated
DNA, while core-
was bound specifically at the primer terminus.
For this reason, we have used in this report a duplex template with a
gap of only 500 nucleotides, which essentially eliminates the
nonspecific background of the
complex bound to SSB-coated DNA. An
apparent competition between
and the
complex has been
observed in which
appeared to compete the
complex off the
DNA
(7) . Although we find that
, like the
complex,
binds nonspecifically to SSB-coated ssDNA
(18) , it is not clear
why
would compete the
complex off DNA since one might
expect a large number of nonspecific binding sites on these SSB-coated
ssDNA templates for both
and the
complex to bind at the
same time. Perhaps there are sites of preferential association, such as
at areas of secondary structure, for which
and the
complex
compete.
, could be assembled
onto DNA
(9) . In the presence of
, the polymerase that
formed was probably core
-
(e.g.Fig. 8B) since
was present in 10-fold molar
excess over core. The function of the two polymerases (+ or
-
) was hypothesized to be in replicating the leading
(+
) or the lagging (-
) strand of a chromosome. The
presence of
made the polymerase more efficient at traversing
obstacles (i.e. duplexes in the path of replication) and thus
was an attractive candidate for action with the leading strand
polymerase. Now, in light of the dimeric structure of
core
-
, it seems more likely that one
protomer is on each core polymerase to increase the efficiency of their
interaction with their respective
clamps on both the leading and
lagging strands. Another important function of the
dimer is to
hold the
complex to the holoenzyme structure
(8) .
40 molecules of core polymerase in E. coli, only half
of which are incorporated into the holoenzyme
(24) . Likewise,
there is an excess of
complex in cell lysates relative to that
within the holoenzyme
(25) . Hence, it seems possible that the
different processive polymerase structures outlined in
Fig. 8
could be present in the cell and may be useful in other
areas of DNA metabolism such as repair, recombination, and mutagenesis.
For example, the core-
polymerase in Fig. 8A may be
useful in short gap repair, where its lower stability on DNA would
allow it to more easily dissociate from DNA upon finishing a gap. The
core-
assemblies that lack the
complex (Fig. 8,
B and C) may be useful in repair of longer gaps such
as in mismatch repair. Furthermore, the ability of
to bind ssDNA
and double-stranded DNA (18) and its dimeric structure (i.e.
may cross-link two DNA molecules) make it an
attractive candidate as a player in replicative recombination.
clamp on DNA during chain extension, but upon completing a
template to the last nucleotide (i.e. upon completing a
lagging strand fragment), it rapidly dissociates from DNA and cycles to
a new primed site
(15, 22) . However, this rapid transfer
of polymerase to another primed site requires that site to be endowed
with a
clamp. Hence, the structure of Pol III* is ideally suited
for chromosome replication (Fig. 8D). The
complex
matchmaker is physically associated with the polymerases by mutual
interaction with
, and therefore, its clamp loading activity is
ever present and available for repeated action in
clamp assembly
onto the multiple RNA primers of the lagging strand.
-SSB interaction
requires no other proteins, and the ssDNA is not absolutely required,
but strengthens the
-SSB interaction considerably (Z. Kelman and
M. O'Donnell, manuscript in preparation).
subunit containing the
kinase recognition sequence and the conditions for labeling it.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.