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INTRODUCTION |
In the Escherichia coli chromosome, the adenine
residues of GATC sequences are recognized and methylated by Dam
methylase (1-3). The protein transfers a methyl group from a methyl
donor, S-adenosylmethionine, to the 6-amino group of adenine
residues in hemimethylated and unmethylated GATC sequences (4). Until methylated by Dam methylase, the newly replicated GATC sequences exist
in a hemimethylated state that in the parental strand is methylated and
in the nascent strand is not. Dam methylase is a monomer with a
molecular mass of 31 kDa (5). Each E. coli cell possesses
approximately 130 molecules of Dam methylase (5). Methylation of GATC
sites by Dam methylase is implicated in regulation of E. coli chromosomal replication (6-10) and methyl-directed mismatch
repair (11-13). Also, Dam function plays a role in regulation of
certain genes such as dnaA (6, 14) and Mu mom
(15).
Regulation of E. coli chromosomal replication is strict and
occurs mainly at the origin of E. coli chromosomal
replication, oriC (16, 17). Within the 245 base pairs of
oriC are 11 repeats of GATC sequences (18); fully methylated
oriC is active for initiation of replication, whereas
hemimethylated oriC is inactive for initiation of
replication (6, 8, 20). In E. coli with a doubling time of
30 min, methylation of oriC by Dam methylase is delayed for
13 min after replication (21). In seqA mutants, methylation
occurs after only 5 min. These results indicate that the 8-min
difference is in part caused by sequestration of oriC by
SeqA protein encoded by seqA gene. SeqA preferentially binds to hemi- compared with fully and unmethylated oriC (22, 25). seqA null mutant exhibits asynchronious and increased
chromosomal initiation, indicating that SeqA protein is a negative
modulator of E. coli chromosomal replication (19, 21, 34).
Also, SeqA protein was independently identified as a protein that bound
to the hemimethylated bacteriophage P1 origin (22).
To understand the in vivo role of SeqA and Dam proteins on
hemimethylated oriC, we studied biochemical functions of the
two proteins on the three AT-rich 13-mer sequences termed L, M, and R
containing four GATC sites at the left end of oriC (23). In addition, footprints demonstrate that SeqA protein binds hemimethylated GATC sequences.
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EXPERIMENTAL PROCEDURES |
Reagent--
Sources were as follows: [
-32P]ATP
(5000 Ci/mmol), Amersham Pharmacia Biotech; poly(dI-dC) and
Sephadex-G50, Amersham Pharmacia Biotech; long ranger polyacrylamide,
FMC Corp. Bioproducts; T4 polynucleotide kinase, New England Biolabs
Inc.; restriction and cloning enzymes, Promega; and nitrocellulose
filter (HAWP, 0.45 µM), Millipore. Unless otherwise
indicated, other reagents were purchased from Sigma.
Bacterial Strains and Plasmid DNAs--
The E. coli
strain DH5
(24) for fully methylated plasmid DNA and
GM3819(dam16:Kmr) (7) for
unmethylated plasmid DNA were used for isolation of plasmid DNA.
E. coli MC1061 (24) and BL21 (24) were used for the
overproduction of Dam and SeqA proteins, respectively. The plasmids
pSS1 (25), pBMA1 (26), pBAD18 (27), and pBluescript SK(+) (Stratagene)
were previously described. To construct pBAD18-Dam, the coding region
of Dam was obtained by polymerase chain reaction and inserted into the
EcoRI and HindIII sites of vector pBAD18.
Proteins--
SeqA protein was purified from BL21(pLys, pSS1) as
described previously (22, 25) with modifications. Following a 2-h
induction after the addition of
isopropyl-1-thio-
-D-galactopyranoside to 0.5 mM at A600 = 0.4, cells were
harvested by centrifugation, resuspended in suspension buffer (25 mM HEPES-KOH (pH 7.5), 1 mM EDTA, 10% sucrose,
and 1 mM DTT,1)
frozen in liquid nitrogen, and stored at
80 °C. On thawing, cells
were autolysed by T7 lysozyme expressed from plasmid pLys. Lysis was
augmented by the addition of 1/10 volume of lysis salts (2.5 M KCl, 0.2 M EDTA, 0.2 M
spermidine, and 10 mM DTT). The autolysed cells were
centrifuged for 45 min at 45,000 rpm in a Beckman 70Ti rotor. The
supernatant was precipitated by the addition of 0.35 g/ml ammonium
sulfate followed by centrifugation for 15 min in a Kontron A8.24 rotor
and resuspended in buffer A (50 mM Tris (pH 7.5), 5 mM EDTA, and 1 mM DTT). The resuspended
material was dialyzed against buffer A containing 50 mM KCl
and loaded onto a heparin-agarose column equilibrated with buffer A
containing 50 mM KCl. SeqA protein was eluted by a linear
gradient of 10 column volumes of 50 mM to 1 M
KCl in buffer A. Active fractions determined in gel-shift assays were
pooled and loaded onto a hydroxyapatite column equilibrated with buffer
D (20 mM potassium phosphate (pH 6.8), 10% glycerol, 1 mM DTT, and 100 mM KCl). The column was washed
with buffer D, then with buffer D containing 0.4 M ammonium sulfate. Near homogeneous and concentrated (1.38 mg/ml) SeqA protein was obtained by elution with buffer D containing 1 M
ammonium sulfate.
Dam methylase was purified from MC1061 (pBAD18-Dam) as described
previously (28) except the blue-Sepharose column was omitted.
Gel-shift Assay--
To obtain differently methylated duplex
13-mers (Fig. 1), synthesized oligonucleotides containing 13-mer
regions were mixed with appropriate combinations,
32P-labeled with T4 polynucleotide kinase and
[
-32P]ATP, heated, and annealed by slow cooling. The
annealed 13-mers were separated from ATP by passing through Sephadex
G-50 column.
Gel-shift assays were performed as described previously (26) with minor
modifications. 20 µl of binding mixture contained 1 µg of
poly(dI-dC) and 1.5 fmol of 32P-labeled hemimethylated
13-mers, unless indicated, in binding buffer (10 mM
Tris-HCl (pH 7.6), 50 mM KCl, 1 mM EDTA, 1 mM DTT, and 10% glycerol). The indicated amounts of
proteins were added and incubated for 20 min at 32 °C. The
subsequent steps were performed as described previously (29).
Filter Binding Assay--
Filter binding assays were performed
as described previously (30) with minor modifications. The indicated
amounts of proteins were added to 20 µl of the binding mixture
described under "Gel-shift Assay" and incubated for 20 min at
32 °C. The reactions were then filtered through nitrocellulose.
Filters were washed with 200 µl of binding buffer, dried, and
quantitated using a liquid scintillation counter.
1,10-Phenanthroline-Copper Ion Nuclease in Situ
Footprinting--
1,10-Phenanthroline-copper (II) (OP-Cu(II)) ion
nuclease footprinting was performed as described previously (31) with
minor modifications. The EcoRI/HindIII fragment
of plasmid pBMA1 from E. coli (DH5
(dam+))or
GM3819(dam-16:Kmr) (7)
were dephosphorylated with calf intestinal alkaline phosphatase and
32P end-labeled either at the EcoRI or
HindIII restriction site as described above. Unmethylated
and methylated fragments were then mixed, and hemimethylated fragments
were generated by heat denaturation and renaturation. A gel-shift assay
described above was performed with 20 fmol of the fragment. After
electrophoresis, the gel was immersed in 200 ml of 10 mM
Tris-HCl (pH 8.0), followed by addition of 20 ml of OP-Cu(II) solution
(2 mM 1,10-phenanthroline and 0.45 mM
CuSO4). The cleavage reaction was initiated by the addition
of 20 ml of 58 mM 3-mercaptopropionic acid, followed by
incubation for 8 min at room temperature. 20 ml of 28 mM
2,9-dimethyl-1,10-phenanthroline was added to quench the cleavage
reaction. Subsequent steps were performed as described previously
(29).
In Vitro Methylation Assay--
The Dam methylation assay was
performed as described previously (32). 500 fmol of indicated 13-mers
was added to 20 µl of Dam assay mixture (0.1 M Tris-HCl
(pH 8.0), 10 mM EDTA, 2.5 mM DTT, and 1.6 µM
S-adenosyl-[methyl-3H]-methionine. Dam
methylase was then added to the mixture, incubated at 37 °C, and
filtered through Whatman DE81 paper. Filters were washed with 0.4 M ammonium bicarbonate, further washed with cold ethanol,
dried, and quantitated using a liquid scintillation counter.
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RESULTS |
Preferential Binding of SeqA Protein to the Hemimethylated
13-mers--
Because there are 11 unique GATC sequences within
oriC (18), it was first necessary to decrease the ambiguity
of analyzing SeqA protein binding to these regions. This was
accomplished by synthesizing un-, hemi-, and fully methylated 13-mer
oligonucleotides containing 4 GATC sequences. Synthesized
oligonucleotides were annealed to each other and used for this study
unless otherwise indicated (Fig. 1). 20 µl of binding reaction mixture contained 0.08 ng (1.5 fmol) of
32P-labeled 13-mers and 1 µg of poly(dI-dC) as competitor
DNA to prevent nonspecific binding.

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Fig. 1.
13-mer region of oriC. The 13-mer
region of oriC studied contains 13-mer L, M, R, and four
GATC sequences (23, 26). Oligonucleotides for the top and bottom
strands were synthesized in forms of adenomethylated and unmethylated
GATCs. For the adenomethylated residues in GATC sequences,
N6-methyl deoxyadenosine
-cyanoethylphosphoramidite (Amersham Pharmacia Biotech) was used for
oligonucleotide synthesis. The oligonucleotides were annealed to
generate duplex DNA containing fully-, hemi- and unmethylated
13-mers.
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In nitrocellulose filter binding assays, SeqA protein preferentially
bound to hemimethylated 13-mers compared with fully or unmethylated
13-mers (Fig. 2A).
Approximately 32 ng of SeqA protein was required to saturate the
binding. Such preference of SeqA protein to hemimethylated 13-mers was
also observed in gel-shift experiments (Fig. 2B). SeqA
protein formed two distinct complexes with hemimethylated 13-mers,
whereas fully or unmethylated 13-mers did not form any complex. Of the
complexes, the fast migrating complex was formed at lower SeqA
concentration, whereas the slow migrating complex was at higher
concentration. Both bands were supershifted by the addition of SeqA
antiserum, indicating that these complexes were in fact formed by
binding of SeqA protein (Fig. 2C).

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Fig. 2.
Preferential binding of SeqA protein to
hemimethylated 13-mers. A, filter binding assays were
performed as described under "Experimental Procedures" using
fully-, hemi-, or unmethylated 13-mers. One unit was defined as the
activity that binds to a quarter of the input labeled probe.
B, gel-shift assays were performed as described under
"Experimental Procedures" using the indicated differently
methylated 13-mers. The resulting complexes of SeqA and hemimethylated
DNA were denoted as either fast migrating complex (Fast MC)
or slow migrating complex (Slow MC). C) SeqA antiserum
raised against rabbit was added to the gel-shift mixture containing
hemimethylated 13-mers. Each lane contained 17 ng of SeqA
protein. lane 1, SeqA protein only; lane 2, SeqA
protein and 0.2 µl of pre-immune serum; lane 3, SeqA
protein and 0.2 µl of SeqA antiserum.
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The binding affinities of SeqA protein for differently methylated
13-mers were compared by challenging the binding of SeqA to labeled
hemimethylated 13-mers with unlabeled and differently methylated
13-mers using filter binding assays (Fig.
3A). Binding of SeqA protein
to hemimethylated 13-mers was reduced by competition with
hemimethylated but not by fully or unmethylated 13-mers. These results
also confirm the preferential binding of SeqA protein to hemimethylated
13-mers. The fast migrating complex, which was formed at lower SeqA
concentrations (Fig. 2B), persisted at higher concentrations
of unlabeled hemimethylated 13-mers compared with the slow migrating
complex formed at higher SeqA concentrations (Fig. 3B). This
result indicates that SeqA protein possesses different affinities for
each binding site of hemimethylated 13-mers.

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Fig. 3.
Competition of SeqA binding to hemimethylated
13-mers with differently methylated 13-mers. Before the addition
of 17 ng of SeqA protein, the indicated amount of unlabeled 13-mers was
added to the reaction mixture containing labeled hemimethylated
13-mers, followed by incubation. The competition was measured using
filter binding (A) or gel-shift assays (B).
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Localization of the Tight Binding Site of SeqA Protein on the
Hemimethylated 13-mers--
To localize SeqA binding sites on the
hemimethylated 13-mers containing four GATC sequences, in
situ footprinting was performed using the OP-Cu(II) complex as a
chemical DNA cleavage agent (31). A gel-shift assay was performed with
the hemimethylated 13-mers in which adenine residues of GATC sequence
were methylated on either the top (Fig.
4A) or bottom strand (Fig.
4B). The 5'-ends of methylated strands were
32P-labeled. After treatment of the gel with OP-Cu(II), the
DNA in each band was isolated and subjected to electrophoresis through a denaturing sequencing gel. Regardless of top or bottom strand methylation, the protected region in the fast migrating complex localized to the GATC sequence of the 13-mer L and its close sequences. In the slow migrating complex, additional protected regions appeared at
the GATC sequences of the 13-mer M and R. These footprinting results
imply that SeqA protein binds first to the hemimethylated GATC sequence
of the 13-mer L and subsequently binds to the remaining hemimethylated
GATC sequences.

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Fig. 4.
The binding of SeqA protein to hemimethylated
GATC sequences. Free DNA (Free), fast migrating complex
(Fast MC), and slow migrating complex (Slow MC)
shown in Fig. 2B were analyzed by OP-Cu(II) footprinting as
described under "Experimental Procedures." Hemimethylated 13-mers
were 32P-labeled at the 5'-end of methylated top strand
(A) or at the 5'-end of methylated bottom strand
(B). L, M, and R indicate 13-mer L, M, and R, respectively.
GATC sequences are denoted as boxes. G and C on the top
indicate Maxam and Gilbert chemical sequencing reactions (24) of G and
C, respectively.
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Dissociation of SeqA Protein from the Hemimethylated 13-mers Is Not
Affected by Dam Methylase--
Methylation of hemimethylated
oriC by Dam methylase is delayed 8 min longer in wild type
cells than in seqA mutants (21). To assess the contribution
of SeqA protein to this delay, the binding stability of SeqA protein to
hemimethylated 13-mers was determined.
After incubation of SeqA protein with 32P-labeled
hemimethylated 13-mers without poly(dI-dC), a large excess of unlabeled
hemimethylated 13-mers was added. At the indicated time, the residual
complex was separated through filter binding (Fig.
5A) or gel-shift assays (Fig.
5, B and C). In filter binding assays, the
half-life of SeqA protein bound to hemimethylated 13-mers was
determined to be 4 min (Fig. 5A). In gel-shift experiments
in which the complexes were separated (Fig. 5, B and
C), the half-life of the fast migrating complex was
determined to be 4 min, whereas the half-life of the slow migrating
complex was 1.5 min. After 10 min, most of the bound SeqA protein was
dissociated from the hemimethylated 13-mers.

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Fig. 5.
The stability of SeqA protein bound to
hemimethylated 13-mers. The binding mixture containing 17 ng of
SeqA protein was incubated for 10 min, and then 500 fmol of unlabeled
hemimethylated 13-mers was added. At the indicated time, the reaction
mixture was analyzed by filter binding (A) or gel-shift
assay (B). C, the fast and slow migrating
complexes (MC) in B were quantitated by using a
FUJIX Bio-Imaging Analyzer (BAS1000). NP, no protein
added.
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Binding specificity of Dam methylase to un-, hemi-, and fully
methylated 13-mers was also determined using filter binding assays
(Fig. 6A). In contrast to SeqA
protein, Dam methylase did not discriminate between the methylation
status of 13-mers and bound equally to hemi-, fully, and unmethylated
13-mers. Also, the differently methylated 13-mers yielded identical
patterns of shifted bands by Dam methylase in gel-shift assays (data
not shown). Binding of Dam methylase was found to be nonspecific
because Dam methylase did not discriminate between the number or
existence of GATC sequences.

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Fig. 6.
Dam methylase cannot dissociate SeqA protein
bound to hemimethylated GATC sequences. A, filter
binding assays were performed with differently methylated 13-mers.
B, the binding reaction mixture containing 18 ng of SeqA
protein, 1.5 fmol of 32P-labeled hemimethylated 13-mers,
1.6 µM of S-adenosylmethionine, and 1 µg
poly(dI-dC) was incubated for 10 min. At the indicated time after the
addition of 250 ng of Dam methylase or 500 fmol of unlabeled
hemimethylated 13-mers, SeqA protein bound to hemimethylated 13-mers
was analyzed in gel-shift experiments. C, the slow migrating
complexes in B were quantitated as described in Fig.
5C. Relative intensity of slow migrating complex indicates
the ratio of the unit of slow migrating complex in a reaction to the
unit of slow migrating complex before the addition of Dam or cold
13-mers.
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Because SeqA and Dam methylase both act on the same substrate, the
possible dissociation of SeqA protein bound to hemimethylated 13-mers
by Dam methylase was examined (Fig. 6, B and C).
Incubation of 17 ng (39 nM) of SeqA protein with
hemimethylated 13-mers and S-adenosylmethionine was followed by the
addition of 250 ng (400 nM) of Dam methylase (Fig.
6B, left panel) or a large excess of unlabeled
hemimethylated 13-mers (Fig. 6B, right panel).
Re-binding of dissociated SeqA protein to the labeled probe was
inhibited by methylation of the SeqA binding sites by Dam methylase.
Re-binding could also be inhibited by the addition of a large excess
hemimethylated 13-mers.
Migration of the fast migrating SeqA-13mer complex was close to one of
the bands shifted by Dam methylase. Therefore, the slow migrating
SeqA-13 mer complex was quantitated (Fig. 6C). The stability
of the slow migrating complex in the presence of Dam methylase was
similar to the stability obtained in the presence of unlabeled
hemimethylated 13-mers. These results indicate that Dam methylase does
not affect the stability of SeqA protein bound to hemimethylated
13-mers, although both SeqA and Dam methylase act on the same
hemimethylated GATC sequence. Because the presence of a large excess
amount of poly(dI-dC) in SeqA binding assays reduced the stability of
both fast and slow migrating complexes in Fig. 6B, the
half-life of the fast migrating complex determined in Fig.
6B was different from that in Fig. 5C.
Delay of Dam Methylation on Hemimethylated GATC Sequences by SeqA
Protein--
Dam methylase catalyzes the conversion of unmethylated
GATC to hemimethylated GATC and of hemi- to fully methylated GATC. Therefore, the effect of SeqA protein on Dam methylation in the 13-mer
region of oriC was examined. Un- or hemimethylated 13-mers were incubated with or without SeqA protein, followed by the addition of Dam methylase and
S-adenosyl-[methyl-3H]methionine (Fig.
7B). At the indicated time,
incorporation of the [3H]methyl group into DNA was
determined. In the absence of SeqA protein, the incorporation of the
methyl group onto hemimethylated 13-mers was half that of unmethylated
13-mers. This is presumably because the available Dam methylation sites
on the hemimethylated DNA were half the available sites on the
unmethylated DNA. In the presence of SeqA protein, Dam methylation of
unmethylated 13-mers was somewhat inhibited (Fig. 7A). This
inhibition could be caused by the binding of SeqA protein to the
intermediate hemimethylated GATC sequences produced by Dam methylase.
When the hemimethylated 13-mers were bound by SeqA protein, Dam
methylation was delayed (Fig. 7B). Because Dam methylase was
unable to dissociate SeqA protein bound to hemimethylated 13-mers (Fig.
6B), the lag before methylation might be attributed to the
time required for dissociation of SeqA protein because of intrinsic
binding stability of the protein.

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Fig. 7.
Delay of Dam methylation by SeqA
protein. In vitro methylation assays were performed
using un- (A) or hemimethylated 13-mers (B) as
described under "Experimental Procedures." The reaction mixture
containing 500 fmol of unmethylated or hemimethylated 13-mers was
incubated for 10 min with or without 700 ng of SeqA protein. At the
indicated time after the addition of 100 ng of Dam methylase,
incorporation of 3H was measured.
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DISCUSSION |
During replication of the E. coli chromosome, newly
synthesized strands exist as unmethylated species until methylated by Dam methylase (21). In most regions of the chromosome, nascent strands
are methylated immediately, but methylation of the nascent oriC strand, which contains 11 GATC sequences, is delayed
for about 13 min, resulting in a hemimethylated state of
oriC (21). Duration of this hemimethylated oriC
is reduced to 5 min in seqA mutants (21). These results,
along with studies demonstrating the preferential binding of SeqA
protein to hemimethylated oriC (25), imply that SeqA protein
is responsible for the maintenance of hemimethylated
oriC.
Because oriC contains 11 repeats of GATC sequence (18), the
complexity of binding kinetic analysis and localization of SeqA protein
to hemimethylated oriC were reduced by using the 13-mer region of oriC containing only 4 GATC sequences (Fig. 1).
Consistent with the results reported with oriC region (25)
and bacteriophage P1 DNA (22), SeqA protein specifically bound to
hemimethylated 13-mers (Fig. 2). Two differently migrating complexes
formed by SeqA and hemimethylated 13-mers were separated in gel-shift
assays, and their formation and stability were analyzed (Figs. 2-5).
The fast migrating complex, which was produced at lower concentrations of SeqA protein, was formed by the binding of SeqA protein to the
hemimethylated GATC sequence of 13-mer L and its close sequences. At
higher concentrations of SeqA protein, additional binding to unoccupied
hemimethylated GATC sites of the fast migrating complex by SeqA protein
produced the slow migrating complex. Regardless of top or bottom strand
methylation, binding modes of SeqA protein appeared to be similar in
the OP-Cu(II) footprinting. Measurement of complex stability indicated
that binding of SeqA protein to the GATC sequence of 13-mer L is
tighter and more stable than to other GATC sequences in hemimethylated
13-mers. These results suggest that the flanking sequences as well as
hemimethylation of GATC duplex DNA is a determinant for the binding of
SeqA protein. It remains to be determined whether additional binding of
SeqA protein is cooperative. Currently, we are analyzing if SeqA
protein preferentially binds certain sites in hemimethylated
oriC and dnaA promoter region.
It has been suggested that Dam methylase binds nonspecifically to DNA
and diffuses along the duplex DNA until a GATC sequence is encountered
(32). Dam methylase then transfers methyl group with equal efficiency
to both hemi- and unmethylated DNA (33). Our studies, using differently
methylated 13-mers (Fig. 6), showed that binding of Dam methylase to
DNA is nonspecific and does not exhibit methylation specificity.
The binding of SeqA protein will compete with methylation by the Dam
protein to the hemimethylated GATC sequences on newly replicated
oriC. A single E. coli contains approximately
1,000 molecules of SeqA protein (25), whereas there are about 130 molecules of Dam methylase (5). Compared with Dam methylase, not only
the abundance of SeqA protein but also the specific binding and higher
affinity of SeqA protein to hemimethylated 13-mers (Figs. 2A
and 6A) implies that SeqA binding dominates Dam methylation during oriC duplication. This process inhibits Dam
methylation, a necessary event for the sequestration of oriC
initiation. SeqA protein dissociated in vitro after 10 min
with a half-life of 4 min (Fig. 5), which roughly matches with the
8-min delay of Dam methylation in seqA wild type compared
with seqA mutant. Also, Dam methylase was unable to
dissociate the bound SeqA protein on hemimethylated 13-mers (Fig. 6,
B and C). These observations suggest that the
spontaneous dissociation of SeqA protein will be followed by Dam methylation.