From the Faculty of Pharmaceutical Sciences, Okayama
University, and § PRESTO, Japan Science and Technology
Corporation, Okayama 700-8530, Japan
Received for publication, October 23, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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DnaA protein, the initiator of chromosomal
DNA replication in Escherichia coli, seems to be
reactivated from the ADP-bound form to its ATP-bound form through
stimulation of ADP release by acidic phospholipids such as cardiolipin.
We previously reported that two potential amphipathic helixes (Lys-327
to Ile-344 and Asp-357 to Val-374) of DnaA protein are involved
in the functional interaction between DnaA and cardiolipin. In relation
to one of these helixes (Asp-357 to Val-374), we demonstrated that
basic amino acids in the helix, especially Lys-372, are vital for this interaction. In this study, we have identified an amino acid in the
second potential amphipathic helix (Lys-327 to Ile-344), which would
also appear to be involved in the interaction. We constructed three
mutant dnaA genes with a single mutation
(dnaAR328E, dnaAR334E, and
dnaAR342E) and examined the function of the mutant
proteins. DnaAR328E, but not DnaAR334E and DnaAR342E, was found to be
more resistant to inhibition of its ATP binding activity by cardiolipin than the wild-type protein. The stimulation of ADP release from DnaAR328E by cardiolipin was also weaker than that observed with the
other mutants and the wild-type protein. These results suggest that
Arg-328 of DnaA protein is involved in the functional interaction of
this protein with acidic phospholipids. We propose that acidic phospholipids bind to two basic amino acid residues (Arg-328 and Lys-372) of DnaA protein and change the higher order structure of its
ATP-binding pocket, which in turn stimulates the release of ADP from
the protein.
DnaA protein is the initiator of chromosomal DNA replication in
Escherichia coli (1). The numerous biochemical activities of
this protein enable it to play important roles not only in the
initiation reaction itself but also in the regulation of the initiation
(1). To fully understand the role of DnaA protein in initiation of DNA
replication in cells, it is important to reveal the functional domain
for each activity (2). To initiate DNA replication, the duplex DNA must
firstly be opened up to enable other replicative proteins to enter the
origin of replication. DnaA protein is responsible for this through
specific binding to the origin of replication (oriC)
followed by self-oligomerization, which causes the duplex DNA to open
up at oriC (1). The N- and C-terminal regions of this
protein are involved in the oligomerization and in the specific DNA
binding, respectively (3-6).
Adenine nucleotides bound to DnaA protein play an important role in the
regulation of replication initiation. DnaA protein has a high affinity
for both ATP and ADP, but although the ATP-binding form is active, the
ADP-binding form is inactive in DNA replication (7, 8). ATP bound to
DnaA protein is hydrolyzed to ADP by its intrinsic ATPase activity, and
this hydrolysis is involved in inactivating DnaA protein following
initiation of DNA replication (9-13). The Lys-178 and Glu-204 amino
acids of DnaA protein have been shown to be essential for ATP binding
and for ATPase activity, respectively (10, 14). The membrane binding
activity of DnaA protein also seems to be involved in the regulation of
replication initiation, through modulation of the adenine-nucleotide
binding capacity of the protein. Acidic phospholipids, in particular
cardiolipin (CL),1 decrease
DnaA protein's affinity for adenine nucleotides and activate the
ADP-bound DnaA protein to the ATP-bound form in the presence of high
concentrations of ATP by stimulating the exchange reaction of ADP with
ATP (15-18). It has been suggested that DnaA protein is activated by
acidic phospholipids to initiate DNA replication in vivo
(19-23). However, the precise molecular mechanism by which acidic
phospholipids interact with DnaA to decrease the affinity of this
protein for adenine nucleotides is still unknown. Identification of
essential amino acids involved in the DnaA-membrane interaction is an
important first step to identifying this mechanism.
A CL protection assay of DnaA protein from trypsin digestion suggested
that a potential amphipathic helix (Asp-357 to Val-374) is involved in
the membrane binding activity of DnaA protein (24). We further
identified that three basic amino acids in the helix (Arg-360, Arg-364,
Lys-372), especially Lys-372, were important for the functional
interaction between DnaA protein and CL, suggesting that this
interaction is mediated by ionic interaction between these basic amino
acids and the acidic moieties of CL (25, 26). A second potential
amphipathic helix (Lys-327 to Ile-344), adjacent to the first, has also
been demonstrated to be involved in the membrane binding activity of
DnaA protein (27). In this paper, we have identified Arg-328 in the
second amphipathic helix (Lys-327 to Ile-344) as playing an important
role in the functional interaction between the DnaA protein and CL. We
propose that a possible molecular mechanism for how acidic
phospholipids decrease the affinity of DnaA protein for adenine
nucleotides is that the interaction of such phospholipids with two
basic amino acids (Arg-328 and Lys-372) of DnaA protein causes a
conformational change of this protein's ATP-binding pocket.
Materials--
A crude extract for an oriC
complementation assay was prepared from the WM433 strain of E. coli as previously described (28). CL was purchased from Sigma.
[ Site-directed Mutagenesis and Plasmid
Construction--
Site-specific mutagenesis was performed using the
method described by Kunkel (30). Briefly, uracil-containing
single-stranded DNA of the M13 phage, containing the coding region of
the dnaA gene, was hybridized with oligonucleotide primers
representing each mutation (Fig. 1). The complementary DNA strand was
synthesized in vitro, and the resultant double-stranded DNA
was introduced into E. coli JM109 cells. The mutation was
confirmed by direct DNA sequencing, and double-stranded DNA containing
the mutation was prepared.
To construct the plasmid for overproduction of each mutant DnaA
protein, the EcoRI-HindIII region of the
double-stranded DNA was ligated with pMZ001 plasmid (10), which
contains the arabinose promoter.
To construct the plasmid for complementation analysis of the mutant
dnaA gene, we introduced the coding regions of the mutant dnaA gene (BamHI-HindIII fragments of
the double-stranded DNA) into pMZ002, which contains the wild-type
promoter of the dnaA gene (10).
Influence of CL on Release of ATP or ADP from DnaA·ATP or
DNA·ADP Complexes--
The stimulation of ATP or ADP release from
DnaA·ATP or DNA·ADP complexes by CL was examined as
described previously (18). Briefly, CL liposomes were prepared from
dried phospholipids on the bottom of glass tubes through vigorous
vortex mixing in water. The amount of phosphorus in the phospholipid
fraction was determined using the method described by Chen et
al. (31). DnaA·ATP or DNA·ADP complexes were formed by
incubation of DnaA with 1 µM [ Filter Binding Assay for ATP or ADP Binding to DnaA
Protein--
The ATP or ADP binding activity of DnaA protein was
determined by a filter binding assay (7). DnaA protein (2 pmol) was incubated with various concentrations of [ Inhibition of ATP Binding to DnaA Protein by CL--
Inhibition
of ATP binding to DnaA protein by CL was examined as described
previously (32). DnaA protein (2 pmol) was preincubated with CL at
0 °C for 5 min and further incubated with 1 µM
[ oriC DNA Replication in a Crude Extract--
Replication of
minichromosomes in a crude extract (Fraction II) was assayed as
described previously (28). Template DNA (M13E10) (200 ng, 600 pmol of
nucleotides), 200 µg of Fraction II from WM433 (dnaA204),
and DnaA protein were incubated in reaction mixtures (28) at 30 °C
for 20 min. The reaction was terminated by chilling on ice and the
addition of 10% trichloroacetic acid. Samples were passed through
Whatman GF/C glass fiber filters. The amount of radioactivity on the
filters was measured with a liquid scintillation counter, and the
amount of synthesized DNA (picomoles of nucleotides) was then calculated.
Strategy for Site-directed Mutagenesis and Purification of Mutant
DnaA Proteins--
To reveal DnaA protein's functional
membrane-binding domain, we constructed a series of mutant DnaA
proteins using site-directed mutagenesis and examined their functional
interaction with CL (25-27). We identified two mutant DnaA proteins,
with a triple substitution for Glu of Arg-360, Arg-364, and Lys-372
(DnaA431), and Arg-328, Arg-334, and Arg-342 (DnaA433), which showed a
decreased ability to interact with CL (25, 27). These results suggest not only that two potential amphipathic helixes (Lys-327 to Ile-344 and
Asp-357 to Val-374) are important in the functional interaction between
DnaA and CL, but also that this interaction is mediated by ionic
interaction between these basic amino acids and acidic moieties of CL.
In relation to the potential amphipathic helix (Asp-357 to Val-374), we
previously identified Lys-372 to be the most important amino acid of
this helix for interaction by constructing three mutant dnaA
genes, each with a single mutation (dnaAR360E, dnaAR364E, or dnaAK372E) (26). In this study, we
tried to identify an additional amino acid in the second potential
amphipathic helix (Lys-327 to Ile-344), which was important for
functional interaction between DnaA protein and CL. We again
constructed three mutant dnaA genes, each with a single
mutation, as shown in Fig. 1. The coding
region of each mutant dnaA gene was conjugated with the promoter of the arabinose operon to construct a plasmid that
overproduced the mutated DnaA protein (DnaAR328E, DnaAR334E, DnaAR342E,
or DnaA433). To avoid contamination of the purified fraction of the mutant DnaA protein with wild-type DnaA protein, the KA450 strain ( Characterization of ATP or ADP Binding Activities of the Mutant
DnaA Proteins--
Any decrease in DnaA protein's affinity for
adenine nucleotides caused by CL was monitored both by CL stimulation
of the release of ADP (or ATP) from DnaA protein and by CL inhibition
of the ATP (or ADP) binding activity of DnaA protein (15). It was
therefore necessary for the mutant DnaA proteins in this study to
maintain their ATP or ADP binding activities to examine their
functional interaction with acidic phospholipids. The ATP binding
activities of these mutant proteins were examined using a filter
binding assay (7) and a Scatchard plot analysis. As shown in Fig.
3, each mutant DnaA protein demonstrated
ATP binding activity. The Kd values of DnaAR328E,
DnaAR334E, DnaAR342E, DnaA433, and the wild-type protein for ATP were
determined to be 77, 101, 78, 127, and 57 nM, respectively.
The number of ATP-binding sites per DnaAR328E, DnaAR334E, DnaAR342E,
DnaA433, and the wild-type protein was calculated to be 0.38, 0.28, 0.30, 0.24, and 0.44, respectively. The Kd value and
the number of ATP-binding sites for the wild-type protein were nearly
the same as reported previously (7).
We also examined the ADP binding activity of these mutant DnaA proteins
in the same manner. As shown in Fig. 4,
each of the mutant DnaA proteins also demonstrated ADP binding
activity. The Kd values of DnaAR328E, DnaAR334E,
DnaAR342E, DnaA433, and the wild-type protein for ADP were determined
to be 135, 151, 151, 184, and 184 nM, respectively. The
number of ADP-binding sites for DnaAR328E, DnaAR334E, DnaAR342E,
DnaA433, and the wild-type protein was calculated to be 0.31, 0.38, 0.30, 0.22, and 0.60, respectively. The Kd value and
the number of ADP-binding sites for the wild-type protein were
nearly the same as reported previously (7).
Because all the mutant DnaA proteins in this study showed both ATP and
ADP binding activity, their functional interactions with acidic
phospholipids could be examined by monitoring either CL-dependent stimulation of the release of ATP (or ADP)
from DnaA protein or CL-dependent inhibition of ADP (or
ATP) binding activity of DnaA protein. The observed slight, but
significant, effects of these mutations, especially R334E, on the
number of ATP- or ADP-binding sites suggests that this helix (Lys-327
to Ile-344) contributes to the adenine nucleotide binding activity of
DnaA protein (see "Discussion").
Inhibition of ATP Binding Activities of the Mutant DnaA Proteins by
CL--
We previously reported that the ATP binding activity of
DnaA433 was found to be less inhibited by CL, suggesting that some of
the mutations in DnaA433 affected the functional interaction between
DnaA and CL (27). In this study we therefore compared the CL inhibition
curve for ATP binding obtained for each single mutation mutant DnaA
protein (DnaAR328E, DnaAR334E, and DnaAR342E) with that of DnaA433 or
the wild-type protein, to identify which of these amino acids was
important for this functional interaction. DnaA protein was
preincubated with CL and further incubated with [ CL-dependent Stimulation of the Release of ADP (or ATP)
from the Mutant DnaA Proteins--
We also examined the functional
interaction between each mutant DnaA protein and CL by monitoring the
release of ADP from DnaA protein in the presence or absence of CL. The
activation of ADP-bound DnaA protein by CL is mediated by CL
stimulation of ADP release from DnaA protein (15).
DnaA·[3H]ADP complex was incubated with CL at 37 °C,
and the remaining ADP-bound DnaA protein was determined by a filter
binding assay. The kapp (apparent rate constant)
can be calculated from the slope of the resultant graph (Fig.
6A). The
kapp values for DnaAR328E, DnaAR334E, DnaAR342E,
DnaA433, and the wild-type protein, in the absence of CL, were 2.2 × 10
We also examined the effect of CL on the release of ATP from these DnaA
proteins, in the same way (Fig. 7). The
results were basically the same as those obtained in relation to ADP
(Fig. 6A). The kapp values for
DnaAR328E, DnaAR334E, DnaAR342E, DnaA433, and the wild-type protein, in
the absence of CL, were 3.2 × 10 Replication Activity of the Mutant DnaA Proteins in Vitro--
We
measured DnaAR328E, DnaAR334E, DnaAR342E, DnaA433, and the wild-type
protein's ability to initiate oriC DNA replication using an
oriC complementation assay in a crude extract (28). As shown
in Fig. 8, both DnaAR328E and DnaAR342E
supported oriC DNA replication in vitro. The
specific activities of DnaAR328E and DnaAR342E, and of the wild-type
protein, were found to be 0.22, 0.09, and 0.38 × 106
units/mg of protein, respectively (1 unit of protein promotes the
incorporation of 1 pmol of nucleotides/min at 30 °C). DnaAR334E showed no oriC DNA replication activity, and neither did
DnaA433 (Fig. 8), as reported previously (27). Thus, the lack of DNA replication activity observed in DnaA433 may be due to the R334E mutation. One possible explanation for this and for the results obtained in relation to DnaAR334E, is the low stability of their complexes with ATP at high temperatures (Fig. 7). They may be unable to
form the ATP-bound form long enough to initiate oriC DNA
replication. DnaAA184V, DnaA46 and DnaA5 required longer incubation periods for expression of their replication activity; however, a time
lag for the DNA replication reaction has been previously reported for
these mutant DnaA proteins (33). For these last mutants, the time
course of DNA replication was approximately linear, as was the case for
the wild-type protein (data not shown).
Replication Activity of the Mutant DnaA Proteins in Vivo--
We
used plasmid complementation analysis with temperature-sensitive
dnaA mutants to examine the DNA replication activities of
these mutant DnaA proteins in vivo. The coding regions of
these mutant dnaA genes, and of the wild-type, were
conjugated with the wild-type dnaA promoter on pMZ002 (10).
Each resultant plasmid was introduced into a high-temperature-sensitive
dnaA46 mutant (KS1003) (34) with mutations in the
ATP-binding site, and incubations were performed at 42 or 30 °C. As
shown in Table I, the ratio of colony
formation efficiency at 42 °C to that at 30 °C of pMZ002 with
both dnaAR328E and dnaAR342E was found to be
~1, as was the case with the wild-type gene, suggesting that these
mutant DnaA proteins were able to initiate oriC DNA
replication in cells. We then performed the same experiments using a
high-temperature-sensitive dnaA508 mutant (KS1007) (34) with
a mutation in DnaA protein's N-terminal region. The results were
similar to those obtained with the dnaA46 mutant (data not
shown). This would appear to rule out the possibility that these mutant
DnaA proteins (DnaAR328E and DnaAR342E) become active by cooperating
with the DnaA46 protein at 42 °C. The colony sizes and growth rates
obtained with the temperature-sensitive dnaA mutants
carrying pMZ002 with dnaAR328E or dnaAR342E were
indistinguishable from those of cells carrying pMZ002 with the
wild-type dnaA gene (data not shown). Thus, we consider that
DnaAR328E protein, which shows a decreased activity for interaction
with CL in vitro, is able to initiate DNA replication in
cells.
In contrast, the mutant dnaA genes encoding DnaAR334E or
DnaA433 could not complement the temperature sensitivities of both the
dnaA46 and dnaA508 mutants (Table I and data not
shown). It would therefore appear that these two mutant DnaA proteins are defective for oriC DNA replication activity both
in vitro and in vivo.
DnaA433, a mutant DnaA protein with three mutations (R328E, R334E,
and R342E), shows two important defects. One such defect is in its
functional interaction with acidic phospholipids, such as CL, and the
other is a defect in oriC DNA replication. In this study, we
constructed three mutant DnaA proteins, each with a single amino acid
mutation (DnaAR328E, DnaAR334E, and DnaAR342E), and we demonstrated
that DnaAR328E shows decreased functional interaction activity with CL
and that DnaAR334E has a defect in oriC DNA replication both
in vitro and in vivo. We have therefore concluded
that the defects described for the DnaA433 mutant in relation to CL
interaction and oriC DNA replication are due to the R328E
and R334E mutations, respectively.
DnaA protein belongs to the AAA+ (ATPases
associated with a variety of cellular
activities) family (35). Sequence alignment analysis of
various AAA+ family proteins have revealed a number of
conserved domains (35). The sensor 2 domain is thought to be important
for the adenine nucleotide binding and ATPase activities of
AAA+ family proteins (35). DNA polymerase III We previously reported that Lys-372 is the most important of the
potential amphipathic helix (Asp-357 to Val-374) amino acids in the
functional interaction between DnaA and CL. In this study, we further
found that Arg-328 is the most important amino acid of the second
potential amphipathic helix (Lys-327 to Ile-344) for this interaction.
We have therefore concluded that DnaA protein may interact with CL
through ionic interactions between the acidic moieties of CL and these
two basic amino acids. Interestingly, Arg-334 (see above) is located
between these two basic amino acids. Based on these facts, we have
considered a possible model for the mechanism by which acidic
phospholipids decrease the affinity of DnaA protein for ADP (Fig.
9). In this model, we predict that CL
binding to Arg-328 and Lys-372 causes a conformational change of the
potential amphipathic helix (Lys-327 to Ile-344). This conformational
change may in turn interfere with Arg-334's ability to interact with
the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]ATP (10 mCi/mmol) and [3H]ADP (40 Ci/mmol) were from Amersham Pharmacia Biotech and DuPont, respectively.
The mutant DnaA protein and the wild-type DnaA protein were purified,
as described previously (29).
-32P]ATP
or [3H]ADP in 40 µl of buffer G (50 mM
HEPES-KOH (pH 8.0), 0.5 mM magnesium acetate, 0.3 mM EDTA, 5 mM dithiothreitol, 17% (v/v)
glycerol, 10 mM ammonium sulfate, and 0.005% Triton X-100)
at 0 °C for 15 min. CL was added, and the mixture was further
incubated at 37 °C. Samples were passed through nitrocellulose
membranes (Millipore HA, 0.45 µm) and washed with ice-cold buffer G. The radioactivity remaining on the filter was counted with a liquid
scintillation counter.
-32P]ATP or
[3H]ADP at 0 °C for 15 min in 40 µl of buffer G. Samples were passed through nitrocellulose membranes and counted as
described above.
-32P]ATP at 0 °C for 15 min in 40 µl of buffer
G. The amount of bound ATP was determined as described above.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
oriC1071::Tn10, rnhA199(Am),
dnaA17(Am), trpE9828(Am), tyrA(Am), thr, ilv, and thyA) of E. coli was used for overproduction of the proteins. Viability was
not dependent on the function of DnaA protein in the KA450 strain. The
addition of 1% arabinose caused significant overproduction of each
mutant DnaA protein (data not shown). Purification of each mutant DnaA
protein was done through ammonium precipitation, precipitation by
dialysis, and gel-filtration column chromatography, as described
previously (29). All mutant DnaA proteins were purified to homogeneity
(Fig. 2), with approximately the same
recoveries (6-9%) as for the wild-type protein (6%). The purity of
each mutant DnaA protein was more than 90%, judging by densitometric
scanning of the gel (Fig. 2). The migration of DnaA433 was a little
slower than for other proteins, as was also the case for DnaA431 (26).
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Fig. 1.
Amino acid and DNA sequences of the potential
amphipathic helix (Lys-327 to Ile-344), and the strategy for
site-directed mutagenesis.
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Fig. 2.
Purification of mutant DnaA proteins.
Active Superose 12 chromatography fractions of each mutant DnaA protein
were pooled, and 0.2 µg of each protein was subjected to
SDS-polyacrylamide gel (10%) electrophoresis and stained with
Coomassie Brilliant Blue R-250.
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Fig. 3.
ATP binding to mutant DnaA protein measured
by a filter binding assay. DnaAR328E, DnaAR334E, DnaAR342E,
DnaA433, and the wild-type protein (2 pmol) were incubated with various
concentrations of [ -32P]ATP for 15 min at 0 °C. The
amount of bound ATP was determined as described under "Experimental
Procedures" and analyzed by the Scatchard plot method.
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Fig. 4.
ADP binding to mutant DnaA proteins measured
by a filter binding assay. DnaAR328E, DnaAR334E, DnaAR342E,
DnaA433, and the wild-type protein (2 pmol) were incubated with various
concentrations of [3H]ADP for 15 min at 0 °C. The
amount of bound ADP was determined as described under "Experimental
Procedures" and analyzed by the Scatchard plot method.
-32P]ATP. The amount of bound ATP was determined
using a filter binding assay. As shown in Fig.
5, DnaAR328E was found to be more
resistant to CL-dependent inhibition of ATP binding than
the wild-type protein, as was DnaA433. The inhibition curve for DnaA433
was much the same as reported previously (27). On the other hand, the
CL inhibition curve for ATP binding to DnaAR334E or DnaAR342E was much
the same as that of the wild-type protein (Fig. 5). These results
suggest that Arg-328 is the most important of these three basic amino
acids located in the amphipathic helix (Lys-327 to Ile-344) for
functional interaction of DnaA protein with CL.
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Fig. 5.
CL inhibition of ATP binding to the mutant
DnaA proteins. DnaAR328E, DnaAR334E, DnaAR342E, DnaA433, and the
wild-type protein (2 pmol) were preincubated with the indicated amounts
of CL for 5 min at 0 °C in 40 µl buffer G, and further incubated
with 1 µM of [ -32P]ATP for 15 min in the
same buffer at 0 °C. The amount of bound ATP was determined as
described under "Experimental Procedures." Amounts of bound ATP are
shown relative to a control (without CL). 100% values of DnaAR328E,
DnaAR334E, DnaAR342E, DnaA433, and the wild-type protein, for ATP
binding are 0.32, 0.28, 0.46, 0.32, and 0.63 pmol, respectively.
4, 6.0 × 10
4, 2.2 × 10
4, 1.4 × 10
3, and 2.4 × 10
4 (s
1), respectively. The
kapp values for DnaAR328E and DnaAR342E in the
absence of CL were nearly the same as that for the wild-type protein,
suggesting that both the DnaAR328E- and DnaAR342E-ADP complexes are
stable at 37 °C. In contrast, the DnaA433-ADP complex was unstable
at 37 °C, even in the absence of CL, as reported previously (27).
The DnaAR334E·ADP complex was found to be partly unstable, suggesting
that Arg-334 is involved in the ADP binding activity of DnaA protein
(see "Discussion"). The kapp values for DnaAR328E, DnaAR334E, DnaAR342E, DnaA433, and the wild-type protein, in
the presence of CL, were 1.4 × 10
3, 2.2 × 10
3, 1.8 × 10
3, 1.7 × 10
3, and 3.4 × 10
3
(s
1), respectively. Compared with the wild-type protein,
CL stimulation of ADP release from DnaAR328E and DnaAR342E was found to
be less, suggesting that these amino acids contributed to the
functional interaction between DnaA protein and CL. We further examined
the effect of various concentrations of CL on the release of ADP from DnaA328, DnaAR342E, and the wild-type protein (Fig. 6B).
DnaAR328E showed the lowest CL stimulation of ADP release (Fig.
6B), again suggesting that Arg-328 is the most important of
the three basic amino acids in the amphipathic helix (Lys-327 to
Ile-344) for the functional interaction between DnaA protein and CL.
The ADP·DnaA433 and ADP·DnaAR334E complexes were unstable in the
presence of CL at high temperatures (Fig. 6A).
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Fig. 6.
The release of ADP from the DnaA·ADP
complex in the presence or absence of CL. The dissociation of ADP
from DnaAR328E, DnaAR334E, DnaAR342E, and DnaA433 (2 pmol) was compared
with that of the wild-type protein (2 pmol) in the presence or absence
of CL (0.01 µg/µl), as described under "Experimental
Procedures." Ct and Co denote the retained and
initial concentrations of ADP·DnaA, respectively. The Co
values obtained for DnaAR328E, DnaAR334E, DnaAR342E, DnaA433, and for
the wild-type protein were 0.62, 0.76, 0.78, 0.54, and 1.12 pmol,
respectively. A, the dissociation of ADP from DnaAR328E,
DnaAR342E, and from the wild-type protein was examined in the presence
of various concentrations of CL. The amounts of ADP remaining on DnaA
after 8 min incubation at 37 °C are shown as values relative to a
control (before the incubation). B, control values
obtained for DnaAR328E, DnaAR342E, and for the wild-type protein, were
0.32, 0.38, and 0.65 pmol, respectively.
4, 7.3 × 10
4, 2.5 × 10
4, 1.7 × 10
3, and 1.6 × 10
4
(s
1), respectively. The kapp
values for DnaAR328E, DnaAR334E, DnaAR342E, DnaA433, the wild-type
protein, in the presence of CL, were 7.5 × 10
4,
2.8 × 10
3, 7.3 × 10
4, 2.6 × 10
3, and 2.1 × 10
3
(s
1), respectively.
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Fig. 7.
The release of ATP from the DnaA·ATP
complex in the presence and absence of CL. The dissociation of ATP
from DnaAR328E, DnaAR334E, DnaAR342E, and from DnaA433 (2 pmol) was
compared with that of the wild-type protein (2 pmol) in the presence or
absence of CL (0.01 µg/ml), as described under "Experimental
Procedures." Ct and Co denote the retained and
initial concentrations of ATP·DnaA, respectively. The Co
values for DnaAR328E, DnaAR334E, DnaAR342E, DnaA433, and for the
wild-type protein, were 0.73, 0.83, 0.85, 0.67, and 1.20 pmol,
respectively.
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Fig. 8.
Replication activity of the mutant DnaA
proteins in a crude extract. DnaAR328E, DnaAR334E, DnaAR342E,
DnaA433, and the wild-type protein were incubated with 1 µM of ATP for 15 min at 0 °C. DNA replication in a
crude extract was performed for 20 min as described under
"Experimental Procedures."
Complementation analysis of temperature sensitivity of a dnaA46 mutant
with plasmids carrying the mutant dnaA genes
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
' and
'
subunits also belong to the AAA+ family (35). The
'
subunit, but not the
' subunit, has both ATPase and adenine
nucleotide binding activities. X-ray structure analysis of the DNA
polymerase III
' subunit has suggested that defects of the
'
subunit in relation to both these activities are partly due to the loss
of an arginine amino acid (corresponding to Arg-215 in the sensor 2 domain of the
' subunit), which is conserved among various
AAA+ family proteins (36). X-ray structure analysis of the
E. coli HslU protein, which also belongs to the
AAA+ family, further suggested that the conserved arginine
of this protein (Arg-393) can interact with the
and
phosphates
of ATP (37). Interestingly, Arg-334 of DnaA protein corresponds to this
arginine (35). From these previous studies, we predicted that DnaAR334E
would have a defect in its adenine nucleotide binding activity. In
fact, both the ATP·DnaAR334E and ADP·DnaAR334E complexes were
unstable at higher temperatures when compared with the wild-type protein (Figs. 6 and 7). We have therefore concluded that the Arg-334
amino acid of DnaA protein is important for the protein's interaction
with adenine nucleotides, as is the case for other AAA+
family proteins.
phosphate of ADP, resulting in the stimulation of ADP release
from the DnaA·ADP complex.
View larger version (19K):
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Fig. 9.
A proposed molecular mechanism
for how CL stimulates ADP release from DnaA protein.
To examine the role played by membrane binding of DnaA protein in
oriC DNA replication in cells, it was useful to study a mutant DnaA protein that showed decreased membrane binding activity but
normal DNA replication activity. DnaAK372E is such a mutant that has
been previously described (26). In this study, we added DnaAR328E to
mutant DnaA proteins showing decreased membrane binding activity and
normal DNA replication activity. If acidic phospholipids activate DnaA
protein in vivo, as discussed above, these mutant DnaA
proteins would be inactive in cells. Because acidic phospholipids inhibited in vitro DNA replication under some conditions
(15), it is also possible that acidic phospholipids negatively regulate the activity of DnaA protein. In this case, these mutant DnaA proteins
defective in acidic phospholipids binding would be hyperactive in DNA
replication activity and possibly cause lethality. As was the case for
DnaAK372E, the mutant dnaA gene encoding for DnaAR328E was
found to be able to normally function for DNA replication in
vivo, which would suggest that the regulation of the activity of
DnaA protein by acidic phospholipids is not essential for DNA replication in cells. However, at present, the possibility that the
remaining membrane interaction activities of these mutant proteins are
enough to activate DnaA protein in cells cannot be dismissed.
Similarly, the possibility that these mutant DnaA proteins can interact
with membranes in cells through the aid of molecular chaperones
cannot be overlooked.
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ACKNOWLEDGEMENT |
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We thank Dr. Y. Ishikawa (Oita University) for his helpful discussions.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture, Japan, and by Ground Research Announcement for Space Utilization promoted by the Japan Space Forum.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.
¶ To whom correspondence should be addressed: Faculty of Pharmaceutical Sciences, Okayama University, 1-1-1, Tsushima-naka, Okayama 700-8530, Japan. Tel./Fax: 81-86-251-7958; E-mail: mizushima@pharm.okayama-u.ac.jp.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M009643200
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ABBREVIATIONS |
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The abbreviations used are: CL, cardiolipin; AAA+, ATPases associated with a variety of cellular activities family of proteins.
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