From the Institute of Molecular Biology and Genetics, School of Biological Sciences, Seoul National University, Seoul 151-742, Korea
Received for publication, September 20, 2000, and in revised form, November 26, 2000
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ABSTRACT |
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Under anaerobic growth conditions,
Escherichia coli operates a two-component signal
transduction system, termed Arc, that consists of ArcB protein, a
transmembrane sensor kinase and ArcA protein, the cognate response
regulator. In response to low oxygen levels, autophosphorylated ArcB
phosphorylates ArcA, and the resulting phosphorylated ArcA (ArcA-P)
functions as a transcriptional regulator of the genes necessary to
maintain anaerobic growth. Under anaerobic conditions, cells maintain a
slow growth rate, suggesting that the initiation of chromosomal
replication is regulated to reduce the initiation frequency. DNase I
footprinting experiments revealed that ArcA-P binds to the left region
of the chromosomal origin, oriC. ArcA-P did not affect the
in vitro replication of plasmid DNA containing the ColE1
origin nor the in vitro replication of viral DNAs; however,
ArcA-P specifically inhibited in vitro E. coli chromosomal
replication. This inhibition was caused by the prevention of open
complex formation, a necessary step in the initiation of chromosomal
replication. Our in vitro results suggest that the Arc
two-component system participates in regulating chromosomal initiation
under anaerobic growth conditions.
Most organisms, including Escherichia coli, are able to
adapt to variable growth conditions and environmental changes by
regulating gene expression (1, 2). Adaptation in bacterial cells is usually achieved through two-component signal transduction systems, which consist of a sensor kinase and a response regulator (2, 3). The
response regulator, phosphorylated by cognate sensor kinase in response
to an external signal, serves as a transcriptional regulator optimizing
gene expression under a given condition. In E. coli, the Arc
(anoxic redox control)
two-component signal transduction system operates in response to a
shift from aerobiosis to anaerobiosis (4). The Arc signal transduction
system consists of the ArcB and ArcA proteins, a transmembrane sensor
kinase and its cognate response regulator, respectively (5, 6). In response to oxygen deficiency or redox change, ArcB autophosphorylates in an ATP-dependent manner and converts to phosphorylated
ArcB (ArcB-P)1 via an
intramolecular phospho-relay of His-292 Anaerobic conditions that induce the Arc two-component signal
transduction system lead to reduction in growth rate (12). Because the
rate of chromosomal replication regulated mainly at the level of
initiation is coupled to growth rate (13), the slow growth rate of
E. coli during times of oxygen deficiency suggests that the
frequency of chromosomal initiation is reduced. It is therefore
probable that regulation of initiation occurs at oriC, the
E. coli origin of chromosomal replication (14). This unique
sequence, which is the highly conserved origin of Gram-negative
bacteria (15), includes four DnaA boxes, AT-rich region containing
three 13-mers, and an IHF binding site, all of which are required for
proper chromosomal initiation of replication (16, 17). Binding of the
initiator protein, DnaA, to the DnaA boxes leads to unwinding of the
AT-rich regions and allows for the entry of DnaB helicase, a required
step for subsequent initiation processes (18, 19). Opening of the
AT-rich region is facilitated by the binding of IHF protein to the IHF
site, thereby bending oriC (20, 21). Although IHF can be
substituted by HU protein (21), in vivo footprinting
experiments suggest that IHF may play a role in determining the timing
of chromosomal initiation during the cell cycle (22).
Here we report that the binding of ArcA-P, phosphorylated by ArcB, to
oriC results in the inhibition of chromosomal initiation. This result suggests that the Arc two-component signal transduction system plays a role in the regulation of chromosomal initiation at
oriC in response to oxygen deficiency.
Reagents and Proteins--
Sources were as follows:
[
Monomeric DnaA protein from HMS174 (pKC596) (23) and HU protein (24)
were purified as previously described.
Bacterial Strains, Primers, and Plasmid and Phage
DNAs--
E. coli strains MC1061 (25), W3110 (26), and
WM433(dnaA204) (27) were previously described. E. coli DH5
The DNAs were as follows: pFToriC (21) for DNase I
footprinting; pSBoriC (29) for nuclease P1 assay;
pBluescript SK(+) (Stratagene Corp.) for cloning; M13RE85 RF
(replicative form) DNA (30), and single-stranded DNAs from phage G4
(31), M13mp19 (Stratagene Corp.), Purification of ArcA and ArcB Proteins--
E. coli
strain MC1061 harboring either pBADarcA or
pBADarcB was grown in LB media to an OD at 595 nm of 0.5 followed by the addition of L(+)-arabinose to a
concentration of 0.2% to induce overproduction of the proteins. 2 h after induction, cells were harvested, resuspended in cell
resuspension buffer (25 mM HEPES-KOH (pH 7.6), 1 mM EDTA, and 2 mM dithiothreitol) to an OD at
595 nm of 200 and frozen in liquid nitrogen. Cell lysis and ammonium sulfate precipitation were performed as previously described (33), except that 0.37 g/ml ammonium sulfate instead of 0.28 g/ml was used.
The pellet was resuspended in buffer A (25 mM HEPES-KOH (pH
7.8), 1 mM EDTA, 10% glycerol, and 2.86 mM
2-mercaptoethanol), which was used throughout the purification
procedure. Subsequently, the activities of ArcA and ArcB were
determined by transphosphorylation reactions as described below.
The resuspended ammonium sulfate-precipitated pellet containing ArcA
protein was dialyzed to the conductivity equivalent of 50 mM KCl then subjected to FastQ column chromatography (60 ml of bed volume, Sigma) using 600 ml of a linear gradient ranging from 50 mM to 1 M KCl in buffer A. The fractions
containing ArcA were pooled and dialyzed to the conductivity equivalent
of 50 mM KCl then loaded onto a heparin-agarose column (12 ml of bed volume, Sigma). A 120-ml gradient ranging from 50 mM to 1 M KCl in buffer A was used for protein
elution, with ArcA eluting at ~150 mM KCl. The fractions
containing ArcA were pooled, diluted with buffer A to the conductivity
equivalent of 50 mM KCl, and loaded onto a FastS column (4 ml of bed volume, Sigma). The fractions containing ArcA were pooled,
diluted to the conductivity equivalent of 50 mM KCl with
buffer A, and subjected to Cibacron Blue column chromatography (2.5 ml
of bed volume, Sigma). A 25-ml gradient ranging from 50 mM
to 2 M KCl in buffer A was run over the column, yielding
near homogeneous ArcA, which eluted at ~0.5 M KCl.
About 17 mg of ArcA protein was obtained from 15 liters
culture of MC1061(pBADarcA).
For ArcB protein purification, the ammonium sulfate precipitate
obtained from 6 liters of MC1061(pBADarcB) culture as
described above was resuspended in buffer A, dialyzed to a conductivity equivalent of 50 mM KCl, and subjected to FastQ column
chromatography (90 ml of bed volume) using a 900-ml gradient ranging
from 50 mM to 1 M KCl in buffer A. Fractions
containing ArcB were pooled, then dialyzed to a conductivity equivalent
of 50 mM KCl and loaded onto a Cibacron blue column (17 ml
of bed volume). A 170-ml gradient ranging from 50 mM to 1 M KCl in buffer A was run over the column. ArcB eluted in a
broad range of fractions, which were pooled, and the protein was
precipitated by the addition of 0.45 g/ml ammonium sulfate followed by
centrifugation at 45,000 rpm for 30 min in a Ti70 rotor
(Beckman). The pellet was resuspended with buffer A and subjected to
Superose-12 gel filtration chromatography (Amersham Pharmacia Biotech,
HR 10/30). ArcB eluted as a single peak. These fractions were pooled
and loaded onto a MonoQ column (Amersham Pharmacia Biotech, HR 5/5).
ArcB eluted from the MonoQ column at near homogeneity and was used in
further experiments. A 6-liter culture of MC1061(pBADarcB)
yielded about 9 mg of homogeneous ArcB.
Transphosphorylation Reaction--
Transphosphorylation
reactions (TP), including purified ArcA and ArcB, were performed as
previously described (34) with minor modifications. 10 µl of the TP
mixture contained 6 µg each of ArcA and ArcB, 0.1 mM ATP,
70 mM KCl, 10 mM MgCl2, 33 mM HEPES-KOH (pH 7.4), 0.1 mM EDTA, and 2 mM dithiothreitol. After incubation at 32 °C for 10 min,
the reaction mixture (2 µl per each assay unless indicated) was
immediately used for further experiments.
To visualize the phosphorylated proteins, [
Phosphorylation reaction of ArcA with carbamyl phosphate (CP) was
performed as previously described (35). 10 µl of the CP mixture
containing the indicated amount of ArcA protein, 40 mM dilithium carbamyl phosphate,125 mM KCl, 10 mM MgCl2, and 100 mM Tris-HCl
(pH 7.0) was incubated at 30 °C for 1 h and immediately used
for experiments.
DNase I Protection Assays--
DNase I protection assays were
performed as previously described (36) with minor modifications. A
435-bp XbaI/XhoI fragment from pFToriC
was labeled at either the XhoI or XbaI
restriction site. 21.5 fmol of labeled fragments was mixed with the
indicated proteins in 25 µl of standard reaction mixture containing
0.1 mM ATP, 50 mM potassium chloride, 10 mM magnesium acetate, 2.5 µg of bovine serum albumin, 40 mM HEPES-KOH (pH 7.6), and 10% glycerol. After incubation
at 32 °C for 10 min, DNase I (5 ng in 1.5 µl of H2O)
was added, incubated for 30 s, then stopped by the addition of 27 µl of 0.6 M sodium acetate, 0.4% SDS, 25 mM
EDTA, and 0.1 mg/ml yeast tRNA. Proteins were removed by
phenol/chloroform extraction, and DNA was precipitated by ethanol,
followed by a 70% ethanol wash. DNA was subjected to electrophoresis
through a 5% Long Ranger polyacrylamide sequencing gel containing 7 M urea. The gel was dried and visualized by autoradiography.
In Vitro oriC Replication Assays--
As previously described
(37, 38), in vitro oriC plasmid replication with fraction II
from WM433 (dnaA204) and purified DnaA protein was performed
using 200 ng of M13RE85 RF DNA as a template.
Single-stranded phage DNAs, such as G4, M13mp19, and Open Complex Formation and P1 Nuclease Assay--
Open complex
formation at oriC was detected using single-strand specific
P1 nuclease as previously described with modifications (21, 39). 25 µl of opening reaction mixture containing the indicated amount of
DnaA, 15 ng of HU, 200 ng of supercoiled pSBoriC, 4 mM ATP, 50 mM potassium glutamate, 2.5 mM magnesium acetate, 2.5 µg of bovine serum albumin, 40 mM HEPES-KOH (pH 7.6), and 17% glycerol, was incubated at
37 °C for 5 min. Then 3 units of P1 nuclease in 3 µl of 30 mM sodium acetate (pH 5.2) was added and incubated at
37 °C for 30 s. The cleavage reaction was quenched by the
addition of 27 µl of stop solution (25 mM EDTA, 0.4 M NaOH). After incubation at room temperature for 10 min,
followed by addition of 6 µl of 3 M sodium acetate (pH
5.2), proteins were removed by phenol/chloroform extraction. With 2.5 µg of yeast tRNA as a carrier for precipitation, DNA samples were
collected by ethanol precipitation followed by a 70% ethanol wash. The
precipitated DNA was resuspended with 6 µl of H2O, and 2 µl was taken to be used as template for primer extension reactions. 6 µl of the primer extension mixture included 1.25 nmol of each dNTP,
0.25 pmol of 5'-end-labeled primer PA1 (21), and 0.42 unit of Vent
(exo ArcA-P Protein Binds to oriC--
To investigate the interaction
of ArcA and ArcB with oriC DNA, ArcA and ArcB proteins were
overproduced and purified to near homogeneity using column
chromatography (Fig. 1A). Due
to poor expression of the ArcB protein, we instead expressed and
purified a truncated ArcB lacking the N terminus from amino acid
residues from 1 to 128 as described previously (34). The ArcA and ArcB proteins used in this study were distinctive from the proteins containing the hexa-histidine (7, 35, 40, 41) or renatured from
SDS-polyacrylamide gels (34) as used in previous studies. The purified
ArcA and ArcB proteins were active in transphosphorylation reactions.
In the presence of ATP, ArcB underwent autophosphorylation and
subsequently transferred the phosphoryl group from itself to ArcA,
forming ArcA-P, whereas ArcA alone was not phosphorylated (Fig.
1B).
Binding of the transphosphorylation mixture (TP mixture), which
contains ArcA, ArcB, and ATP, to the oriC region of the
E. coli chromosome was detected using a DNase I protection
assay (Fig. 2). Increasing the amount of
TP mixture added to the footprinting reaction revealed that an
~150-bp region located at the left end of oriC, including
the three 13-mer AT-rich regions, DnaA box R1, and IHF binding site,
was protected from DNase I cleavage (Fig. 2). The omission of ArcA,
ArcB, or both from the TP mixture abolished the protection pattern
observed at oriC, indicating that the oriC
binding activity requires both ArcA and ArcB proteins.
Neither ADP nor the nonhydrolyzable ATP-analogue AMPPNP was able to
substitute ATP for the oriC protection activity of the TP
mixture, implying the requirement of ATP hydrolysis for binding (Fig.
3A). Efficient oriC
protection activity required more than 50 µM ATP. Without
ArcB and ATP, it has been shown that the phosphoryl group donors
carbamyl phosphate and acetyl phosphate can phosphorylate ArcA (35).
Therefore, we incubated ArcA with carbamyl phosphate in the absence of
ArcB and ATP, and found an identical DNase I cleavage protection
pattern (Fig. 3B). These results indicate that ArcA-P,
produced by phosphorylation of ArcA protein either by ArcB and ATP or
by carbamyl phosphate, binds to the left end of oriC.
ArcA-P Inhibits in Vitro oriC-dependent Initiation of
Chromosomal Replication--
The AT-rich regions and DnaA box R1 found
at oriC are indispensable for both in vitro and
in vivo initiation of chromosomal replication (18, 29, 42).
Therefore, the effect of ArcA-P, which was found to bind those regions
of oriC, on chromosomal initiation was examined using an
in vitro oriC plasmid replication assay (37, 38). This assay
resembles in vivo chromosomal initiation in many aspects,
including dependence upon the oriC sequence, requirement of
replicative proteins, and bidirectional replication from
oriC (37, 43).
Addition of purified DnaA protein to the oriC plasmid
replication assay sustained the replication of oriC plasmid
M13RE85 RF DNA, in which the oriC region has been inserted
into M13mp8 RF DNA (Fig. 4A).
The presence of the TP mixture in the assay, however, inhibited
DnaA-dependent oriC plasmid replication.
Omission of ArcA, ArcB, or both from the TP mixture eliminated the
inhibitory activity of the TP mixture (Fig. 4B), indicating
that the inhibitory activity is dependent on both ArcA and ArcB.
However, incubation of ArcA protein with carbamyl phosphate instead of
ArcB and ATP also inhibited oriC plasmid replication,
whereas carbamyl phosphate or ArcA alone was not inhibitory (Fig.
4C). These results imply that ArcA-P, formed either by ArcB
and ATP or by carbamyl phosphate, inhibits the oriC plasmid
replication, and ArcB is not a requirement.
To determine whether ArcA-P specifically inhibits initiation at
oriC, the effect of ArcA-P on other origins was studied.
Single-stranded viral DNAs ArcA-P Does Not Affect the Binding of DnaA and IHF Protein to
oriC--
Because DNase I footprinting revealed the binding region of
ArcA-P at a 150-bp region of the left end of oriC containing
both DnaA box R1 and the IHF binding site, DNase I footprinting was further performed to determine whether ArcA-P inhibits initiation of
replication at oriC by blocking the interaction of DnaA or IHF with oriC (Fig. 5).
Addition of increasing amounts of DnaA to oriC (Fig.
5A, lanes 2-4) resulted in the protection of
DnaA boxes R1 to R4 and IHF bound to the IHF binding site (Fig.
5B, lanes 2-4) both as previously described
(21). DnaA and IHF added prior to or after ArcA-P did not allow the
binding of ArcA-P to DnaA box R1 nor the IHF site, respectively;
however, neither protein affected the binding of ArcA-P to the AT-rich
region (Fig. 5, A and B, lanes 5-11).
These results indicate that the binding of DnaA to DnaA box R1 and IHF
to the IHF site is preferred over the binding of ArcA-P to those sites.
However, DnaA and IHF do not affect ArcA-P binding to the AT-rich
region.
To further study the binding of ArcA-P to the AT-rich region of
oriC, competition experiments using IciA were performed.
IciA protein specifically binds to the three 13-mers in the AT-rich region and inhibits the initiation stage of in vitro oriC
replication (52). Interestingly, addition of IciA to a preformed
ArcA-P·oriC complex displaced ArcA-P to generate a
footprint similar that of IciA only. Conversely, ArcA-P displaced IciA
bound to oriC (Fig. 5B, lanes
14-16).
ArcA-P Blocks Open Complex Formation in oriC-dependent
Initiation--
At the onset of initiation, DnaA protein unwinds the
AT-rich regions of oriC with the aid of HU or IHF, forming
the open complex, a step that is prerequisite for the subsequent stages
of initiation (18, 19). DnaA-dependent strand opening of
the AT-rich region can be observed using single-stranded-specific P1
nuclease and primer extension assays (Fig.
6), as previously described (21, 39).
Addition of the TP mixture prior to DnaA protein in the assays resulted
in inhibition of open complex formation. However, the addition of the
TP mixture after open complex formation did not significantly inhibit
open complex formation. These results imply that ArcA-P functions prior
to the DnaA-dependent strand opening of the AT-rich region.
The amounts of TP mixture required for oriC binding (Fig.
2), inhibition of oriC replication (Fig. 4B), and
inhibition of open complex formation (Fig. 6) were comparable to each
other. These results suggest that binding of ArcA-P to oriC
inhibits oriC initiation by blocking open complex
formation.
ArcA protein phosphorylated by ArcB and ATP or by carbamyl
phosphate binds the left end of oriC. This bound region
includes the AT-rich 13-mers, DnaA box R1, and the IHF binding site,
regions all highly conserved in chromosomal replication origins of
Gram-negative bacteria (15) and all of which are essential for
initiation of E. coli chromosomal replication (18, 29, 42).
In chromosomal initiation events, the AT-rich region of oriC
is unwound upon binding of DnaA proteins to the DnaA boxes (18, 19).
Interaction of DnaA protein with the AT-rich region, leading to strand
opening, is facilitated by the bending of oriC by IHF
protein bound to the IHF site (20, 21). Binding of ArcA-P blocks
DnaA-dependent strand opening of the AT-rich regions (Fig.
6), a step required for subsequent stages of chromosomal initiation to
occur. Binding of ArcA-P also results in the inhibition of in
vitro oriC plasmid replication (Fig. 4). Because ArcA-P does not
appear to affect the binding of DnaA and IHF to their loci (Fig. 5),
such inhibition may be caused by ArcA-P binding to the AT-rich regions
of oriC, thereby inhibiting a proper interaction between
DnaA protein and the AT-rich region, an interaction which is thought to
be required for the opening of the AT-rich region by DnaA (29,
44).
Aside from our reported binding of ArcA-P to oriC, ArcA-P
binds to a number of promoter regions (35, 44-46), including the pfl promoter that controls expression of pyruvate formate
lyase (46). We observed that ArcA-P possessed similar affinities to the
oriC and pfl promoters using gel-shift assays
(data not shown). However, we could not match the suggested ArcA-P
binding consensus sequence (49) with the oriC sequences, the
only commonality we found was a richness of A and T. Although
unphosphorylated ArcA binds to pfl and sodA
promoter regions in a several- to 10-fold excess over ArcA-P, with the
same pattern of ArcA-P binding (46, 47), we could not detect any
binding of unphosphorylated ArcA to oriC, even when using a
15- to 20-fold excess (up to 15 µM) as compared with
ArcA-P in the DNase I footprinting assay (data not shown). ArcA-P, even
with a molecular mass of 27 kDa, protected a wide region, ~150
bp, of oriC from DNase I cleavage (Figs. 2 and 3). This
broad binding pattern was also observed at other ArcA-P binding sites
(35, 46, 48). The increased amount of ArcA-P enhanced the intensity of
protected regions, rather than resulting in the broadening of the bound
region, which would be evident if sequential bindings of ArcA-P were
taking place (Fig. 2). These binding patterns suggest that multimerized
or oligomerized ArcA-P, formed prior to DNA binding, as suggested from
other binding sites (35, 46, 47), may bind to the left half of
oriC. The DNase I-hypersensitive sites with ~10-bp
intervals (Figs. 2 and 3) suggest that multimerized ArcA-P wraps the
left end of oriC.
Chromosomal replication is regulated mainly at the level of initiation;
the rate of chromosomal replication is coupled to growth rate (13).
Different physiological conditions determine unique growth rates,
reflected by the frequency of chromosomal initiation. Slow growth
conditions require a reduction in the frequency of chromosomal
initiations to meet the retarded growth. However, it has been scarcely
documented how organisms set the initiation frequency in response to
various physiological conditions or environmental stresses. In E. coli, it was reported that UV irradiation inhibits the
initiation of chromosomal replication from oriC (50),
however, there are no further studies showing the regulatory factors
involved or underlying mechanisms.
The pleiotrophic effects of arcA mutations (4-6, 41, 51,
52) present challenges in performing in vivo experiments and make interpretation of the results difficult. Even under aerobic condition, arcA mutants possess reduced chromosome numbers
compared with the wild type (data not shown). Although there is no
clear in vivo data available, our in vitro
results suggest that Arc, a two-component signal transduction system
operated under anaerobic conditions in E. coli, plays a role
in regulation of chromosomal initiation. Under oxygen depletion stress,
the response regulator ArcA, phosphorylated by sensor kinase ArcB, may
bind to oriC and reduce chromosomal initiation.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Asp-576
His-717 (7).
Subsequently, ArcB-P phosphorylates Asp-54 of ArcA, and the resulting
ArcA-P (phosphorylated ArcA) functions as a transcriptional repressor
for the sdh, gltA, lld,
cyo, and sodA genes (4, 6, 8, 9) and as an
activator for cyd, pfl, and traY genes
(4, 8, 10, 11) to sustain anaerobic growth.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (5000 Ci/mmol), [
-32P]dCTP
(3000 Ci/mmol), and deoxynucleotides, Amersham Pharmacia Biotech; Long
Ranger polyacrylamide, FMC BioProducts; DNase I, Life Technologies Inc.; nuclease P1, Roche Molecular Biochemicals; T4 polynucleotide kinase and Vent (exo
) DNA polymerase, New England
BioLabs; calf intestinal alkaline phosphatase and restriction enzymes,
Promega Corp. Unless indicated, reagents were purchased from Sigma
Chemical Co.
(28) was used for isolation of plasmid DNA.
X174 (31) for in vitro
complementation assays. For the construction of pBADarcA and
pBADarcB, coding regions of arcA containing the
whole polypeptide and arcB containing from the amino acid
residue 129 to the stop codon were amplified using polymerase chain
reaction and inserted into the EcoRI/HindIII site
of pBAD18 (32) and the KpnI/XbaI site of pBAD24
(32), respectively.
-32P]ATP
was added to the above mixture. The reaction was terminated by addition of gel-loading buffer (10% glycerol, 3% SDS, 3%
-mercaptoethanol, and 0.3% bromphenol blue). After incubation at 55 °C for 3 min, the
mixture was subjected to 12% SDS-polyacrylamide gel electrophoresis. The gel was dried and visualized by autoradiography.
X174, and
pBluescript containing the ColE1 replication origin, 200 ng each, were
used for DnaA-independent replication. In these assays, all conditions
were identical to oriC plasmid replication reactions, except
that DnaA was omitted.
) DNA polymerase in Vent (exo
) buffer
with 5 mM MgSO4. The primer was 5'-end-labeled
with [
-32P]ATP and T4 polynucleotide kinase;
unincorporated radioactivity was removed using a Bio-Gel 6 spin column
(Bio-Rad). The mixture was subjected to primer extension reactions in a
thermocycler, for 20 cycles (95 °C for 1 min except for 4 min in the
first cycle, 55 °C for 1 min, and 72 °C for 1 min except for 6 min in the last cycle). The reaction was stopped by the addition of 4 µl of sequencing gel loading buffer, then subjected to
electrophoresis through a 5% Long Ranger polyacrylamide sequencing gel
containing 7 M urea. The gel was dried and visualized by autoradiography.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Phosphorylation of ArcA by ArcB.
A, 3 µg each of purified ArcB and ArcA were run on
SDS-polyacrylamide gel electrophoresis and visualized after staining
with Coomassie Blue. B, transphosphorylation reactions with
the indicated proteins and [ -32P]ATP were performed as
described under "Materials and Methods." Phosphorylated proteins
were visualized by autoradiography. M indicates molecular
mass markers as kDa.
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Fig. 2.
Binding of Arc to the left half of
oriC. DNase I protection assays were performed
with the XbaI/XhoI fragment 5'-end-labeled at the
XbaI site (A) or XhoI site
(B) as described under "Materials and Methods." Unless
indicated, 2 µl of the TP mixture was used for assays. Lane
1, free DNA; lanes 2-4, 2 µl of the TP mixture with
the indicated proteins; lanes 5-8, 0.25, 0.5, 1, and 2 µl
of the TP mixture containing both ArcB and ArcA. The locations of three
AT-rich 13-mers (LMR), four DnaA boxes (R1-R4),
and the IHF binding site (IHF) in oriC are
indicated to the left of the figure. A,
G, C, and T indicate dideoxy
sequencing of pFToriC. The numbers to the
right correspond to the numbers of the oriC
nucleotide sequence (17).
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Fig. 3.
Binding of ArcA-P to the
oriC. DNase I protection assays were performed
using the XbaI/XhoI fragment 5'-end-labeled at
the XhoI site as described in Fig. 2 and "Materials and
Methods." A, lane 1, free DNA; lane
2, TP mixture without nucleotide; lanes 3 and
4, TP mixture containing 100 µM each of ADP
and AMPPNP respectively; lanes 5-7, TP mixture containing
100, 50, and 25 µM ATP. The same concentrations of each
nucleotide was included in footprinting reaction. B, instead
of ArcB and ATP, 40 mM carbamyl phosphate was added in 10 µl of CP mixture containing 3 µg of ArcA. Lane 1, free
DNA; lane 2-4, 0.5, 1, and 2 µl of CP mixture containing
ArcA and carbamyl phosphate; lane 5, 2 µl of CP mixture
containing ArcA alone. CP indicates carbamyl
phosphate.
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Fig. 4.
Inhibition of in vitro
oriC replication by ArcA-P. In vitro
DNA replication assays with oriC plasmid M13RE85 RF DNA
(A-C) or indicated DNAs (D) were performed as
described under "Materials and Methods." A, in the
presence (+TP) or absence ( TP) of 2 µl of TP
mixture, the indicated amount of DnaA was added to each oriC
replication assay. B, TP mixture or TP mixture with
indicated proteins omitted were titrated in oriC replication
assays containing 100 ng of DnaA protein. C, CP mixture
containing 6 µg of ArcA, 40 mM carbamyl phosphate, or
both, was titrated in oriC replication assays containing 100 ng of DnaA. D, TP mixture was titrated in replication assay
containing the indicated single-stranded DNAs or plasmid DNA.
X174, M13, and G4 replicate from single-
to double-stranded RF DNA using unique initiation processes, with each
viral origin using varying proteins (16). In the absence of DnaA, the
soluble proteins of fraction II, used for oriC plasmid
replication, are sufficient to replicate single-stranded viral DNAs to
RF DNAs (37). Plasmid pBluescript, which contains the ColE1 origin, can
also be replicated by fraction II in the absence of DnaA, but with less
efficiency than the DnaA-dependent replication of oriC plasmid DNAs (37). Addition of the TP mixture to these reactions did not affect replication of any of the tested
single-stranded viral DNAs or pBluescript DNA (Fig. 4D),
indicating that ArcA-P specifically inhibits the initiation of
replication at oriC.
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Fig. 5.
Binding of ArcA-P, DnaA protein, IHF, and
IciA protein to oriC. DNase I protection assays
were performed with the XbaI/XhoI fragment
5'-end-labeled at the XbaI site (A) or
XhoI site (B) as described under "Materials and
Methods." Lane 1, free DNA; lanes 2-4, 25, 50, 100 ng of DnaA protein (A) or 15, 30, 60 ng of IHF
(B); lanes 5-6, 100 ng of DnaA (A) or
60 ng of IHF (B) followed by addition of 0.5 and 1 µl of
TP mixture; lanes 7-8, 0.5 and 1 µl of TP mixture;
lanes 9-11, 1 µl of TP mixture followed by addition of
25, 50, and 100 ng of DnaA protein (A) or 15, 30, and 60 ng
of IHF; lane 12, 600 ng of ArcA followed by addition of 100 ng of DnaA (A) or 60 ng of IHF (B); lane
13, 600 ng of ArcB followed by addition of 100 ng of DnaA
(A) or 60 ng of IHF (B); lane 14, 1 µl of TP mixture followed by addition of 75 ng of IciA protein;
lane 15, 75 ng of IciA protein; lane 16, 75 ng of
IciA protein followed by addition of 1 µl of TP mixture
View larger version (40K):
[in a new window]
Fig. 6.
Inhibition of open complex formation by
ArcA-P. P1 nuclease cleavage and primer extension assays were
performed as described under "Materials and Methods." In
A: lanes 1-4, the indicated amount of DnaA was
added to the opening reaction; lanes 5-8, TP mixture was
added after open complex formation, then incubated 5 min more;
lanes 9-11, TP mixture, instead of DnaA, was added to the
open complex formation reaction, incubated for 5 min, followed by
addition of DnaA and another 5-min incubation. In B: The
extent of P1 cleavage at the 13-mer M in A was measured
using a FUJIX Bio-Imaging Analyzer (BAS 1000) and normalized to the
value of lane 5.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Edmund C. C. Lin and Dr. Ohsuk Kwon for helpful discussions and Gillian Newman for careful editing of this manuscript.
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FOOTNOTES |
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* This work was supported in part by the Basic Research Program of Korea Science and Engineering Foundation (Grant 1999-1-209-004-5) and by a grant from Life Phenomena and Function Research of Korea Institute of Science and Technology Evaluation and Planning.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.
Supported by a BK21 Research Fellowship from the Korean Ministry
of Education.
§ To whom correspondence should be addressed: Tel.: 82-2-880-7524; Fax: 82-2-874-1206; E-mail: dshwang@plaza.snu.ac.kr.
Published, JBC Papers in Press, December 22, 2000, DOI 10.1074/jbc.M008629200
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ABBREVIATIONS |
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The abbreviations used are:
ArcB-P, phosphorylated ArcB;
ArcA-P, phosphorylated ArcA;
CP mixture, phosphorylation mixture containing carbamyl phosphate;
RF, replicative
form;
TP mixture, transphosphorylation reaction mixture;
bp, base pair(s);
AMPPNP, adenosine 5'-(,
-methylenetriphosphate).
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REFERENCES |
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