From the Molecular Biology Program, Memorial
Sloan-Kettering Cancer Center, New York, New York 10021 and the
§ Department of Biochemistry, Biophysics, and Genetics,
University of Colorado Health Sciences Center,
Denver, Colorado 80262
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ABSTRACT |
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The DNA polymerase III holoenzyme is composed of
10 subunits. The core of the polymerase contains the catalytic
polymerase subunit, , the proofreading 3
5
exonuclease,
,
and a subunit of unknown function,
. The availability of the
holoenzyme subunits in purified form has allowed us to investigate
their roles at the replication fork. We show here that of the three
subunits in the core polymerase, only
is required to form
processive replication forks that move at high rates and that
exhibit coupled leading- and lagging-strand synthesis in
vitro. Taken together with previous data this suggests that the
primary determinant of replication fork processivity is the interaction
between another holoenzyme subunit,
, and the replication fork
helicase, DnaB.
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INTRODUCTION |
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The replisome of Escherichia coli is a complex protein machine composed of the DNA polymerase III holoenzyme (pol III HE),1 which synthesizes the nascent DNA, and the primosome, which unwinds the parental duplex and synthesizes primers for the initiation of Okazaki fragment synthesis (1). The composition of the primosome can vary depending on the manner in which the replication fork helicase, DnaB (2), is introduced to the DNA. Primosomes loaded at oriC are composed of only DnaB and DnaG (3), the primase (4), whereas primosomes loaded at recombination intermediates (5) are likely to also include PriA, PriB, PriC, and DnaT (6, 7). The function of these latter four proteins at the replication fork have yet to be established.
The pol III HE is itself composed of 10 subunits (8). , the
catalytic polymerase subunit (9),
, the 3
5
proofreading exonuclease (10), and
, a subunit of unknown function, associate to
form the polymerase core (11).
binds
but not
, whereas
binds
but not
(12), suggesting a linear array of
. The
association of
and
acts to improve the catalytic efficiency of
each polypeptide, increasing the polymerase activity of
by 2-3-fold (13, 14) and the exonuclease activity of
by 8-fold on a
mispaired substrate and 32-fold on a paired substrate (13). It was also
suggested that
increased the processivity of
during DNA
synthesis on primed single-stranded DNA (15).
The DnaX complex is composed of six subunits organized as
2
2
(16, 17).
acts to
dimerize two core assemblies via an interaction with
(18, 19). At
the replication fork, this results in a physical coupling of the
leading- and lagging-strand polymerases in space (20). The other
subunits in the DnaX complex, which have been referred to as the
complex (21), are likely involved in loading and unloading the
processivity subunit,
(22, 23), from the DNA (24, 25). The
dimer encircles the DNA and associates with
to topologically lock
the polymerase onto the template, thereby enabling processive synthesis
(26, 27).
also plays a central role at the replication fork where, as a result of a protein-protein interaction with DnaB, it cements the
replisome together, allowing rapid replication fork movement (28). This
interaction also results in the protection of
on the leading-strand
side from premature recycling by the
-complex (29), thus defining
which of the two polymerase cores becomes the leading-strand polymerase
at the replication fork (29, 30).
We have been studying the action of the pol III HE at replication forks
formed during rolling circle DNA replication on specialized tailed form
II (TFII) DNA templates in the presence of the single-stranded DNA-binding protein (SSB) and the X-type primosomal proteins. In
this report, we have analyzed the contributions of the three subunits
of the polymerase core to replication fork function. We find that only
the
subunit is required to form replication forks that are as
processive and which move at the same rate as those formed with the
intact HE. In addition, replication forks containing only
responded
to control of Okazaki fragment synthesis as mediated by the primase
(31-33), elaborated coupled leading- and lagging-strand synthesis, and
were as stable as replication forks formed with the complete polymerase
core.
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MATERIALS AND METHODS |
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Reagents, DNAs, Enzymes, and Replication Proteins--
NTPs and
dNTPs were from Pharmacia Biotech Inc. [-32P]dATP was
from Amersham. Alkaline phosphatase was from Boehringer Mannheim. Single-stranded circular DNAs from bacteriophages f1AY-7/M and f1R229-A/33 were prepared as described previously (34). pBROTB form I
DNA was prepared as described (35).
Rolling Circle DNA Replication Assay--
TFII DNA was prepared
as described by Mok and Marians (41). Reaction mixtures (12 µl)
containing 50 mM HEPES (pH 7.9), 12 mM MgOAc,
10 mM dithiothreitol, 5 µM ATP, 80 mM KCl, 0.1 mg/ml bovine serum albumin, 1.1 µM SSB, 0.42 nM TFII DNA, 3.2 nM
DnaB, 56 nM DnaC, 240 nM DnaG (or as
indicated), 28 nM DnaT, 2.5 nM PriA, 2.5 nM PriB, 2.5 nM PriC, and the pol III core,
, or
as indicated and all other HE subunits at 28 nM, were preincubated at 30 °C for 2 min. NTPs were
added to final concentrations of 1 mM ATP, 200 µM GTP, 200 µM CTP, and 200 µM UTP, and dNTPs to 40 µM and the reaction
was incubated for 2 min at 30 °C. [
-32P]dATP
(2000-4000 cpm/pmol) was added to the reaction mixture and the
incubation was continued at 30 °C for an additional 10 min. DNA
synthesis was quenched by addition of EDTA to 40 mM. Total
DNA synthesis was determined by assaying an aliquot of the reaction
mixture for acid insoluble radioactivity. The reaction mixtures were
treated with alkaline phosphatase (3 units) at 37 °C for 45 min and
the DNA products analyzed by alkaline gel electrophoresis as described
(31). The
X-type primosomal proteins were used rather than just
DnaB, DnaC, and DnaG because the former group of proteins assembles
replication forks about 15-fold more efficiently than the latter
(41).
Determination of Coupling of Leading- and Lagging-strand
Synthesis--
Standard rolling circle reaction mixtures containing
core, , or
as indicated were incubated for 2 min at 30 °C
in the absence of label to establish active replication forks. Aliquots (1 µl) were then transferred to a prewarmed dilution reaction mixture
(90 µl) containing all buffer components, NTPs, dNTPs, [
-32P]dATP, SSB, and primase at their standard
concentrations, but that lacked DNA template, all other HE subunits,
and the preprimosomal proteins (PriA, PriB, PriC, DnaT, DnaB, and
DnaC). The incubation was continued at 30 °C for 10 min and
terminated by the addition of EDTA. As a control for Okazaki fragment
size, the original reaction mixture was incubated at 30 °C for 10 min in the presence of [
-32P]dATP and in the absence
of any diluent. Reactions were then processed and analyzed as described
above.
Determination of Replication Fork Processivity--
Standard
rolling circle reaction mixtures were assembled either in the presence
of core, , or
, as indicated. The effect of the anti-
antibody (42) on initiation was assessed by including it in the
reaction mixture from the start, before the ATP concentration was
raised and before the dNTPs and other NTPs were added. The effect of
the antibody on elongation was determined by adding the antibody along
with the [
-32P]dATP 2 min after the reactions had been
initiated. Reactions were then processed and analyzed as described
above.
Determination of Replication Fork Rates--
Standard rolling
circle replication reactions containing core, , or
were
increased in size 4-fold. Reaction mixtures were incubated for 6 min at
30 °C after the addition of [
-32P]dATP.
5-Methyl-dCTP was then added to a final concentration of 0.4 mM and aliquots (7 µl) were withdrawn every 10 s for
the next minute. Each aliquot was mixed with 1 µl of 20 mM ddTTP to terminate the elongation reaction. Aliquots
were then heated at 68 °C for 10 min and then treated with 10 units
each of the AluI, HaeIII, HhaI, and
HpaII restriction endonucleases for 2 h at 37 °C. DNA products were analyzed by electrophoresis through alkaline-agarose gels (20 × 25 × 0.5 cm) at 40 V for 48 h. The
electrophoresis buffer was changed once after 24 h.
Determination of Replication Fork Stability--
oriC
DNA replication was reconstituted as described by Hiasa and Marians
(43) using pBROTB DNA as the template in the presence of DnaA, DnaB,
DnaC, DnaG, SSB, HU, and the pol III HE reconstituted with either core,
, or
as indicated. Replication reactions (75 µl) were
initiated in the absence of any topoisomerase. After a 2-min
incubation, [
-32P]dATP was added and the incubation
continued for 1 min. Under these conditions only an early replication
intermediate (ERI) is formed. Subsequent nascent chain elongation
requires the release of the accumulated topological constraint. The
label was chased by the addition of an 100-fold excess of cold dATP
(time 0) and the incubation continued. Aliquots (15 µl) were removed
at the indicated times, mixed with the SmaI restriction
endonuclease (20 units), and incubated an additional 10 min. DNA
products were then analyzed by electrophoresis through denaturing
alkaline-agarose gels as described (43).
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RESULTS |
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Okazaki Fragment Synthesis Is Modulated Properly at Replication
Forks Containing only --
Incubation of the TFII DNA template,
SSB, the primosomal proteins, and the pol III HE generates replication
forks that support rolling circle DNA replication. These forks produce
multigenome length double-stranded tails that are composed of a long,
continuous leading strand and short (about 2 kb) Okazaki fragments
(31). We have demonstated that the forks formed in vitro
possess many of the characteristics of bona fide E. coli
replication forks. They are highly processive, synthesizing leading
strands in excess of 0.5 megabase in length (31), move at rates
comparable to that of the fork in vivo (31, 41), exhibit
coupled leading- and lagging-strand DNA synthesis (20, 44), and
regulate the size of the Okazaki fragments produced in response to
various reaction parameters (31, 44-47). To examine the roles of
the three subunits of the polymerase core at the replication fork, we
therefore assessed the ability to form replication forks with
and
alone, as well as the characteristic properties of the forks formed.
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Replication Forks Containing Only Are Processive, Move at High
Rates, and Elaborate Coupled Leading- and Lagging-strand
Synthesis--
The replication forks that form at oriC are
extraordinarily processive. Each fork presumably synthesizes a leading
strand of about 2.3 × 106 nt in length in one
polymerase binding event. We have developed a protocol that allows us
to test replication fork processivity (41) that makes use of the
observation that once formed in an elongation complex with
,
is
no longer accessible to antibody (42). Thus, replication forks that are
processive are resistant to inhibition by anti-
antibody.
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Replication Forks Formed with ,
, or
Exhibit
Identical Stabilities--
The presence or absence of a 3
5
proofreading exonuclease function might only affect the stability of
the polymerases when the fork was paused. Thus, we considered that
we might not observe any differences between replication forks in the
presence and absence of
when they were moving, as they were in all
the experiments described above. Because it is difficult to pause replication forks in the rolling circle replication assay, we used the
oriC replication system, where forks can be paused as a
result of accumulated positive overwindings in the template DNA (51),
to investigate this issue.
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DISCUSSION |
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The replication fork is a complex structure where upward of 30 protomers combine to execute ordered, semi-conservative DNA replication
in a rapid and highly efficient fashion. Understanding the role of the
polypeptides at the fork is the key to understanding how the replisome
functions. We have been using a rolling circle DNA replication system
reconstituted with purified proteins in an effort to contribute to this
understanding. The availability of all the protein components in highly
purified form has allowed an analysis of the replication fork functions
that are disrupted when various polypeptides are omitted from
replication fork assembly. Such analyses revealed, for example, the
requirement for a protein-protein interaction between the subunit
of the HE and DnaB that literally defined the replisome, cementing the
polymerase and the helicase together, enabling rapid replication fork
movement (28), and determining which of the two polymerase cores will
be the leading-strand polymerase (29, 30).
In this report we have considered the contributions of the three
subunits of the polymerase core to replication fork function. ,
,
and
purify as a tight 1:1:1 complex when polymerase activity is
scored by, e.g. using nicked salmon sperm DNA as the
template (11). When processive DNA synthesis is used as the assay, the core is isolated as a component of the HE (8).
is the product of
holE (12, 52), which can be disrupted in E. coli
without any apparent affect (53). Biochemical analyses have also failed to attribute a catalytic activity to
.
is the proofreading 3
5
exonuclease (10) encoded by dnaQ (54). It can form a
tight complex with both
and
, apparently forming the bridge between them in the core (12). Strains deficient in
show the expected mutator effect (55).
is the polymerase subunit (9), encoded by dnaE (56) and is, of course, essential for
viability.
Because these three proteins form such a tight complex, it is expected
that they are present at the replication fork. On one level, the
contribution of is obvious. At a different level, we have
investigated whether
and
played additional roles that affected
replication fork function directly. We have assessed the distinguishing
characteristics of replication forks that contained
,
,
or only
. We found that replication forks that contained only
performed in a fashion indistinguishable from those containing the
complete core. Thus, neither
nor
were required to either maintain high rates of processive replication fork movement, to couple
the leading- and lagging-strand polymerases, or to ensure proper
recycling of the lagging-strand polymerase from the just completed
Okazaki fragment to the new primer terminus. This makes it unlikely
that these subunits are required to maintain the structural integrity
of the replisome. Replication fork assembly did require higher
concentrations of
than
. This reduced efficiency suggests that
may facilitate the interaction of
with another protein at
the replication fork.
In a study examining processivity of ,
, and
on
primed single-stranded phage DNAs in the presence of
and the
-complex, Studwell-Vaughan and O'Donnell (12) concluded that
,
while maintaining a reasonable processivity in the range of 1-3 kb, was still significantly less processive than the combination of
and suggested that highly processive DNA synthesis by the HE was
contingent on the exonuclease subunit. Kim and McHenry (14) did not
observe this difference when they used
complex to load
onto the
DNA and an anti-
antibody challenge similar to the one described in
this report to test processivity. This latter report did note that HE
reconstituted with only
synthesized DNA at about one-fifth the rate
of HE reconstituted with
or
. Thus, the results
observed at bona fide replication forks are somewhat different from
each of these reports, underscoring the importance of assessing
function in the proper context. Presumably, the major difference is
that the
-DnaB interaction that occurs at replication forks is the
overriding factor in the determination of fork rate and
processivity.
However, a structural role for at the replication fork under
certain conditions cannot be completely ruled out. dnaQ
disruptions in Salmonella typhimurium display two
phenotypes, a mutator defect and a slow growth defect (57). Spontaneous
suppressor mutations of the slow growth phenotype that have been mapped
to
arise very rapidly in these strains. The purified supressor
polymerase was shown to have 3-5-fold higher activity than the
wild-type polymerase (58). This suggests that
activates
in vivo. It is interesting to consider that this apparent
difference may reflect the basic difference between the rolling circle
replication system and replication forks on the chromosome of E. coli. That is, the in vitro system proceeds in an
unimpeded fashion around the template, whereas in the cell, replication
forks encounter all sorts of protein roadblocks on the DNA as well as
damaged bases in the template. It may be that under these circumstances
is required for stability of the fork. This possibility awaits
further investigation.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM34557 (to K. J. M.) and GM36255 (to C. S. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 The abbreviations used are: pol III HE, the DNA polymerase III holoenzyme; SSB, the single-stranded DNA-binding protein; TFII, tailed form II; ERI, early replication intermediate; kb, kilobase pair(s); nt, nucleotide(s).
2 D. Langley and K. J. Marians, unpublished data.
3 M. Olson, J. Carter, H. G. Dallmann, and C. S. McHenry, unpublished data.
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REFERENCES |
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