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J. Biol. Chem., Vol. 277, Issue 21, 18483-18488, May 24, 2002
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From the Department of Molecular and Cellular Biology, Faculty of
Biotechnology, University of Gdansk, 24 Kladki,
80 822 Gdansk, Poland
Received for publication, August 8, 2001, and in revised form, February 12, 2002
The Escherichia coli molecular
chaperone protein ClpB is a member of the highly conserved Hsp100/Clp
protein family. Previous studies have shown that the ClpB protein is
needed for bacterial thermotolerance. Purified ClpB protein has been
shown to reactivate chemically and heat-denatured proteins. In this
work we demonstrate that the combined action of ClpB and the DnaK,
DnaJ, and GrpE chaperones leads to the activation of DNA replication of
the broad-host-range plasmid RK2. In contrast, ClpB is not needed for
the activation of the oriC-dependent
replication of E. coli. Using purified protein components
we show that the ClpB/DnaK/DnaJ/GrpE synergistic action activates the
plasmid RK2 replication initiation protein TrfA by converting inactive
dimers to an active monomer form. In contrast, Hsp78/Ssc1/Mdj1/Mge1,
the corresponding protein system from yeast mitochondria, cannot
activate the TrfA replication protein. Our results demonstrate for the
first time that the ClpB/DnaK/DnaJ/GrpE system is involved in protein
monomerization and in the activation of a DNA replication factor.
The Escherichia coli protein ClpB is a member of the
universally conserved Hsp100/Clp protein family (1, 2). Based on the
growth defect phenotype of E. coli clpB mutants, it has been proposed that ClpB is involved in thermotolerance (3, 4). In agreement
with this, thermally aggregated proteins were shown to be disaggregated
in wild-type cells but not in a clpB mutant strain upon a
shift to lower temperature (5, 6). Recently, the cooperative action of
ClpB with the DnaK, DnaJ, and GrpE chaperone system has been reported.
It has been shown that ClpB cooperates in vitro with the
DnaK chaperone system in the reactivation of chemically and
heat-denatured substrate proteins (7-10).
Other members of the Hsp100/Clp protein family, namely the E. coli proteins ClpA, ClpX, and ClpY, have been shown to be involved in the ATP-dependent protein degradation through a
transient association with the catalytic protease components ClpP and
ClpQ (ClpP for ClpX and ClpA; ClpQ for ClpY) (11). In the absence of
ClpP, ClpX and ClpA display chaperone activities (12, 13). Several substrates have been identified both for the chaperone and proteolytic activities of these proteins (1, 2, 14-18).
Previous work has shown that the broad-host-range plasmid RK2 can
replicate and maintain itself in a wide variety of Gram-negative bacteria (19). This property has made RK2 a particularly interesting system for studying fundamental questions about the cellular processes involved in DNA replication. Only two factors required for RK2 replication are encoded by the plasmid DNA: (i) the replication origin
(oriV) sequence consisting of five iterons (17-bp iterated sequences) (20) and four DnaA boxes (9-bp DnaA protein binding sites)
(21), and (ii) the plasmid replication initiation protein TrfA
(22-24). Other of the proteins required for plasmid DNA replication are supplied by the bacterial host (25). It has been demonstrated in a
purified system that the DnaA initiation protein, DnaB helicase, DnaC,
DNA gyrase, DnaG primase, SSB, HU, and Pol III proteins of E. coli are required for RK2 replication (26). The binding of the
TrfA protein to the iteron sequences is the most crucial step during
RK2 replication initiation. Only the monomeric form of the TrfA protein
can bind to the iteron sequences and initiate DNA replication (27). The
binding of TrfA in the presence of HU results in strand opening in the
AT-rich oriV region, specifically at the four 13-mers
present within this region (21). This open structure serves as the
entry site for the DnaB helicase complex, thus leading to initiation of
DNA replication (26).
The TrfA initiation protein exists in two forms, 44-kDa and 33-kDa, as
a result of an internal translational start in the trfA gene
(22-24). It has been shown that either protein can function in DNA
replication in E. coli. However, the 44-kDa TrfA is
indispensable during RK2 replication initiation in Pseudomonas
aeruginosa (24, 28, 29). Although the purified TrfA protein is
found largely in the dimeric form, it binds iteron sequences as a
monomer (30-32). The involvement of the chaperone protein ClpX in the
activation of TrfA for iteron binding and consequently for initiating
oriV replication has been previously studied in detail (33).
It has been shown that the ClpX chaperone protein can convert TrfA
dimers into active monomers in vitro.
In previous work, evidence was obtained that in addition to ClpX other
chaperone(s) may also play a role in TrfA activation in E. coli (33). Here, we demonstrated that ClpB in combination with
DnaK chaperone system plays a major role in the replication of the RK2
plasmid. Our work is in contrast with studies with plasmid P1 where it
was shown in vitro that the replication initiation protein
of this plasmid can be activated to a monomer form by either the DnaK
chaperone machinery alone or the ClpA protein alone (12, 34).
Bacterial and Yeast Strains, Proteins, Plasmids, and
Reagents--
The E. coli strains used in this study were:
MC1000 and SG20250, the parent strains of MC1000
(clpB::kan), and SG22100
(clpB::kan), respectively (3, 35). Published
protocols were utilized for the purification of E. coli
chaperone proteins ClpB (36), DnaK, DnaJ, and GrpE (37). The yeast
chaperones Hsp78, Ssc1, Mdj1, and Mdg1 were purified as described (38,
39). Purification of the E. coli DnaA, DnaB, and DnaC
proteins were carried out following the established protocols described
previously (26). The 33-kDa histidine-tagged version of the RK2 plasmid
replication initiation protein TrfA was purified as previously
described (27). The histidine-tagged monomeric mutant form of the
33-kDa TrfAG254D/S267L was purified as described (40). This protein,
has been used in previous RK2 biochemical analyses (21, 26, 33, 41-45) and is known to initiate replication in vitro with kinetics
similar to those of the largely dimeric wild-type protein. Unlike the wild-type, the mutant protein is present largely in the form of the
monomer, which is the active form of TrfA for binding to the iterons at
the RK2 replication origin (27). Plasmids pTJS42 and pKD19L1 carrying
the minimal RK2 origin region and plasmid pBSoriC carrying the
oriC DNA origin fragment have been previously described (41,
46). HU, SSB, and DNA gyrase proteins were obtained from Enzyco, Inc.,
creatine kinase, bovine serum albumin (Fraction V), creatine phosphate,
and rNTPs from Sigma, dNTPs from Pharmacia,
[methyl-3H]dTTP from ICN Radiochemicals, and goat
anti-rabbit IgG from Bio-Rad.
RK2 oriV and E. coli oriC DNA Replication Using Crude
Extracts--
Preparation of E. coli crude extracts (FII)
and reaction conditions were essentially as previously described (47).
The standard reactions contained 300 ng of plasmid template and
histidine-tagged versions of wild-type 33-kDa TrfA or the largely
monomeric preparations of the 33-kDa TrfA mutant protein TrfA 254D/267L
(42) at the indicated concentrations. For
oriC-dependent reactions, the pBSoriC template
was used, and the TrfA protein was omitted. Incubation was at 32 °C
for 60 min. Reactions were stopped by trichloroacetic acid
precipitation and total nucleotide incorporation (pmol) was measured by
liquid scintillation counting following filtration onto Whatman
GF/C glass fiber filters.
Template Unwinding Assay (FI*)--
Helicase unwinding assays
(FI* formation) were performed as previously described (26). Plasmid
pKD19L1 (300 ng) or pTJS42 (300 ng), containing the RK2 replication
origin, was used in the DNA template. Unless noted otherwise, proteins
were added at the following amounts: DnaA (320 ng), DnaB (600 ng), DnaC
(120 ng), TrfA 254D/267L (500 ng), HU (5 ng), gyrase (120 ng), and SSB
(230 ng).
Glycerol Gradient Centrifugation--
To analyze the monomeric
and dimeric forms of TrfA protein, glycerol gradient centrifugation was
performed essentially as described in (48). Reaction mixtures (60 µl)
with proteins in buffer were assembled on ice and contained 40 mM Hepes/KOH, pH 8.0, 25 mM Tris/HCl, pH 7.4, 80 µg/ml bovine serum albumin, 4% sucrose, 4 mM
dithiothreitol, 11 mM magnesium acetate, and 2 mM ATP. The mixture was incubated for 10 min at 32 °C
and then loaded onto a 3.2-ml linear 15-35% (v/v) glycerol gradient
in buffer (50 mM Hepes, pH 8.0, 10 mM
MgCl2, 200 mM KCl, 4 mM
dithiothreitol) and centrifuged at 46,000 rpm for 24h at 2 °C in a
Beckman SW60 rotor. Fractions were collected from the top of the tube
and analyzed by SDS/PAGE, followed by protein transfer and
immunoblotting with rabbit antisera against the TrfA protein. Bound
rabbit antibody was detected by a colorimetric reaction with an
horseradish peroxidase-conjugated goat anti-rabbit IgG.
The ClpB Chaperone Plays a Role in RK2 DNA
Replication--
Previously published in vitro results
demonstrated that the plasmid RK2 replication initiation protein TrfA
can be activated by a chaperone (33). ClpX, the molecular chaperone in
the ATP-dependent reaction, converts TrfA dimers to active
monomers. In agreement, the mutant protein TrfA 254D/267L, which exists
mainly in monomeric form does not require the presence of ClpX for
replication activity. Because we were not able to observe a significant
effect of the deletion of the clpX gene on the maintenance
of the RK2 plasmid in E. coli, we previously proposed that
other chaperone system(s) are also involved in TrfA activation (33).
Based on the previous analysis of RK2 DNA replication in protein
extracts prepared from E. coli strains carrying mutations in
different chaperones (33), ClpB was found to be the most attractive candidate.
The effect of ClpB on the initiation of DNA replication was tested
using a bacterial crude extract replication system (see "Materials
and Methods"). Extracts obtained either from wild-type E. coli cells or the clpB deletion mutant were used in two
different replication reactions. The first one,
oriV-dependent, utilizes the plasmid RK2 minimal
replication origin and the plasmid replication initiation protein TrfA.
The second one, oriC-dependent, represents DNA
replication from the E. coli minimal replication origin
dependent on the presence of the DnaA initiation protein.
oriV DNA replication activity in crude Fraction II extracts
prepared from the clpB-deficient strain was significantly
lower (80% reduction) when compared with the same reaction carried out
in extracts prepared from wild-type bacteria (Fig.
1A). In contrast, the absence
of ClpB in the crude extract had only a limited effect on
oriC DNA replication (25% reduction).
We also analyzed the effect of the addition of purified ClpB protein to
the oriV and oriC replication reactions using
crude Fraction II extracts prepared from the E. coli
clpB-deficient strain (Fig. 1, B and
C). In the presence of ClpB, the level of oriV-dependent replication increased 2-fold
(Fig. 1B). In contrast, oriC DNA replication was
somewhat diminished (Fig. 1C). The reduction in
oriC replication observed in these experiments may be due to the nonspecific disaggregation of particular replication machinery components. A similar nonspecific decrease in the DNA replication has
been observed during experiments with the monomeric form of the TrfA
protein (Fig. 2B). Overall the
results indicate that plasmid RK2 DNA replication but not bacterial DNA
replication is activated by the ClpB chaperone.
ClpB Is Involved in the Activation of the Dimeric Wild-type TrfA
but Not Monomeric Mutant--
Using the
oriV-dependent in vitro replication
system and utilizing crude Fraction II extracts prepared from the
E. coli clpB mutant, we investigated the replication
activity of the 33-kDa dimeric wild-type TrfA protein and the TrfA
monomeric mutant TrfA 254D/267L. After purification, the 33-kDa TrfA
wild-type protein is found mainly in a dimeric form. However, it is the
TrfA monomeric form that binds iterons within the RK2 replication
origin and initiates DNA replication (30-32). The His6-TrfA
254D/267L mutant protein contains two amino acid replacements resulting
in a high copy RK2 mutant phenotype in vivo. The mutant
His6-TrfA 254D/267L protein is fully functional in vivo (40)
and in vitro (21, 26, 33, 41-45), and is present largely in
the monomeric form (27). It was found that the replication activity of
wild-type TrfA in crude extracts prepared from the E. coli
clpB mutant strain was significantly reduced in comparison to the
activity of monomeric TrfA 254D/267L (33). Experiments performed in the
course of this work are consistent with the previous observation. The
addition of the purified ClpB chaperone to the replication mixture
containing dimeric TrfA protein increased the level of replication
3-fold as compared with the reaction without ClpB (Fig. 2A).
The observed level of replication activity of wild-type TrfA in the
absence of ClpB protein is due to activation by the ClpX chaperone that is present in the bacterial extract (33). Also, possibly other not yet
identified chaperones that are present in the extract, bring about a
limited amount of activation of the wild-type TrfA protein. In
contrast, the TrfA 254D/267L-dependent replication was not
activated by the addition of ClpB. Instead, a slight reduction of DNA
synthesis was observed (Fig. 2B). The experiments with both
wild-type TrfA and monomeric TrfA 254D/267L were performed at low and
high TrfA concentrations (Fig. 2). The results obtained were very
similar, showing that ClpB-dependent stimulation of replication is observed only with wild-type TrfA regardless of its concentration.
Activation of TrfA Requires Both ClpB and the
DnaK/DnaJ/GrpE Chaperone
Machinery--
The crude Fraction II extract used for oriC
and RK2 replication contains not only DNA replication proteins but also
other cellular components including ClpX and other
molecular chaperones. To investigate whether ClpB acts by itself or in
combination with other intracellular factors, we measured the
activation of TrfA in a system reconstituted with purified proteins. It
was previously published that the replication activity of TrfA can be
determined by analyzing FI* formation, an extensively unwound
covalently closed circular DNA replication intermediate (26). In a
control reaction when TrfA 254D/267L was used, FI* formation was easily detectable in the absence of chaperone proteins (Fig.
3, lane 2). The addition of
dimeric wild-type TrfA did not result in template conversion to the FI*
form (Fig. 3, lane 3). Surprisingly, the addition of the
ClpB chaperone to this reaction mixture did not facilitate FI*
formation indicating that additional cellular components are required
to activate TrfA.
Recently, it has been demonstrated that ClpB cooperates with the
DnaK/DnaJ/GrpE chaperone machinery in the refolding of stable protein
aggregates (7, 8). These results raised the possibility that ClpB might
cooperate with the DnaK, DnaJ, and GrpE chaperones in plasmid RK2
replication activation. The addition of purified ClpB, DnaK, DnaJ, and
GrpE chaperones to the reaction mixture containing wild-type TrfA,
oriV template, and the other required components resulted in
FI* formation, presumably because of activation of the TrfA protein
(Fig. 3, lane 6). The addition of ClpB alone or the DnaK,
DnaJ, and GrpE chaperones alone was not sufficient for TrfA activation
(Fig. 3, lanes 4 and 5).
We characterized in detail the requirements for individual chaperone
machinery components in the TrfA activation reaction. Experiments with
omissions of one or two chaperone proteins were performed (Fig.
4). Clearly, the full system, using all
four chaperones was the most efficient (Fig. 4, lanes 6 and
11). FI* formation was slightly reduced when GrpE was
omitted from the reaction (Fig. 4, lane 5). The absence of
DnaJ resulted in a large reduction of FI* formation but did not
abolished it (Fig. 4, lane 4). In contrast, the presence of
both DnaK (Fig. 4, lane 3) and ClpB (Fig. 3, lane
5) is indispensable for TrfA activation. In the absence of both
DnaJ and GrpE, reduced TrfA activation was observed (Fig. 4, lane
8). These results clearly show that ClpB and DnaK are the most
crucial components; however, TrfA activation is most efficient when the
ClpB, DnaK, DnaJ, and GrpE proteins are all present.
We also tested whether the E. coli DnaK/DnaJ/GrpE chaperone
machinery affects RK2 replication in crude extracts. The addition of
antibodies against DnaK substantially reduced the RK2 replication using
wild-type TrfA and bacterial crude extracts (Fig. 3B). In contrast the same reaction with monomeric TrfA 254D/267L was not significantly affected (data not shown). Addition of DnaK protein substantially compensates the inhibitory effect of DnaK antibody (Fig.
3B).
Activation of TrfA Is Specific for Bacterial Chaperone
Proteins--
RK2 is a broad-host-range plasmid able to transfer and
replicate its DNA in a wide spectrum of Gram-negative bacteria (19). In
contrast, RK2 DNA can be transformed to, but is not stably maintained
in, Gram-positive bacteria and Saccharomyces sp. (50). Therefore, it is an attractive candidate system for investigating the
substrate specificity of various chaperones from various organisms. Purification of the Hsp78, Ssc1, Mdj1, and Mge1 yeast mitochondrial homologs of E. coli ClpB, DnaK, DnaJ, and GrpE proteins,
allowed us to address the question regarding host specificity for the chaperone-mediated activation of TrfA protein. We analyzed FI* formation after addition of Saccharomyces cerevisiae
chaperone proteins to the reaction with wild-type TrfA. The results
presented in Fig. 5, showed that in
contrast to the E. coli system, the yeast
Hsp78/Ssc1/Mdj1/Mge1 homologous system was not sufficient for TrfA
activation (compare lanes 3 and 4). In contrast
to the TrfA activation, both the bacterial and yeast chaperone proteins were capable of reactivation of chemically denatured luciferase (data
not shown).
The
ClpB/DnaK/DnaJ/GrpE
Chaperone System Activates TrfA by Converting Inactive Dimers to Active
Monomers--
The experiments using the analysis of FI* formation
clearly showed that the ClpB/DnaK/DnaJ/GrpE system activates RK2 DNA
replication initiated by the largely dimeric TrfA protein. Moreover, it
was shown that only dimers of wild-type TrfA, but not monomeric form (TrfA 254D/267L) require chaperone activation. Using glycerol gradient
sedimentation we analyzed the TrfA quaternary structure before and
after incubation with ClpB, DnaK, DnaJ, and GrpE. In control
experiments, in the absence of chaperone proteins, TrfA was detected in
fractions corresponding to the position of the dimeric form of the
protein (approximate molecular mass of 74.8 kDa)(Fig.
6A). When TrfA was incubated
in the presence of the ClpB/DnaK/DnaJ/GrpE chaperones prior to
centrifugation, the TrfA protein was found in fractions corresponding
to the size of a monomer (approximate molecular mass of 37.4 kDa) since
very little, if any, TrfA protein was detected in the fraction
corresponding to that of the TrfA dimer (Fig. 6B). The
conversion of TrfA dimers to monomers appears to be very efficient.
After sedimentation, any residual protein in the bottom of each tube
was solubilized with 10% SDS to analyze for the presence of aggregated
proteins. Insignificant amounts of pelleted protein were found (data
not shown).
A surprising observation made during the past few years is that
many of the plasmid-encoded proteins that specifically recognize the
origin of replication bind to their corresponding DNA sites as monomer
(51). Yet, the purified protein is found mainly in the inactive dimeric
form. The pioneering studies of Wickner et al. (34)
demonstrated in vitro that the DnaK/DnaJ/GrpE chaperone machinery is capable of monomerization of the dimeric RepA replication protein of plasmid P1. These studies were motivated by earlier in
vivo observations, demonstrating that mutations in either
dnaK, dnaJ, or grpE resulted in
plasmid P1 instability (52, 53). Subsequent work demonstrated that in
addition to DnaK/DnaJ/GrpE the ClpA chaperone alone is capable of
monomerizing the P1 RepA protein (12). Our previous studies had
uncovered an analogous reaction, i.e. the monomerization of
the RK2 replication protein. In our original study the ClpX chaperone
protein was shown to be sufficient for the monomerization of the TrfA
replication protein (33). An accompanying result in this study was the
fact that the clpX deletion mutation was unaffected in the
maintenance of the RK2 plasmid. The work described here was motivated
not only by this observation, but also by our previous demonstration of a substantial reduction in TrfA activity in replication assays performed in crude extracts prepared from an E. coli clpB
mutant strain (33). Interestingly, suggestions of the possible
involvement of ClpB in DNA replication come also from investigations of
the bacteriophage Mu replication
system.1
Our results clearly showed that indeed ClpB is involved in TrfA
activation. ClpB was sufficient to stimulate RK2 replication in crude
extracts prepared from clpB mutant that also contained other
chaperones (Figs. 1 and 2). Experiments with a fully reconstituted system, where all components were added in purified form (Figs. 3A and 4), revealed that in addition to ClpB other factors
are required in TrfA activation. We found that, in addition to ClpB, the DnaK, DnaJ, and GrpE chaperones are necessary for this reaction. The TrfA activation was shown to be the consequence of the conversion of its dimeric form to an active monomeric one. In agreement with this
conclusion the TrfA 254D/267L mutant did not require the presence of
either ClpB or the DnaK/DnaJ/GrpE chaperones for activity in the
in vitro replication system.
Our results showing that these two chaperone systems cooperate are
consistent with recently published observations. It was demonstrated
that in vitro E. coli ClpB together with DnaK,
DnaJ, and GrpE facilitate the disaggregation of stable protein
aggregates (7-10). The exact role of particular proteins in this
bi-chaperone system as well as molecular events in the reaction are not
clear. A sequential mechanism with ClpB initially binding to the
substrate protein, followed by Hsp70 protein action, was postulated
(7). The function of ClpB has been defined to some extent. Based on the
growth defect phenotype of E. coli clpB mutants, it has been proposed that ClpB is a factor involved in thermotolerance (3, 4).
Thermally aggregated proteins were shown to be dissolved in wild-type
cells but not in a clpB mutant strain upon shifting the
cells to lower temperature (5, 6).
Our observation that the ClpB/DnaK/DnaJ/GrpE bi-chaperone system plays
a role in plasmid RK2 DNA replication initiation defines a novel
function of ClpB. Thus, the ClpB/DnaK/DnaJ/GrpE system is involved not
only in solubilization and refolding of protein aggregates induced by
heat or other stress conditions (6), but also, like certain other
chaperone systems (12, 13, 33, 34, 37, 51, 53), it is involved in the
activation of a DNA replication factor. Detailed analysis showed that
all four proteins from the bi-chaperone system are needed for efficient TrfA activation; however, limited activation could still be obtained when GrpE and/or DnaJ were omitted (Fig. 4). Thus, the ClpB and DnaK
chaperones are the key elements of the activation system. Interestingly, in contrast to P1 plasmid replication, the Hsp70 machinery alone is not able to activate the RK2 replication initiation protein. Also, TrfA could not be converted to the active form solely by
the action of ClpB. This reveals an important difference between the
ClpB and ClpX chaperone function.
The ClpB/DnaK/DnaJ/GrpE system displays specificity for the RK2 plasmid
replication system. We did not observe a significant effect of ClpB on
replication initiation from oriC. Previous genetic and
biochemical evidence had suggested that molecular chaperones could be
involved in bacterial chromosomal DNA replication. For example, it had
been demonstrated in vitro that either DnaK or GroEL/ES
chaperones, in the presence of ATP, can protect the DnaA protein from
aggregation. Furthermore, DnaA aggregates could be dissociated by DnaK,
thus allowing the initiation of oriC DNA replication
in vitro (54, 55). In our experiments we observe only
limited reduction in oriC-dependent DNA
replication in crude Fraction II extracts prepared from the E. coli clpB mutant. This suggests that the ClpB chaperone is not an
essential factor for DnaA protein activation. Further investigations
must be performed to verify this preliminary conclusion.
It is likely that the TrfA protein, as the replication initiation
factor of the broad-host-range plasmid RK2, requires the chaperone
system for activation in diverse bacterial species. It has been
proposed that TrfA activation in so many distantly related species can
be performed by redundant chaperone systems (33). We further speculate
that in E. coli ClpB-dependent TrfA activation
plays a more important role than ClpX-dependent activation since replication in FII extract prepared from a
clpB-deleted strain is more impaired than that from a
clpX deletion strain (33). The chaperone requirements for
activation of TrfA possibly vary depending on the bacterial species. On
the other hand homologous chaperone systems isolated from diverse
organisms can perform the same biological reactions utilizing the same
substrates. For example, heat- or chemically denatured firefly
luciferase is a substrate for reactivation by the E. coli
ClpB/DnaK/DnaJ/GrpE, yeast mitochondrial Hsp78/Ssc1p/Mdj1p/Mge1p
chaperones as well as cytosolic Hsp104/Ssa1p/Ydj1p (8, 39, 49). Our
results show that TrfA is activated by bacterial but not by yeast
chaperones. Interestingly, RK2 DNA can be transferred to, but not
maintained, in Gram-positive bacteria and Saccharomyces sp.
(50).
We thank Drs. Debbie Ang and Jaroslaw
Marszalek for critical reading of the manuscript.
*
This work was supported by the Polish State Committee for
Scientific Research Grants 3P04A01422 and 6P04B02317 and
Ministry of National Education National Institutes of Health
Grant 98-349 from the United States-Polish Maria Sklodowska-Curie Fund
II.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.
Published, JBC Papers in Press, March 11, 2002, DOI 10.1074/jbc.M107580200
1
T. Baker, personal communication.
Cooperative Action of Escherichia coli ClpB Protein
and DnaK Chaperone in the Activation of a Replication Initiation
Protein*
and
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
ClpB chaperone plays a role in
oriV DNA replication. In vitro
replication reactions using oriV (pKDL19) and
oriC (pBSoriC) as DNA templates were carried out in
bacterial crude Fraction II extracts. A, extracts prepared
from an E. coli clpB deletion mutant and its wild-type
isogenic strain have been used to test oriC- and
oriV-dependent replication. The wild-type
His6-TrfA (200 ng) was added to the
oriV-dependent reaction. Relative DNA synthesis
was calculated as a percentage of nucleotide incorporation obtained in standard
reactions using extract prepared from wild-type E. coli (240 pmol for oriC and 120 pmol for oriV).
B, the wild-type His6-TrfA (200 ng) was used in the
oriV- dependent replication mixture supplemented with
various amounts of ClpB protein as indicated. C, various
amounts of ClpB protein were added to the oriC- dependent
replication mixture as indicated. Reactions shown in B and
C were carried out in extracts prepared from an E. coli clpB deletion mutant under the conditions described under
"Material and Methods."
View larger version (10K):
[in a new window]
Fig. 2.
ClpB increases the replication activity of
dimeric wild-type TrfA but not monomeric TrfA 254D/267L.
A, the wild-type His6-TrfA in the low amount of 200 ng
(filed circles) or the high amount of 2 µg (open
circles) was tested for replication activity in crude Fraction II
extract prepared from the E. coli clpB mutant. Reactions
were carried out as described under "Materials and Methods" except
that the extracts were supplemented with the indicated amounts of ClpB.
Stimulation of DNA synthesis in the reactions was calculated relative
to nucleotide incorporation obtained in the reaction without ClpB
protein i.e. 50 pmol with wild-type His6-TrfA at 200 ng or
169 pmol with wild-type His6-TrfA at 2 µg. The data has been
normalized so that 1 represents the level of DNA synthesis
measured in the absence of ClpB. B, the monomeric mutant
His6-TrfA 254D/267L in the low amount of 50 ng (filed
squares) and the high amount of 500 ng (open triangles)
was tested as described for wild-type His6-TrfA. Stimulation of DNA
synthesis was calculated relative to nucleotide incorporation obtained
in the reactions without ClpB protein, i.e. 290 pmol with
His6-TrfA 254D/267L at 50 ng or 600 pmol and with His6-TrfA 254D/267L
at 500 ng).
View larger version (21K):
[in a new window]
Fig. 3.
Both ClpB and the DnaK/DnaJ/GrpE
chaperone machinery are required for TrfA activation.
A, TrfA replication activity was determined using a
reconstituted purified protein system and a
helicase-dependent DNA unwinding assay with the supercoiled
plasmid DNA template pTJS42 as described under "Materials and
Methods." Formation of FI* (marked by a black arrow), an
extensively unwound covalently closed circular DNA form, depends upon
TrfA-dependent origin opening and helicase loading. All
reactions contained E. coli DnaA, DnaB, DnaC, gyrase, SSB,
HU, and creatine kinase. Proteins His6-TrfA (1 µg), His6-TrfA
254D/267L (500 ng), ClpB (2.5 µg), DnaK (2.2 µg), DnaJ (400 ng),
and GrpE (800 ng) were added as indicated. B, the effects of
the addition of polyclonal DnaK antibodies (serum) on oriV
DNA replication in crude extract prepared from wild-type E. coli was determined to assess the role of the DnaK protein in TrfA
activation. A wild-type His6-TrfA (2 µg) was used in the experiment.
Reactions were carried out under the conditions described under
"Materials and Methods" except that anti-DnaK antibodies and
purified DnaK protein (1.5 µg) were added to the replication
reactions when indicated.
View larger version (48K):
[in a new window]
Fig. 4.
Requirements of chaperone proteins for TrfA
activation. The formation of FI* was analyzed in vitro
using the system reconstituted with purified proteins described under
"Materials and Methods." Wild-type His6-TrfA (1 µg), His-TrfA
254D/267L (500 ng), and molecular chaperones ClpB (2.5 µg), DnaK (2.2 µg), DnaJ (400 ng), and GrpE (800 ng) were added to the reactions as
indicated.
View larger version (31K):
[in a new window]
Fig. 5.
The activation of TrfA is specific for
bacterial chaperone proteins. The activity of wild-type His6-TrfA
protein (1 µg) was analyzed by the FI* assay after addition to a
standard reaction mixture of E. coli proteins ClpB (2.5 µg), DnaK (2.2 µg), DnaJ (400 ng), and GrpE (800 ng) or their yeast
mitochondrial homologues Hsp78 (3.7 µg), Ssc1 (1.7 µg), Mdj1 (220 ng), and Mge1 (60 ng). Reactions were carried out and analyzed as
described under "Materials and Methods." His-TrfA 254D/267L (500 ng) was used as a positive control.
View larger version (40K):
[in a new window]
Fig. 6.
The ClpB/DnaK, DnaJ, GrpE chaperone system
activates TrfA by converting inactive dimers to active monomers.
Glycerol gradient analytical centrifugation was performed as described
under "Materials and Methods" after incubation of dimeric wild-type
His6-TrfA (10 µg) with (B) or without (A) ClpB
(15 µg), DnaK (11 µg), DnaJ (2 µg), and GrpE (4 µg). After
centrifugation, portions of collected fractions were analyzed by
SDS-PAGE electrophoresis followed by Western blot analysis with
polyclonal anti-TrfA antibodies. the arrow indicates the
position of the DnaK monomer (70 kDa) used as a standard during
sedimentation analysis. A wild-type His6-TrfA protein as a molecular
weight marker is shown on the far right lane.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Recipient of EMBO Young Investigator Program. To whom
correspondence should be addressed. Tel./Fax: 48-58-301-9222; E-mail: igor@biotech.univ.gda.pl.
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