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J. Biol. Chem., Vol. 276, Issue 42, 39186-39191, October 19, 2001
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From the Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021
Received for publication, May 15, 2001, and in revised form, July 30, 2001
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Initiation of DNA replication at oriC
occurs bidirectionally both in vivo and in
vitro. Although the proteins involved in establishing the
replication forks are known, little is known about the events that
ensure that initiation is bidirectional. We show here that in the
absence of DNA gyrase, replication fork progression from
oriC on a plasmid template in vitro is
unidirectional, although both replication forks have formed at the
origin. There was no bias in the release of one fork or the other,
ruling out protein blockage of one fork as a possible reason for the
asymmetric release. Timely release of both forks required the presence
of either DNA gyrase or topoisomerase IV, suggesting that modulation of
the topology of the origin region is the governing factor.
Replication of circular bacterial genomes occurs bidirectionally
from the origin of chromosomal DNA replication, oriC. In Escherichia coli, replication from the origin has been
reproduced in vitro using small plasmid DNA templates
carrying the E. coli oriC (1). DnaA recognizes and binds to
a number of repeated sequences in the minimal origin, "DnaA boxes"
(2), organizing the origin DNA into a nucleoprotein structure that
enables the interaction of some of the DnaA protomers with A + T-rich
13-mers that are just counterclockwise to the minimal origin. In the
presence of the double-stranded DNA-binding protein HU, these 13-mers
become denatured, presumably as a result of a weak affinity of DnaA for the single-stranded 13-mers (3).
Once this region of localized denaturation is established, replication
fork assembly can proceed, governed by a series of protein-protein
interactions. DnaB, the replication fork DNA helicase, is transferred
to the DNA at a stoichiometry of two DnaB hexamers per oriC
(4) from DnaB-DnaC complexes in solution (5) as a result of a
protein-protein interaction between DnaA and DnaB (6). Once on the DNA,
DnaB recruits DnaG, the Okazaki fragment primase, also via a
protein-protein interaction (7), to the DNA. This interaction
determines where DNA replication will start and whether two replication
forks will form (8).
Subsequent replication fork assembly requires the binding of the DNA
polymerase III holoenzyme (pol III
HE),1 the replicative
polymerase, to the primers that are synthesized by DnaG. It is likely
that the first primer synthesized by a DnaB-DnaG primosome moving in
the clockwise direction away from oriC becomes the leading
strand for the counterclockwise moving replication fork and vice versa.
Subsequent replication fork establishment then requires the pol III HE
synthesizing the leading strand moving in, e.g. the
counterclockwise direction, to establish a protein-protein interaction
with the DnaB that is moving in the same direction. The This description of the events during initiation of replication from
oriC proceeds under the tacit assumption that these events can account for both formation and release of two replication forks
from the origin. We show here that this assumption is too simplistic.
Whereas two replication forks do form at oriC in the presence of the proteins mentioned, DnaA, DnaB, DnaC, DnaG, HU, and the
single-stranded DNA-binding protein (SSB), timely release of both of
these replication forks from the origin requires the presence of either
DNA gyrase or topoisomerase IV (topo IV). This requirement for a
topoisomerase could reflect the formation of some type of a complex at
the origin that is necessary for proper bidirectional replication.
However, although we have been able to detect an interaction between
DnaB and GyrB, we cannot detect one between DnaB and DNA gyrase. We
therefore conclude that topological modulation of the origin region is
necessary for timely release of both replication forks during initiation.
Replication Proteins and Antisera--
Proteins for
oriC replication, DnaA, DnaB, DnaC, DnaG, HU, SSB, the DNA
pol III HE (reconstituted from preparations of pol III* and the oriC DNA Replication--
Standard oriC DNA
replication reaction mixtures (15 µl) were as described by Hiasa and
Marians (11) except that incubation was at 37 °C. When present, DNA
gyrase was at 30 nM, topo IV at 22 nM, Tus at
50 nM, DNA polymerase I at 19 nM, RNase H at
0.12 nM, and DNA ligase at 14 nM. Reactions
mixtures that contained DNA ligase also contained 50 µM
NAD. Template DNA was plasmid pBRTO1.2, a 6.7 kb-long
molecule carrying two Ter sites, one 1.2 kb counterclockwise
of the origin and the other 1.4 kb clockwise of the origin (Fig.
1C). Gel electrophoretic analyses of replication products
were performed as described previously (11).
Protein-Protein Cross-linking and Western Blot
Analysis--
Proteins (5-6 µM) were mixed in buffer
containing HEPES-KOH (pH 7.8) and 100 mM NaCl. Reaction
mixtures (15 µl) were incubated at 30 °C for 10 min.
Bis(sulfosuccinimidyl) suberate (freshly diluted in 50 mM
HEPES-KOH (pH 7.8) and 100 mM NaCl) was added to a final
concentration of 0.004%, and the reactions were incubated at 30 °C
for 30 min. Cross-linking was stopped by the addition of 1 M glycine to a final concentration of 66 mM,
and the incubation was continued for an additional 10 min. After
addition of 25 µl of a 2× loading buffer (100 mM
Tris-HCl (pH 6.8), 200 mM Only One Replication Fork Is Active in Early Replication
Intermediates--
Plasmid DNA templates carrying oriC that
are replicated in vitro by a combination of DnaA, DnaB,
DnaC, DnaG, HU, SSB, and DNA gyrase produce replication products that
are characteristic of bidirectional replication initiating at or near
the origin sequence (Fig. 1A).
On these templates, which are 6.7 kb in length (Fig. 1C),
bidirectional replication produces two distinct populations of DNA when
analyzed by denaturing alkaline-agarose gel electrophoresis (Fig.
1A, lane 2) as follows: a leading strand population centered about 3 kb in length, indicative of the fact that the two replication forks that form at oriC meet roughly half-way around the
template; and a lagging strand population centered about 0.5 kb in
length. The addition of RNase H, DNA ligase, and DNA polymerase I
(subsequently denoted RLP) to the reaction converts the products to
full-length material (Fig. 1A, lane 3), indicating that the
clockwise-moving leading strand could be ligated to the
counterclockwise moving lagging strand on the same template molecule,
for example (Fig. 1B). Circular nascent DNA is not observed
because with gyrase as the only topoisomerase present, the bulk of the
replicated DNA accumulates as a late replication intermediate, where
there is still, on average, about 150 base pairs of unreplicated
parental DNA (14). We have shown previously that either topoisomerase III (15) or IV (16) is required to support the terminal stages of
replication when the late intermediate is converted to two daughter
molecules.
In the absence of gyrase, initiation can occur because the template DNA
is negatively supercoiled; however, replication fork progression
proceeds only until it is inhibited by the accumulation of excess
positive linkages in the template. The DNA product formed is called an
early replication intermediate (ERI). We have shown that the ERI is a
true kinetic intermediate in the replication pathway (15). Subsequent
completion of replication requires the release of the topological
strain, which can be accomplished by either the addition of a
topoisomerase or by cutting the template with a restriction enzyme.
Leading strands present in the ERI are about 1 kb in length (Fig.
1A, lane 1). At first glance, this size seems far too great. The plasmid template DNA contains about 40 negative supercoils; thus,
if two replication forks formed and proceeded away from oriC
in the ERI, one would expect the leading strands to be about 350 nucleotides in length, indicative of removal of the negative supercoils
as the template was unwound as a result of replication, followed by
generation of about 30 positive supercoils before replication ceased
because of the accumulated topological strain. However, as we will show
here, only one replication fork releases from the origin in the ERI,
and as we have shown elsewhere (17), origin-proximal regression of the
nascent DNA in the ERI allows more fork progression than one would have
predicted based on the superhelical density of the starting template DNA.
That only one replication fork was active in the ERI is shown by the
pulse-chase analysis displayed in Fig. 1A (lanes
4 and 5). Here the ERI was formed first and then the
[
To investigate the possibility that only one replication fork was
active in the ERI more closely, an assay was devised to score release
of both replication forks on one DNA molecule. This assay used the same
template DNA as in the experiment shown in Fig. 1A, but we
now included the replication fork arrest protein Tus. This protein will
bind to the Ter sequences that are present 1.2 kb
counterclockwise and 1.4 kb clockwise of oriC. The basis of
the assay is shown in Fig. 1D. If two replication forks
initiated, released from the origin, and progressed to the
Ter sites, leading strands of 1.2 and 1.4 kb should be
observed. In the presence of RLP, on any one molecule that has two
active forks, the clockwise-moving leading strand should be joined to
the counterclockwise-moving lagging strand, and vice versa, generating
a 2.6-kb nascent DNA. If only one fork has released, the addition of
RLP will not have any affect on the length of the nascent products.
In the absence of gyrase, two populations of nascent DNA were observed
as follows: a leading strand population centered about 1.2 kb in length
and a smaller lagging strand population (Fig. 1E, lane 1).
The length of the leading strand population argues strongly that on
most template molecules only one fork had progressed. Bidirectional
leading strand synthesis on one DNA template would have been expected
to result in leading strands that were, at best, half of the observed
length when the superhelical density of the template and nascent strand
regression were factored in. The addition of Tus led to the production
of leading strand DNA products of defined length (Fig. 1E, lane
3). However, in the presence of RLP, although ligation of the
nascent lagging strand fragments together could be observed, in neither
case were any products larger than 1.2-1.4 kb observed (Fig. 1E,
lanes 2 and 4). On the other hand, when gyrase was
present, the two defined leading strands were again observed in the
presence of Tus, but now the addition of RLP converted the majority of
the nascent DNA to a 2.6-kb product (Fig. 1E, compare
lanes 5 and 6).
The kinetics of fork release from the origin in the absence and
presence of gyrase was investigated using the assay described above
(Fig. 2). In the presence of gyrase, the
2.6-kb band could be detected within 1 min after the start of the
reaction, whereas even after 15 min of incubation in the absence of
gyrase only a small minority of the nascent DNA was converted to this
product (Fig. 2B). Therefore, we conclude that in the
absence of gyrase only one replication fork progresses to any
significant extent. This is also supported by the observation that
roughly twice as much DNA was synthesized in the presence of gyrase
compared with the absence of gyrase (Fig. 2C).
Even in the Absence of Gyrase, both Replication Forks Form at the
Origin
In either case, the 2.6-kb product diagnostic of bidirectional
replication formed (Fig. 3A)
with the same kinetics (Fig. 3B). These observations
indicate that during the initial incubation in the presence of the
labeled dATP, both replication forks had, in fact, formed at the origin
in the absence of gyrase. The fact that identical results were observed
using either DNA gyrase or SmaI ruled out the possibility
that, on any one molecule, nascent DNA made by one fork,
e.g. the clockwise one, that had formed during the
pulse-label became joined to nascent DNA made by a counterclockwise
fork that had formed during the chase period. This is because
initiation at oriC requires negatively superhelical DNA
(15). Digestion by SmaI prevents subsequent initiation by relaxing the template. Thus, it seemed clear that the reason that both
forks were not observed in Fig. 1A was that there was a
delay in the release of one of the forks from the origin. Because the plasmid template is so small and the replication fork is known to move
at roughly 1 kb/s, any delay in the neighborhood of 6 s will, in
the absence of Tus, allow the first fork that released to transit all
the way around the template before the release of the second fork. We
conclude that gyrase is neither required for formation of replication
forks during initiation nor for their progression.
Timely Release of Both Replication Forks from the Origin Requires
Modulation of Template Topology
To examine the possibility of blockage, we reasoned that such an event
would be manifested as a bias in which of the forks appeared to release
first. Indeed, cursory examination of the data in Figs. 1 and 2
supports this idea, because there appears to be a bias for the release
of the counterclockwise moving fork in the absence of gyrase. This
possibility resonates with the fact that because initiation of both
forks occurs just to the left of the origin, the clockwise-moving fork
would have to presumably transit through the region of the template
where DnaA was bound. This could cause significant delay. Indeed, in
the
To examine this possibility more closely, replication reactions were
performed in the presence of Tus and in either the presence or absence
of gyrase. The replication proteins were then inactivated by heating at
65 °C, and the DNA products were digested with either the
EcoRI or SacII restriction enzymes, which cut the
template once between the origin and the Ter sites in the
counterclockwise and clockwise directions, respectively (Fig.
4A). The amount of radioactivity in the bands at 1.2 and 1.4 kb was then determined. This
restriction enzyme digestion was necessary to remove a background that
complicates the analysis, i.e. in the absence of this
digestion, for example, incomplete leading strands from the
clockwise-moving fork that were in excess of 1200 nucleotides would
appear to contribute to the amount of radioactivity on the gel that
could be attributed to the counterclockwise-moving fork. The
restriction enzyme digestion removes these leading strands from that
region of the gel.
The results of this analysis are shown in Fig. 4. In the presence of
gyrase, the amount of radioactivity is roughly the same for each fork
(Fig. 4, B and D). In the absence of gyrase,
there is less than a 2-fold bias for the counterclockwise-moving fork at the earliest time points in the analysis (Fig. 4, C and
E). We think that this is unlikely to indicate a significant
bias in fork release because, as described above, the analysis is
somewhat subjective with respect to how regions of the gel are chosen
to represent one fork or the other and the data have not been
normalized to the size of the DNA products (which would further reduce
the apparent bias). If there actually were a 2-fold bias in fork
release, we would have expected to observe a 2.6-kb band representing
half the radioactivity in lanes 8-10 of Fig. 2A,
for example. We therefore conclude that whereas there may be a slight
bias to which fork releases in the ERI in the absence of gyrase, it is
insufficient to account for the data shown in Figs. 1 and 2.
We also considered that gyrase could be playing an architectural role
at the origin, contributing to the formation of a replisome complex at
the origin that was necessary for proper bidirectional replication. We
reasoned that this might be manifested by protein-protein interactions
between gyrase and a component of the replication fork. An examination
by cross-linking using the bifunctional cross-linking agent
bis(sulfosuccinimidyl) suberate revealed an interaction between GyrB
and DnaB (Fig. 5). Mixtures of these
proteins at high concentrations (>1 µM) were treated
with the cross-linking agent for 30 min and then analyzed by
electrophoresis through 6.5% polyacrylamide gels containing SDS. A
silver-stained gel (Fig. 5A) as well gels that were
Western-blotted using either anti-DnaB antisera (Fig. 5B) or
anti-GyrB antisera (Fig. 5C) are shown. In each case, a band
(labeled with an asterisk) could be detected with a mobility
of about 220 kDa that was dependent on the presence of both GyrB and
DnaB. This is a clear indication of an interaction between these two
proteins. From the estimated size of the cross-linked band, we suspect
that the molecular species involved are GyrB2 and
DnaB3, and both forms are known to exist in solution.
Although this is a promising and interesting observation, we have been
unable to demonstrate an interaction between DnaB and native gyrase;
thus, because we cannot eliminate the possibility that the interaction
surface on GyrB is unavailable for DnaB when GyrB is formed into native
gyrase, we cannot ascribe any significance to the observed
interaction.
To explore whether the requirement for gyrase for timely release of
both replication forks from oriC related to timely
modulation of the topology about the origin, we repeated the experiment
described in Fig. 2 replacing gyrase with topo IV (Fig.
6). Although topo IV cannot supercoil DNA
as gyrase does, it will relax positive supercoils and can support
replication fork progression (19). We found that both replication forks
released from the origin, allowing the formation of the 2.6-kb DNA
fragment. Thus, we conclude that the observed requirement for gyrase
for timely release of both replication forks from the origin relates to
a requirement for topological modulation of the template DNA and is
unlikely to be a specific requirement for the presence of gyrase
itself.
Replication of the E. coli chromosome occurs
bidirectionally from oriC (20, 21). Although this reaction
has been reproduced in vitro, thus identifying the enzymes
required, little is known of the molecular events that actually occur
at the origin to ensure that two replication forks have formed and can
proceed around the chromosome. In this report, we have shown that in
the absence of topological modulation of the template DNA, even though
two replication forks can be detected as having initiated at the
origin, only one fork is released.
Because the plasmid template DNA used in vitro is negatively
supercoiled, we have been able to develop a pulse-chase protocol that
allows us to observe the action of a particular set of replication forks. In this procedure, an ERI is formed by initiating replication in
the absence of a topoisomerase. During this period
[ To investigate this possibility further, we developed an assay that
could definitively score bidirectional replication on the template DNA.
This assay utilizes the replication fork arrest protein, Tus. Template
DNAs were engineered such that the binding sites, Ter, for
Tus were positioned close to and on either side of the origin in an
orientation that would result in replication fork arrest in both
directions when Tus was present. In addition, DNA polymerase I, RNase
H, and DNA ligase were included in the reaction. This ensured that in
the case of bidirectional replication, the leading strand that was
being synthesized, e.g. in the clockwise direction from the
origin, would be ligated with the Okazaki fragments being made in the
counterclockwise direction from the same template strand. Thus,
if these circumstances prevailed, a DNA fragment equal in length to the
distance between the two Ter sites on the template would be
produced. On the other hand, if one replication fork had moved
clockwise on one template and another replication fork had moved
counterclockwise on a different template, no such fragment could be generated.
By using this assay, it was clear that only one replication fork had
progressed to any significant extent in ERIs. Interestingly, however,
it could be demonstrated that both replication forks had formed during
initiation. We considered three possible explanations for these
observations. One obvious possibility was that one of the replication
forks was blocked and prevented from moving a significant distance
because of DnaA bound to the origin. This is because initiation at
oriC actually occurs in an A + T-rich zone just
counterclockwise of the minimal origin sequence (4). Thus, the
clockwise-moving fork has to transit across the origin, a region of DNA
that is presumably bound to multiple DnaA molecules in a
nucleoprotein-like, protein-DNA complex (22). Indeed, during Another possibility that was considered was that formation of a
replisome complex at the origin might require gyrase as a component,
rather than require its activity as a topoisomerase, to be assembled
correctly. This would be consonant with recent cell biological findings
in vivo in Bacillus subtilis (23, 24) that
suggest that replication of the chromosome occurs at a fixed point in
the cell, where the template DNA is presumably drawn through a protein
complex rather than the individual replication forks traversing each
chromosome arm. Along these lines, we were able to demonstrate that one
of the subunits of gyrase, GyrB, could be cross-linked to DnaB.
However, a similar interaction between DnaB and native gyrase could not
be demonstrated. Even so, if such an interaction occurred, we have no
independent means for placing it at the origin. Thus, the possibility
that a single replication factory synthesizes the DNA in a bacterial
cell remains an attractive hypothesis.
In fact, we could demonstrate that gyrase was neither required for the
assembly of the two forks at the origin nor for timely release of both
forks. Topo IV worked just as well. This observation leads us to the
conclusion that we were observing a requirement for timely topological
modulation of the template DNA that allowed both forks to proceed. We
do not know what aspect of origin structure this topological modulation
is required to relieve. One possibility is that as both forks proceed
from the origin, negative supercoils might become trapped in the region
between them, distorting the DNA and preventing elongation by the fork
that released second. In support of this, we have also observed that
inclusion of topoisomerase III, which will relax only negative, not
positive, supercoils under these conditions, also allows both
replication forks to proceed (data not shown).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit of
the HE mediates this interaction (9, 10). Note that the DnaB that is
presumably captured by protein-protein interactions by the HE moving,
as a point of this illustration, in the counterclockwise direction is
not the same DnaB that mediated an interaction with the primase to
synthesize the primer for leading strand synthesis in that direction.
Based on mapping of initiation points in the region of oriC,
it has been suggested that the two DnaBs loaded to the chromosome must
pass each other during establishment of the replication forks (4).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit), topo IV, and DNA gyrase, were purified as described
previously (11). Antisera to DnaB was described previously (12).
Antisera to GyrA and GyrB was raised in rabbits. E. coli DNA
polymerase I, RNase H, and ligase were from Roche Molecular
Biochemicals. Tus was prepared as described by Hill and Marians
(13).
-mercaptoethanol, 20%
glycerol, 0.2% bromphenol blue, and 4% SDS), the samples were heated
at 100 °C for 5 min and then chilled on ice for 5 min. Proteins were
electrophoresed through a 6.5% SDS-polyacrylamide gel at 220 V for
4 h and visualized by silver staining. For Western blotting,
proteins were separated as described above except that the amount of
protein loaded was reduced by half. Proteins were transferred overnight
at 4 °C at 25 V onto nitrocellulose membrane. Blots were probed with
rabbit polyclonal antibodies raised against either DnaB or GyrB and
were developed using an alkaline phosphatase-conjugated system.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Only one replication fork is active in early
replication intermediates. A, denaturing alkaline
agarose gel of DNA replication products. Lane 1, products
from an early replication intermediate formed in the absence of DNA
gyrase. Lane 2, products from a standard replication
reaction containing DNA gyrase. Lane 3, products from a
standard replication reaction that also contained RNase H, DNA ligase,
and DNA polymerase I (RLP). Lanes 4 and 5,
products from a pulse-chase reaction where the ERI was formed first in
the absence of DNA gyrase and in the presence of
[ 
-32P]dATP and the replication forks were then
released by adding either the SmaI restriction endonuclease
(lane 4) or DNA gyrase (lane 5) at the same time
as a 100-fold excess of unlabeled dATP. B, possible modes of
DNA replication and the result of treating the reaction products with
RLP. C, map of the template DNA, pBRTO1.2.
D, assay for bidirectional replication. Only when two forks
have released from oriC will a 2.6-kb species be formed in
the presence of RLP. E, only in the presence of gyrase are
two forks released from oriC. Reaction mixtures containing
the indicated proteins in addition to DnaA, DnaB, DnaC, DnaG, SSB, HU,
and the DNA polymerase III holoenzyme were incubated for 15 min at
37 °C, and the products were analyzed by denaturing alkaline agarose
gel electrophoresis. B and D,
arrowheads denote direction of fork movement only, not
active 3'-ends.
-32P]dATP was diluted out by a factor of 100 as the
topological strain was released by the addition of either
SmaI, a restriction enzyme that cuts the template DNA just
to the left of oriC (Fig. 1C), or DNA gyrase. In
each case the result was the same in that full-length DNA was the
predominant product. If the ERI contained two paused replication forks
with leading strands of about 1 kb, the final product length observed
after the chase should have been somewhere intermediate between 5.7 and 3.3 kb.
View larger version (33K):
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Fig. 2.
Kinetics of fork release in the presence and
absence of gyrase. Standard replication reaction mixtures
containing Tus either in the presence or absence of gyrase, as
indicated, were increased in volume 6-fold and incubated at 37 °C.
Aliquots (15 µl) were removed at the indicated times and the samples
processed and analyzed as described under "Materials and Methods."
A, denaturing alkaline agarose gel. B,
PhosphorImager analysis was used to quantitate the fraction of the
total radioactivity incorporated that was represented by the 2.6-kb DNA
species. C, PhosphorImager analysis was used to quantitate
the total radioactivity incorporated at each time point.
PSL, photostimulated luminescence.
We next investigated whether the reason that only one fork
progressed in the ERI was because only one fork, rather than the two
required for bidirectional replication, was initiated under the
conditions used. To do so, we used pulse-chase analysis as described
above, except that Tus was present from the beginning of the reaction
so that the conditions of the assay described in Fig. 1D
applied. Thus, the ERI was formed first in the presence of
[-32P]dATP. The label was then diluted out by the
addition of a 100-fold excess of cold dATP at the same time that the
topological constraint was released by adding either DNA gyrase or
SphI, which cuts the template once outside of the region
flanked by the Ter sites (Fig. 1C).
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Fig. 3.
Both forks form at the origin during
initiation. A, pulse-chase analysis of replication from
pre-formed ERIs. Standard replication reaction mixtures increased in
volume 6-fold containing Tus but not gyrase were incubated for 1 min in
the presence of [ -32P]dATP at 37 °C. A 100-fold
excess of unlabeled dATP and either gyrase (lanes 1-5) or
SphI (lanes 6-10) was then added and the
incubation continued. Aliquots (15 µl) were withdrawn at the
indicated times, and the samples were processed and analyzed as
described under "Materials and Methods." An autoradiogram of the
denaturing alkaline agarose gel is shown. B, PhosphorImager
analysis was used to quantitate the fraction of the total radioactivity
incorporated that was represented by the 2.6-kb DNA species.
We considered three possible reasons for the observed delay in release of both replication forks
from oriC in the absence of gyrase: (i) that in fact both forks did release, but there was a roadblock on one side or the other
and close to the origin that prevented progression of one of the forks;
(ii) that an interaction between gyrase and one of the proteins of the
replisome was required; and (iii) that it was release of
topological strain across the origin that was the determining factor.
Of course, a combination of these is also possible.
replication system, such a delay appears to be caused by the
O protein bound to the origin (18).
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Fig. 4.
Fork release in the absence of gyrase is not
significantly biased. Standard reaction mixtures increased in
volume 6-fold containing Tus were incubated at 37 °C either in the
presence (B and D) or absence (C and
E) of gyrase. Aliquots (15 µl) were withdrawn at the
indicated times, and the reactions were terminated by heating at
65 °C for 5 min. Samples either received no further treatment or
were digested with either the SacII or EcoRI
restriction enzymes as indicated. Analysis was by denaturing alkaline
agarose gel electrophoresis (B and C). Total
radioactivity incorporated was determined by PhosphorImager
analysis (D and E). PSL,
photostimulated luminescence.
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Fig. 5.
Protein-protein cross-linking detects an
interaction between GyrB and DnaB. Protein-protein cross-linking
was as described under "Materials and Methods." Three gels are
shown. A, silver-stained gel. B and C,
Western blots of gels using either anti-DnaB antisera (B) or
anti-GyrB antisera (C) as probes. The asterisk in
all panels shows the position of the DnaB-GyrB cross-linked
species.
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Fig. 6.
Topological modulation of the origin is
required for the timely release of both replication forks. A
standard replication reaction mixture containing Tus and topoisomerase
IV was increased in volume 6-fold and incubated at 37 °C. Aliquots
(15 µl) were removed at the indicated times, and the samples were
processed and analyzed as described under "Materials and Methods."
Shown for comparison are the products of replication after a 5-min
incubation in the presence of gyrase.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]dATP is included in the reaction. Replication
fork progression proceeds in the ERI until excess positive supercoils
have accumulated. These forks are stalled, but active, and replication
fork progression can be resumed by the provision of either a
topoisomerase or by cutting the template DNA with a restriction enzyme.
In the latter run out, part of the procedure, excess unlabeled
dATP is included to dilute the radioactive label. By using this
procedure we have been able to show that either DNA gyrase (15), topo
IV (19), or topoisomerase III (15), but not topoisomerase I (15), can support replication fork progression during 
-type DNA replication, for example. We had noted, however, that the replication run out product under these conditions was invariably full length. This suggested that only one replication fork was active in the ERI. If both
forks were active, it would not be possible to observe a full-length
leading strand DNA; instead, one would expect two half-length leading strands.
-type
replication of plasmid templates carrying the bacteriophage origins
of replication, such a blockage has been observed (18). Although we
obtained evidence for a slight bias in fork release in the anticipated
direction, it could not account for the data.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM34557.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.
N.S. dedicates this paper to the memory of Elena Smelkova.
Published, JBC Papers in Press, August 14, 2001, DOI 10.1074/jbc.M104411200
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ABBREVIATIONS |
|---|
The abbreviations used are: pol III HE, polymerase III holoenzyme; topo IV, topoisomerase IV; kb, kilobase pair; SSB, single-stranded DNA-binding protein; ERI, early replication intermediate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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