Site-directed Mutational Analysis for the ATP Binding of DnaA
Protein
FUNCTIONS OF TWO CONSERVED AMINO ACIDS (LYS-178 AND ASP-235)
LOCATED IN THE ATP-BINDING DOMAIN OF DnaA PROTEIN IN
VITRO AND IN VIVO *
Tohru
Mizushima
§,
Tohru
Takaki
,
Toshio
Kubota
,
Tomofusa
Tsuchiya§,
Takeyoshi
Miki
,
Tsutomu
Katayama
, and
Kazuhisa
Sekimizu
¶
From the
Faculty of Pharmaceutical Sciences, Kyushu
University, Fukuoka 812-8582 and the § Faculty of
Pharmaceutical Sciences, Okayama University, Okayama 700-8530, Japan
 |
ABSTRACT |
DnaA protein, the initiator of chromosomal DNA
replication in Escherichia coli, is activated by binding to
ATP in vitro. We introduced site-directed mutations into
two amino acids of the protein conserved among various ATP-binding
proteins and examined functions of the mutated DnaA proteins, in
vitro and in vivo. Both mutated DnaA proteins
(Lys-178
Ile or Asp-235
Asn) lost the affinity for both ATP and
ADP but did maintain binding activity for oriC. Specific
activities in an oriC DNA replication system in
vitro were less than one-tenth those of the wild-type protein. Assay of the generation of oriC sites sensitive to P1
nuclease, using the mutated DnaA proteins, revealed a defect in
induction of the duplex opening at oriC. On the other hand,
expression of each mutated DnaA protein in the temperature-sensitive
dnaA46 mutant did not complement the temperature
sensitivity. We suggest that Lys-178 and Asp-235 of DnaA protein are
essential for the activity needed to initiate oriC DNA
replication in vitro and in vivo and that ATP
binding to DnaA protein is required for DNA replication-related
functions.
 |
INTRODUCTION |
Initiation of DNA replication, a key step in the regulation of
cell proliferation, seems to be regulated by the control of activity of
initiator proteins. DnaA protein, the initiator of chromosomal DNA
replication in Escherichia coli (1-4), has a high affinity
for adenine nucleotides; the ATP-binding form of DnaA protein is
active, whereas the ADP-binding form is inactive in an oriC
DNA replication system in vitro (5). Synthesized organic
compounds designed to block the ATP binding to DnaA protein specifically inhibited oriC DNA replication in
vitro (6). These observations mean that adenine nucleotide binding
to DnaA protein may regulate the activity of the protein in
vivo.
There is genetic evidence to support the notion that initiation of DNA
replication is regulated by adenine nucleotide binding to DnaA protein
in vivo; (i) spontaneous dnaA46 and
dnaA5 mutants show recessive lethality and DnaA46 and DnaA5
proteins have a decreased affinity for ATP and ADP (7, 8); (ii) a
spontaneous dnaAcos mutant shows dominant lethality due to
overinitiation of DNA replication and DnaAcos protein loses the
affinity for ATP and ADP (9, 10); (iii) site-directed mutation (Glu-204
Gln) also causes dominant lethality, and the mutated protein, DnaA
E204Q, has decreased intrinsic ATPase activity (11).
DnaA protein has Walker A and B motifs that are common to a number of
nucleotide-binding proteins (12). Analysis by site-directed mutation
for conserved amino acids located in Walker A and B motifs of these
nucleotide-binding proteins revealed the necessity of these amino acids
for nucleotide binding and also for functions related to
nucleotide-binding activity. Substitution of conserved amino acids
located in Walker A and B motifs of SecA, an essential protein for
protein translocation across cytoplasmic membranes in E. coli (13), showed that ATP binding and ATPase activity of this
protein are essential for protein translocation, in vitro and in vivo (14-17). To obtain more direct evidence that
the activity of DnaA protein is regulated by ATP binding, analysis by
site-directed mutation of amino acids essential for the ATP-binding
activity should be rewarding. We introduced site-directed mutations
into two amino acids (Lys-178 or Asp-235) of DnaA protein located in Walker A or B motif, respectively, and examined functions of the mutated DnaA proteins in vitro and in vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Wild-type DnaA protein was purified, as described
(18), but with some modifications (19-21). Specific activity of the
protein was 0.3 × 106 units/mg. Purity of the
fraction exceeded 90%, as determined by SDS-polyacrylamide (10%) gel
electrophoresis. A crude extract was prepared from WM433 strain as
described (22, 23).
[
-32P]ATP (3000 Ci/mmol) and [3H]ADP (40 Ci/mmol) were purchased from Amersham Pharmacia Biotech and DuPont. P1
nuclease and trypsin were from Yamasa Co. and Worthington Biochemical
Co., respectively.
Bacterial Strains--
JM109 (recA1, endA1, gyrA96, thi,
hsdR17, supE44, relA1,
(lac-proAB)/F'[traD36,
proAB+, lacIq, lacZ
M15]), KS1001
(W3110, lacZ), KS1003 (KS1001, dnaA46) (24, 25),
and WM433 (dnaA204, leu19, pro19, trp25, his47, thyA59, arg28,
met55, deoB23, lac11, strA56, sul1, hsdSK12) were from our
laboratory stock.
Site-directed Mutagenesis and Plasmid
Construction--
Site-specific mutation was carried out using the
methods of Kunkel (26). In brief, uracil-containing single-stranded DNA of M13 phage, which contains the coding region of the dnaA
gene1 was hybridized with the
oligonucleotide primer, 5'-CGGGTCTGGGTATCACTCACCTGCTG-3', or 5'-CAAAAAACTGAATATCGTTGATCAGCAGTG-3, containing a
mismatch sequence for replacement of Lys-178 with Ile or Asp-235 with
Asn, respectively (the changed bases are underlined). The complementary DNA strand was synthesized in vitro, and the resultant
double-stranded DNA was introduced into JM109 cells. The mutation was
confirmed by DNA sequencing, and double-stranded DNA (pMZ000-3 or
pMZ000-4), with a mutation for replacement of Lys-178 with Ile or
Asp-235 with Asn, respectively, was prepared.
For overproduction of the mutant DnaA protein, we used the pMZ001
plasmid (11), which contains the arabinose promoter (9). The
EcoRI-HindIII region of pMZ000-3 or pMZ000-4 was
ligated with pMZ001. The resultant plasmid was named pMZ001-3 or
pMZ001-4, respectively, and used for overproduction of the mutant DnaA
protein.
For analysis of the function of the mutant dnaA gene
in vivo, we introduced the coding region of the mutant
dnaA gene under the promoter of the wild-type
dnaA gene. The BamHI-HindIII fragment of pMZ000-3 or pMZ000-4 was ligated to pMZ002 (11), which contains the
wild-type promoter of the dnaA gene to construct pMZ002-3 (dnaA401) or pMZ002-4 (dnaA402),
respectively.
Filter-binding Assay for ATP and ADP Binding to DnaA
Protein--
ATP- and ADP-binding activity of DnaA protein was
determined by the filter-binding assay (5). DnaA protein (2 pmol) was incubated with [
-32P]ATP or [3H]ADP at
0 °C for 15 min in 40 µl of buffer G (50 mM
Tricine2-KOH (pH 8.25), 0.5 mM magnesium acetate, 0.3 mM EDTA, 7 mM dithiothreitol, 20% (v/v) glycerol, and 0.007% Triton
X-100). Samples were passed through nitrocellulose membranes (Millipore
HA, 0.45 mm) and washed with ice-cold wash buffer (50 mM
Tricine-KOH (pH 8.25), 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). The radioactivity remaining on the filters was counted in a
liquid scintillation counter.
Limited Digestion of DnaA Protein by Trypsin--
Trypsin
cleavage of DnaA protein was performed as described (27), but with some
modifications. DnaA protein (8 pmol) was preincubated with ATP or ADP
for 15 min at 0 °C and further incubated with 160 ng of trypsin in
buffer G at 30 °C for 20 min. The incubation was terminated by
addition of SDS-sample buffer, and samples were heat-treated. After
SDS-polyacrylamide gel electrophoresis, DnaA protein was visualized by
staining with Coomassie Brilliant Blue R-250.
oriC DNA Replication in a Crude Extract--
Replication of
minichromosomes in a crude extract (Fraction II) was assayed, as
described (22, 23). Template DNA (M13E10) (200 ng, 600 pmol as
nucleotides), 240 µg of Fraction II from WM433 (dnaA204),
and DnaA protein were mixed with reaction mixtures (22, 23) and
incubated for replication at 30 °C for 20 min. The reaction was
terminated by chilling on ice and adding 10% trichloroacetic acid.
Samples were passed through Whatman GF/C glass-fiber filters. The
amount of radioactivity on the filters was measured in a liquid
scintillation counter, and the amount of DNA synthesized (picomoles of
nucleotides) was calculated (22, 23).
Assay for Generation of oriC Sites Sensitive to P1
Nuclease--
Generation of oriC sites sensitive to P1
nuclease was measured as described (4), but with some modifications.
DnaA protein (5 pmol) was incubated with 150 fmol of pBSoriC
(4) and 67 ng of HU protein in buffer (50 µl) containing 60 mM HEPES/KOH (pH 8.0), 320 µg/ml bovine serum albumin, 8 mM magnesium acetate, 30% (v/v) glycerol, and 5 mM ATP for 1 min at 38 °C. P1 nuclease (4.5 units,
Yamasa) was added, and the P1 cleavage reaction was terminated after
25 s by adding 20 µl of 50 mM EDTA and 1% SDS. Samples were used for agarose (1.2%) gel electrophoreses followed by
ethidium bromide staining.
 |
RESULTS AND DISCUSSION |
Construction and Purification of Mutant DnaA Proteins--
Yoshida
and Amano (28) compared amino acid sequences of various ATP-binding
proteins and predicted that Lys-178 and Asp-235 of DnaA protein,
located in Walker A and B motifs, respectively, are essential for its
ATP-binding activity (28). They also predicted that Glu-204 of DnaA
protein is a catalytic residue for intrinsic ATPase activity of the
protein (28). These amino acids are conserved among DnaA proteins from
various species of bacteria (1). As for Glu-204, when we replaced
glutamic acid with glutamine, we found that the residue is required for
the intrinsic ATPase activity of DnaA protein (11). Characterization of
the mutant DnaA protein (DnaA E204Q) revealed the role of the ATPase
activity, that is negative regulation of initiation of DNA replication
in cells (11). We introduced mutation into Lys-178 or Asp-235 of DnaA protein and examined the function of the mutated DnaA proteins in
vitro and in vivo, the objective being to better
understood functions of these amino acids and the role of the
ATP-binding activity in DNA replication.
Site-directed mutation in the dnaA gene was done to change
Lys-178 to Ile or Asp-235 to Asn in order to construct the mutant dnaA gene, dnaA401 or dnaA402,
respectively. A coding region of the dnaA401 or
dnaA402 gene was conjugated with the promoter of the
arabinose operon to construct a plasmid for overproduction of the
mutated DnaA protein (DnaA K178I or DnaA D235N). To avoid contamination
of the wild-type DnaA protein in the fraction of the mutant DnaA
protein, the KA450 strain with deletion of the dnaA gene on
chromosome DNA (9) was used as the host for overproduction. Addition of
1% arabinose led to overexpression of each mutant DnaA protein.
Through purification procedures, we examined the mutated DnaA proteins
by immunoblotting with affinity-purified anti-DnaA serum (Table
I). Since mutant DnaA proteins (DnaA
K178I and DnaA D235N) were recovered in insoluble fractions after
centrifugation of the cell lysate (data not shown), the mutant DnaA
proteins were solubilized with guanidine HCl, followed by gel
filtration column chromatography. Aggregated and monomer forms of the
mutant proteins were recovered (data not shown) as in the case of the wild-type DnaA protein (18). The monomer form of DnaA K178I and DnaA
D235N was purified to apparent homogeneity, with a 13% recovery (Table
I). Purity of each final fraction (fraction III) exceeded 90%, as
determined by SDS-polyacrylamide gel electrophoresis (Fig.
1).
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Table I
Purification of DnaA K178I and DnaA D235N
Strain KA450 transformed with pMZ001-3 or pMZ001-4 was grown in 20 liters of LB medium containing 25 µg/ml thymine at 37 °C until the
optical density at 595 nm reached 0.5, then arabinose was added to a
level of 1%. After 1 h of incubation at 37 °C, the cells were
harvested by centrifugation, resuspended in buffer C (18) containing
250 mM KCl to an optical density at 595 nm of 220, and
stored at 80 °C. Thawed cell suspension was diluted 2-fold with
buffer C containing 250 mM KCl, and spermidine-HCl and egg
white lysozyme were added to the final concentrations of 20 mM and 400 mg/ml, respectively. After incubation at 0 °C
for 30 min, the sample was frozen in liquid nitrogen. The lysate was
thawed and centrifuged for 30 min at 20,000 rpm in a Beckman TL100.3
rotor. The precipitates (Fraction I) were washed twice with buffer C
containing 250 mM KCl and finally resuspended in buffer C
containing 4 M guanidine HCl, 250 mM KCl.
Insoluble materials were removed by centrifugation for 30 min at 50,000 rpm in a rotor Beckman TLA100.3. The supernatant (Fraction II) was
gel-filtered on a Superose 12 column (Pharmacia fast protein liquid
chromatography HR10/30) equilibrated with buffer D (18) at a flow rate
of 0.3 ml/min, and DnaA fractions were pooled (Fraction III). Through
purification, DnaA protein was monitored and determined by
immunoblotting (18).
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Fig. 1.
Isolation of DnaA K178I and D235N.
Protein fractions from Table I were applied on SDS-polyacrylamide
(10%) gel and stained with Coomassie Brilliant Blue R-250
(A, DnaA K178I; B, DnaA D235N).
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|
Characterization of ATP- and ADP-binding Activity of DnaA K178I and
DnaA D235N--
DnaA protein (DnaA K178I, DnaA D235N, or the wild-type
protein) and various concentrations of [
-32P]ATP were
incubated, and the amount of ATP bound to the protein was determined by
filter-binding assay (5). As shown in Fig. 2A, the mutant DnaA proteins
were defective in high affinity binding to ATP; even in the presence of
1 µM ATP, a negligible amount of ATP bound to the mutant
DnaA proteins. As for the wild-type DnaA protein, the
Kd value of the protein for ATP was 30 nM, a value much the same as noted earlier (5). We also examined high affinity binding of the mutant DnaA proteins for ADP. As
shown in Fig. 2B, both mutant DnaA proteins were inert for
the high affinity binding for ADP. The Kd value of the binding of ADP to the wild-type DnaA protein was 100 nM, that is much the same as reported elsewhere (5).

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Fig. 2.
ATP and ADP binding to DnaA K178I and D235N,
measured by filter-binding assay. DnaA K178I and DnaA D235N or the
wild-type DnaA protein (2 pmol) was incubated with various
concentrations of [ -32P]ATP (A) or
[3H]ADP (B) for 15 min at 0 °C. The amount
of bound ATP or ADP was determined by filter-binding assay, as
described under "Experimental Procedures."
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|
Since the low affinity binding of ATP to DnaA protein could not be
detected by the filter-binding assay we used, the observations described in Fig. 2 did not exclude the possibility that mutant DnaA
protein can bind to ATP or ADP in the presence of high concentrations of ATP or ADP. Since ATP concentrations in cells and in an
oriC DNA replication system in vitro are
relatively high (more than 1 mM), ATP binding of these
proteins should be examined in the presence of high concentrations of
ATP. We investigated the ATP- or ADP-binding activity of the mutant
DnaA proteins (DnaA K178I and DnaA D235N) by the ATP- or
ADP-dependent formation of trypsin-resistant peptide (27,
29). DnaA K178I, DnaA D235N, or the wild-type protein was preincubated
with various concentrations of ATP or ADP at 4 °C and then further
incubated with trypsin. Limited trypsinolysis of the wild-type DnaA
protein produced a predominant 30-kDa peptide, in an ATP- or
ADP-dependent manner (Fig.
3), much the same as noted elsewhere (27,
29). In the case of DnaA K178I and DnaA D235N, the 30-kDa peptide was
not detected, even in the presence of 2 mM ATP or ADP (Fig.
3). Therefore, the mutant DnaA proteins apparently could not bind to
ATP or ADP, even in the presence of high concentrations of ATP or
ADP.

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Fig. 3.
Limited trypsin digestion of DnaA K178I and
D235N in the presence of ATP or ADP. K178I, D235N, or the
wild-type DnaA protein (4.8 pmol) was preincubated with ATP (1 µM or 2 mM) or ADP (1 µM or 2 mM) at 0 °C for 15 min and further incubated with
trypsin, as described under "Experimental Procedures." Samples
applied to SDS-polyacrylamide gel (10%) electrophoresis were
visualized by staining with Coomassie Brilliant Blue R-250.
|
|
Replication Activity of DnaA K178I and DnaA D235N in Vitro--
We
measured the replication activity of DnaA K178I and DnaA D235N in an
oriC complementation assay (22, 23). As shown in Fig.
4, DnaA K178I and DnaA D235N were less
active than the wild-type protein for DNA replication. The specific
activity of these mutant proteins was less than one-tenth that of the
wild-type protein. DnaA A184V, DnaA46, and DnaA5 required longer
incubation periods for expression of replication activity; the time lag
for DNA replication reaction for these mutant DnaA proteins has been reported (16, 17, 27). In the case of DnaA K178I and DnaA D235N, the
time course of DNA replication is approximately linear, as is the case
for the wild-type protein (Fig. 4B). Preincubation of the
wild-type DnaA protein with 1 µM ADP (but not the mutant proteins) inhibited replication activity (Fig. 4A), findings
consistent with our observations that these mutant proteins cannot bind
to ADP (Figs. 2 and 3). Therefore, these amino acids (Lys-178 and Asp-235) are apparently needed for replication activity of DnaA protein
in vitro.

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Fig. 4.
Replication activity of DnaA K178I and DnaA
D235N in a crude extract. K178I, D235N, or the wild-type DnaA
protein (A: indicated amounts; B:
DnaA+, 1 pmol; DnaA K178I and DnaA D235N, 1.5 pmol) was
incubated with 1 µM ATP (A, B) or
ADP (A) for 15 min at 0 °C. DNA replication in a crude
extract was done (A, 20 min; B, indicated
periods), as described under "Experimental Procedures."
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oriC-binding Activity of DnaA K178I and DnaA D235N--
DnaA
protein, which specifically binds to the oriC sequence,
causes duplex opening (4). The binding of ATP or ADP to wild-type DnaA
protein does not affect binding to oriC (5). When we
examined oriC-binding activity of the mutant DnaA proteins
by filter-binding assay (20), the binding activity of DnaA K178I and
DnaA D235N was indistinguishable from that of the wild-type protein, as
shown in Fig. 5. Binding of the mutant
DnaA proteins to the oriC DNA was competed for by nonlabeled
oriC DNA but not by
X174 DNA (data not shown), which has
no DnaA box, as in the case of the wild-type DnaA protein. Thus, mutant
DnaA proteins can specifically bind to oriC DNA. These
observations suggest that mutation (K178I or D235N) in the ATP-binding
domain does not affect the oriC-binding activity of DnaA
protein. These results were also interpreted to mean that the mutation
(K178I or D235N) specifically affects the ATP-binding activity of DnaA
protein, the result being low replication activity of the proteins.

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Fig. 5.
oriC-binding activity of DnaA K178I and
D235N. K178I, D235N, or the wild-type DnaA protein was incubated
for 5 min at 30 °C with 25 fmol (10000 cpm) of 3'-end-labeled
pBSoriC (4). Samples were passed through membranes
(Millipore, HAWP), and the retained radioactivity was counted. The
amount of bound DNA to DnaA protein was determined as described
(10).
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Activity of DnaA K178I and DnaA D235N for Duplex Opening at
oriC--
Because our observations suggested that the mutant DnaA
proteins showed low replication activity as a result of loss of the affinity for adenine nucleotides we asked which stage of DNA
replication would defects in the mutant DnaA (DnaA K178I and DnaA
D235N) be evident. It was reported that the ATP- but not ADP-binding
form of DnaA protein is active for duplex opening, as monitored by generation of a sensitive site at oriC to P1 nuclease (4,
5). We compared the potential of mutant DnaA proteins to that of the wild-type protein with regard to duplex opening. As shown in Table II, generation of oriC sites
sensitive to P1 nuclease (proportion of linear molecules) was
stimulated by adding wild-type DnaA protein but not by exposure to DnaA
K178I and DnaA D235N, which suggested that these mutant proteins are
not active in duplex opening at oriC. When the wild-type
DnaA protein was preincubated with ADP, the generation of
oriC sites sensitive to P1 nuclease was not stimulated by
the protein (Table II), as described (5). Preincubation of the mutant
DnaA proteins with ADP did not alter their potential for generation of
oriC sites sensitive to P1 nuclease (Table II). Based on
these observations, we attribute the low activity of the mutant
proteins (DnaA K178I and DnaA D235N) for DNA replication in
vitro to their lack of potential for duplex opening at
oriC. Thus, the thesis that the ATP binding to DnaA protein
is required for duplex opening at oriC is given support.
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Table II
Activity of DnaA K178I and DnaA D235N involved in duplex opening at
oriC
K178I, D235N, or the wild-type DnaA protein was preincubated with 1 µM ATP or ADP for 15 min and further incubated with
pBSoriC (4) and P1 nuclease, as described under
"Experimental Procedures." Samples were applied to agarose (1.2%)
gel electrophoresis, and densitometric scanning of ethidium
bromide-staining gels was performed. Relative amounts of linear
pBSoriC to that of input DNA are given.
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Activity of DnaA K178I and DnaA D235N for DNA Replication in
Vivo--
To examine functions of these amino acids (Lys-178 and
Asp-235) of DnaA protein in vivo, we did plasmid
complementation experiments with the mutant dnaA genes
(dnaA401 and dnaA402), using a
temperature-sensitive dnaA46 mutant. Since results from
biochemical studies on DnaA K178I and DnaA D235N revealed that both
mutant proteins lose the affinity for ATP, examination of the activity
of the mutant DnaA proteins in cells may yield important information on
the physiological roles of ATP-binding activity of DnaA protein. The
coding region of dnaA401 (DnaA K178I), dnaA402
(DnaA D235N), or the wild-type dnaA gene was conjugated with
the wild-type dnaA promoter on pMZ002 (11), and each
resultant plasmid was introduced into a temperature-sensitive dnaA46 mutant (KS1003) followed by incubation at 42 °C or
30 °C. As shown in Table III, the
ratio of transformation efficiencies at 42 °C to that at 30 °C of
pMZ002-3 (dnaA401) or pMZ002-4 (dnaA403) was less
than 1/105, whereas that of pMZ002-2 (wild-type) (11) was
0.65. We confirmed that approximately the same amount of the mutant and
wild-type DnaA proteins was expressed in the KA450 strain (9), as based on immunoblotting analysis (data not shown). These results suggest that
DnaA K178I and DnaA D235N cannot initiate DNA replication at 42 °C
in the dnaA46 mutant and that Lys-178 and Asp-235 of DnaA
protein are essential for activity for DNA replication in cells. The
ATP-binding capacity of DnaA protein is apparently essential for
replication activity of DnaA protein.
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Table III
Complementation analysis of temperature sensitivity of a dnaA46 mutant
with plasmids carrying the mutant dnaA gene
KS1003 (dnaA46) cells were transformed with pMZ002-2
(dnaA+), pMZ002-3 (dnaA401), or
pMZ002-4 (dnaA402) plasmids, which have the dnaA
gene encoding the wild-type, K178I, or D235N DnaA protein,
respectively. Cultures were diluted and spread on LB agar plates
containing 50 µg/ml ampicillin. Plates were incubated at 42 °C or
30 °C, the number of colonies was counted, and the transformation
efficiency was determined.
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|
 |
ACKNOWLEDGEMENT |
We thank M. Ohara for comments on the
manuscript.
 |
FOOTNOTES |
*
This work was supported in part by grants-in-aid for
scientific research from the Ministry of Education, Science, Sports and Culture, Japan.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, Kyushu University, Fukuoka 812-8582, Japan. Tel.: 81-92-642-6641; Fax: 81-92-642-6646; E-mail:
sekimizu{at}bisei.pharm.kyushu-u.ac.jp.
The abbreviation used is:
Tricine, N-[2-hydroxy-1,1-bis
(hydroxymethyl)ethyl]glycine.
1
L. Guo, T. Miki, and K. Sekimizu, unpublished
data.
 |
REFERENCES |
-
Skarstad, K.,
and Boye, E.
(1994)
Biochim. Biophys. Acta
1217,
111-130[Medline]
[Order article via Infotrieve]
-
Hirota, Y.,
Mordoh, J.,
and Jacob, F.
(1970)
J. Mol. Biol.
53,
369-387[Medline]
[Order article via Infotrieve]
-
Fuller, R. S.,
Funnell, B. E.,
and Kornberg, A.
(1984)
Cell
38,
889-900[Medline]
[Order article via Infotrieve]
-
Bramhill, D.,
and Kornberg, A.
(1988)
Cell
52,
743-755[Medline]
[Order article via Infotrieve]
-
Sekimizu, K.,
Bramhill, D.,
and Kornberg, A.
(1987)
Cell
50,
259-265[Medline]
[Order article via Infotrieve]
-
Mizushima, T.,
Sasaki, S.,
Ohishi, H.,
Kobayashi, M.,
Katayama, T.,
Miki, T.,
Maeda, M.,
and Sekimizu, K.
(1996)
J. Biol. Chem.
271,
25178-25183[Abstract/Free Full Text]
-
Hupp, T. R.,
and Kaguni, J. M.
(1993)
J. Biol. Chem.
268,
13128-13136[Abstract/Free Full Text]
-
Hwang, D. S.,
and Kaguni, J. M.
(1988)
J. Biol. Chem.
263,
10633-10640[Abstract/Free Full Text]
-
Katayama, T.
(1994)
J. Biol. Chem.
269,
22075-22079[Abstract/Free Full Text]
-
Katayama, T.,
and Kornberg, A.
(1994)
J. Biol. Chem.
269,
12698-12703[Abstract/Free Full Text]
-
Mizushima, T.,
Nishida, S.,
Kurokawa, K.,
Katayama, T.,
Miki, T.,
and Sekimizu, K.
(1997)
EMBO J.
16,
3724-3730[Abstract/Free Full Text]
-
Walker, J. E.,
Saraste, M.,
Runswick, M. J.,
and Gay, N. J.
(1982)
EMBO J.
1,
945-951[Medline]
[Order article via Infotrieve]
-
Matsuyama, S.,
and Mizushima, S.
(1995)
Advances in Cell and Molecular Biology of Membrane and Organelles, Vol. 4, pp. 61-84, JAI Press, Greenwich, CT
-
Wolk, J. V. d.,
Klose, M.,
Breukink, E.,
Demel, R. A.,
Kruijff, B. D.,
Freudl, R.,
and Drissen, A. J. M.
(1993)
Mol. Microbiol.
8,
31-42[Medline]
[Order article via Infotrieve]
-
Mitchell, C.,
and Oliver, D.
(1993)
Mol. Microbiol.
10,
483-497[Medline]
[Order article via Infotrieve]
-
van der Wolk, J. P. W.,
Klose, M.,
de Wit, J. G.,
den Blaauwen, T.,
Freudl, R.,
and Drissen, A. J. M.
(1995)
J. Biol. Chem.
270,
18975-18982[Abstract/Free Full Text]
-
Sato, K.,
Mori, H.,
Yoshida, M.,
and Mizushima, S.
(1996)
J. Biol. Chem.
271,
17439-17444[Abstract/Free Full Text]
-
Sekimizu, K.,
Yung, B. Y.,
and Kornberg, A.
(1988)
J. Biol. Chem.
263,
7136-7140[Abstract/Free Full Text]
-
Mizushima, T.,
Ishikawa, Y.,
Obana, E.,
Hase, M.,
Kubota, T.,
Katayama, T.,
Kunitake, T.,
Watanabe, E.,
and Sekimizu, K.
(1996)
J. Biol. Chem.
271,
3633-3638[Abstract/Free Full Text]
-
Mizushima, T.,
Katayama, T.,
and Sekimizu, K.
(1996)
Biochemistry
35,
11512-11516[CrossRef][Medline]
[Order article via Infotrieve]
-
Kubota, T.,
Katayama, T.,
Ito, Y.,
Mizushima, T.,
and Sekimizu, K.
(1997)
Biochem. Biophys. Res. Commun.
232,
130-135[CrossRef][Medline]
[Order article via Infotrieve]
-
Fuller, R. S.,
Kaguni, J. M.,
and Kornberg, A.
(1981)
Proc. Natl. Acad. Sci. U. S. A.
78,
7370-7374[Abstract]
-
Fuller, R. S.,
and Kornberg, A.
(1983)
Proc. Natl. Acad. Sci. U. S. A.
80,
5817-5821[Abstract]
-
Shinpuku, T.,
Mizushima, T.,
Guo, L.,
Miki, T.,
and Sekimizu, K.
(1995)
Biochem. Biophys. Res. Commun.
212,
84-89[CrossRef][Medline]
[Order article via Infotrieve]
-
Mizushima, T.,
Yokoyama, K.,
Mima, S.,
Tsuchiya, T.,
and Sekimizu, K.
(1997)
J. Biol. Chem.
272,
21195-21200[Abstract/Free Full Text]
-
Kunkel, T. A.
(1985)
Proc. Natl. Acad. Sci. U. S. A.
82,
488-492[Abstract]
-
Carr, K. M.,
and Kaguni, J. M.
(1996)
Mol. Microbiol.
20,
1307-1318[Medline]
[Order article via Infotrieve]
-
Yoshida, M.,
and Amano, T.
(1995)
FEBS Lett.
359,
1-5[CrossRef][Medline]
[Order article via Infotrieve]
-
Yung, B. Y.,
and Kornberg, A.
(1989)
J. Biol. Chem.
264,
6146-6150[Abstract/Free Full Text]
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