Minichromosome maintenance (MCM) proteins play an
essential role in eukaryotic DNA replication and bind to chromatin
before the initiation of DNA replication. We reported that MCM protein complexes consisting of MCM2, -4, -6, and -7 bind strongly to a
histone-Sepharose column (Ishimi, Y., Ichinose, S., Omori, A., Sato,
K., and Kimura, H. (1996) J. Biol. Chem. 271, 24115-24122). Here, we have analyzed this interaction at the molecular
level. We found that among six mouse MCM proteins, only MCM2 binds to histone; amino acid residues 63-153 are responsible for this binding. The region required for nuclear localization of MCM2 was mapped near
this histone-binding domain. Far-Western blotting analysis of truncated
forms of H3 histone indicated that amino acid residues 26-67 of H3
histone are required for binding to MCM2. We have also shown that mouse
MCM2 can inhibit the DNA helicase activity of the human MCM4, -6, and
-7 protein complex. These results suggest that MCM2 plays a different
role in the initiation of DNA replication than the other MCM
proteins.
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INTRODUCTION |
Minichromosome maintenance
(MCM)1 proteins play an
essential role in eukaryotic DNA replication. Six MCM proteins
(MCM2-7) have been identified from yeast to mammals, and each of them
plays a distinct role in DNA replication (reviewed in Refs. 1-3).
Several lines of evidence suggest that MCM proteins are required for
the initiation of DNA replication (4-6). Consistent with this idea, MCM proteins associate with the chromatin before the onset of DNA
replication and detach from the chromatin during DNA replication (7-9). Biochemical analyses have indicated that these six MCM proteins
interact. In extracts from Schizosaccharomyces pombe (10),
mouse cells (11) and mitotic human cells (12), a complex of
approximately 600 kDa, containing all six MCM proteins, have been
identified. Based on the molecular mass of each MCM protein, this
complex is probably a hexamer containing a single molecule of the six
MCM proteins. However, sub-complexes containing MCM2, -4, -6, and -7 or
MCM3 and -5 have also been isolated from human cell extracts (13-17),
Xenopus egg extracts (18), and mouse cell extracts (19). Our
group recently found that the DNA helicase activity was associated with
the human MCM4, -6, and -7 protein complex (20). The 600-kDa complex
containing the six MCM proteins is detected in soluble cell extracts;
however, the structures of the chromatin-bound MCM protein
heterocomplexes (21) remain to be elucidated.
Origin recognition complex (ORC) and CDC6 protein are both required for
the initiation of DNA replication and are necessary for the binding of
MCM proteins to chromatin in Xenopus egg extracts (22, 23).
CDC6-dependent loading of MCM proteins onto chromatin (24,
25) and origins (26, 27) has also been observed in Saccharomyces
cerevisiae. The genetic interaction between MCM proteins and ORC
in S. cerevisiae supports these findings (28, 29). These
results suggest that MCM proteins form a complex with ORC and CDC6 at
the replication origin. In S. cerevisiae the number of MCM
molecules recovered is much greater than the number of ORC molecules;
however, over half of these MCM molecules are not bound to chromatin
(24, 30). In addition ORC6 and CDC6 can be released from the chromatin
under conditions where almost all of MCM5 remains associated with the
chromatin (24). Therefore, the molecular basis of the interaction
between MCM proteins and the chromatin remains to be determined. Here,
we report that an amino-terminal portion of MCM2 has an affinity for
histone and that MCM2 can inhibit the DNA helicase activity of the
human MCM4, -6, and -7 protein complex.
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EXPERIMENTAL PROCEDURES |
Synthesis of MCM Proteins and Binding to Histone--
Six mouse
Mcm genes, Mcm2 (accession no. D86725) (11),
-3 (8), -4 (19), -5 (19),
-6 (11), and -7 (31), had been cloned in
pBluescript II plasmid (Stratagene, La Jolla, CA) at the
EcoRI site, as reported. Truncated forms of the
Mcm2 gene were amplified by polymerase chain reaction (PCR)
using primers from each site. Forward primers starting from nucleotide
(nt) 153 (for deleting (
) amino acid residues 1-34), 236 (
1-62), 279 (
1-76), 327 (
1-92), 399 (
1-116), and 536 (
1-166) and a reverse primer ending at nt 3323 were used as PCR
primers to truncate an amino-terminal region. A forward primer starting
from nt 51 and reverse primers ending at nt 326 (for amino acid
residues 1-92), nt 398 (residues 1-116), nt 452 (residues 1-134), nt
509 (residues 1-153), nt 731 (residues 1-227), and nt 896 (residues 1-282) were used to truncate a carboxyl-terminal region of the Mcm2 gene. To construct Mcm2 genes that were
deleted from amino acid residues 1-34 or 1-92, an ATG sequence was
added to the 5'-ends of the forward primers to create a methionine at
the amino terminus of the MCM2 proteins, and stop codons were added to
the 3'-end of the reverse primers to produce MCM2 truncated at the
carboxyl-terminal region. A forward primer starting from nt 236 and a
reverse primer ending at nt 509 were used to construct an
Mcm2 gene that encodes amino acid residues 63-153. The
resulting DNAs were cloned into pBluescript II SK
at the
EcoRI site (
1-34,
1-62,
1-76,
1-116,
1-166, 1-116, and 1-282), between the SalI and
EcoRI sites (
1-92), or between the EcoRI and
BamHI sites (1-92, 1-134, 1-153, 1-227, and 63-153) and
purified by anion exchange (Qiagen, Hilden, Germany). MCM proteins were
synthesized in vitro in the presence of
[35S]methionine in a reticulocyte lysate system, as
suggested by the manufacturer (TNT®-coupled reticulocyte
lysate system, Promega, Madison, WI). The reaction mixture was diluted
with 400 µl of 40 mM Hepes, pH 7.5, and 40 mM
dithiothreitol and then concentrated to about 50 µl using a
Microcon-10 column (Amicon, Bevely, MA) to remove free [35S]methionine. An aliquot of the sample was diluted to
10 µl with 0.3 M NaCl in buffer A (20 mM
Tris-HCl, pH 7.5, 0.5 mM EDTA, 1 mM
dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and
10% glycerol) containing 0.05% Nonidet P-40 and then incubated with 10 µl of the histone-Sepharose beads in the same buffer for 1 h
at 4 °C with vibration. The supernatant was saved after
centrifugation, and the beads were washed three times with the above
buffer. Proteins bound to the beads were eluted twice with 20 µl of 2 M NaCl in buffer A plus Nonidet P-40. The bound and unbound
proteins were electrophoresed in a 10, 15, or 20% polyacrylamide gel
(32), and the radioactivity on the gel was analyzed with a Bio-Image Analyzer (Fuji, Tokyo, Japan).
Expression of MCM2-GFP Fusion Protein in HeLa
Cells--
Truncated forms of the mouse Mcm2 gene were
constructed by PCR using forward primers starting from nt 327 (for
deleting (
) amino acid residues 1-92) and 509 (
1-153) and the
reverse primer ending at nt 2762. In the cases of
1-92 and
1-153, ATG sequences were added to the 5'-ends of the forward
primers. The native form as well as the truncated forms of the mouse
Mcm2 gene were cloned into pEGFP-N1
(CLONTECH, Palo Alto, CA) at the HindIII
site (native,
1-34) or between the HindIII and
EcoRI (
1-153) sites, to synthesize MCM2-GFP fusion
proteins where the carboxyl-terminal ends of the MCM2 proteins were
fused to the amino-terminal end of GFP. The cloned DNAs were
transfected into HeLa cells using Tfx 20 (Promega). The cells of
105 were grown on coverslips in a 35-mm dish with 2 ml of
DMEM supplemented with 5% FCS and antibiotics (50 units/ml penicillin
and 50 µg/ml streptomycin) for 1 day. The cloned DNA (1.4 µg) was
added to 1 ml of DMEM without FCS and antibiotics, and the solution was mixed well. Tfx 20 (4.2 µl) was then added to this DNA solution (final ratio of Tfx 20: DNA = 2:1). The solution was mixed well and left for 15 min at room temperature. The culture medium was removed
from the dish, and the DNA/lipid solution was added. The dish was
incubated at 37 °C in a CO2 incubator for 1 h. Two
ml of DMEM with 10% FCS and antibiotics was added, and the dish was returned to the incubator. The next day, the medium was replaced with
fresh DMEM with 5% FCS plus antibiotics after washing three times with
the medium. After 8 h (24 h after transfection), the cells were
fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for
30 min at room temperature and then permeabilized with 0.5% Triton
X-100 in PBS. After washing three times with PBS, cells were stained
with 4',6'-diamidino-2-phenylindole. Photographs were taken to Kodak
TMY400 film using Zeiss Axioplan with 63 × objective.
Far-Western Blotting of H3 Histone Fusion Protein--
The
histone H3 gene of HeLa cells was cloned by reverse transcription-PCR
(RNA LA PCR Kit, Takara, Tokyo). The H3 histone gene was cloned into
pTrxFus vector (Invitrogen, NV Leek, Netherlands), and the DNA sequence
was determined using a DNA sequencing kit (Dye Termination Cycle
Sequencing Ready Reaction, Perkin-Elmer). The amino acid sequence of H3
histone, deduced from the nucleotide sequence, was the same as the
published sequence (accession no. X0090) (33). Truncated forms of the
histone H3 gene, constructed by PCR, were cloned between the
KpnI and BamHI sites of the TrxFus vector.
Thioredoxin-H3 fusion proteins were overexpressed in transformed Escherichia coli. The fusion proteins were electrophoresed
on a 15% polyacrylamide gel and then transferred to a membrane
(Immobilon-PSQ, Millipore, Bedford, MA). The proteins on
the membrane were visualized by staining with 0.1% Ponceau S in 1%
acetic acid for 10 min. After destaining with water, the membrane was
incubated in 0.05% Tween 20 in PBS for 2 h and then incubated in
PBS containing 5% calf serum and 0.3% bovine serum albumin (34). The
membrane was incubated with the [35S]methionine-labeled
amino-terminal fragment (amino acid residues 1-282) of the mouse MCM2
protein in the same solution for 2 h at room temperature. After
washing with PBS, the radioactivity on the membrane was measured using
the Bio-Image Analyzer.
Production of MCM2 Protein in Insect Cells--
The full size
mouse Mcm2 gene was cloned into an HLTA vector between the
EcoRI and NotI sites (PharMingen, San Diego), and recombinant baculovirus encoding histidine-tagged Mcm2 gene
was prepared as the manufacturer suggested. Soluble His-MCM2 protein was recovered after lysis of the virus-infected Sf9 cells. The extracts were mixed with nickel nitrilotriacetic acid-agarose beads,
and the beads were washed with 50 mM sodium phosphate, pH
8.0, 0.3 M NaCl, and 10% glycerol in the presence of 20 mM imidazole. Proteins bound to the beads were eluted with
0.3 M imidazole in 50 mM sodium phosphate, pH
6.0, 0.3 M NaCl, and 10% glycerol. The partially purified
His-MCM2 protein was then loaded onto a histone-Sepharose column in 0.3 M NaCl, and the fusion protein was eluted by 2 M NaCl (16). Purified His-MCM2 protein was diluted to 0.2 M NaCl and then concentrated with a Centricon-30 column
(Amicon).
DNA Helicase Assay and Protein Analysis--
The MCM protein
fraction that contains mainly MCM4, -6, and -7 proteins was purified
from HeLa cells by histone H3/H4-Sepharose column chromatography and
then by glycerol gradient centrifugation (20). DNA helicase activity
was measured by displacement of 17-mer oligonucleotides annealed to
M13mp18 DNA. Proteins were electrophoresed in 10% polyacrylamide gels
containing SDS, or 4 or 5% polyacrylamide gels in 50 mM
Tris-HCl and 50 mM glycine, pH 8.8 (35), and then stained
with silver.
SV40 T Antigen Signal--
A plasmid (pSV78-T-lacZ) encoding the
SV40 T antigen nuclear localization signal (PPKKKRKV) was kindly
provided by M. Tanaka (Mitsubishi Kasei Institute of Life Sciences). An
HindIII site was created at the carboxyl end of a fragment
containing the signal by PCR, and the resultant HindIII
fragment
(5'-AGCTTACC/ATG/GCC/AAG/ATC/CCT/CCT/AAG/AAG/AAG/CGC/AAA/GTC/ GAG/A-3')
containing the signal was inserted into a construct producing the
truncated form (
1-153) of mouse MCM2 protein that had been fused
with GFP. Slashes indicate frames of translated amino acids. As the
result, a peptide (MAKIPPKKKRKVE) was added to the amino terminus of
the fusion protein.
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RESULTS |
Binding of MCM2 Protein with Histone--
Among the six MCM
proteins that were detected in 0.2 M NaCl-soluble HeLa
whole cell extracts, MCM protein complexes containing MCM2, -4, -6, and
-7 proteins bound strongly to a histone-Sepharose column (16). These
four MCM proteins from mouse FM3A cells also bound to the
histone-Sepharose column (data not shown). Each of the six mouse MCM
proteins was synthesized in vitro in the presence of
[35S]methionine using a reticulocyte lysate system (Fig.
1A). The most slowly migrating
bands of each MCM protein synthesized corresponded to full-size
products, since the electrophoretic mobilities of these bands were
coincident with those of native MCM proteins. The ability of the MCM
proteins to bind histone was examined (Fig. 1B). Each of the
labeled MCM proteins was incubated with histone-Sepharose beads at 0.3 M NaCl, and bound proteins were eluted from the Sepharose by increasing the NaCl concentration to 2 M. Approximately
90% of the recovered MCM2 was detected in the bound fraction, but the
other five MCMs did not bind to the histone-Sepharose under these
conditions (less than 10% was recovered in the bound fraction). These
results suggest that MCM2 is mainly responsible for the strong binding
of MCM2, -4, -6, and -7 complexes to the histone-Sepharose. This
conclusion was supported by the finding that the elution of MCM2 from
the histone H3/H4-Sepharose column was slightly retarded compared with
that of the MCM4, -6, and -7 (16).

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Fig. 1.
MCM2 binds to histone. A, mouse
MCM proteins (MCM2-7) were synthesized in vitro in the
presence of [35S]methionine and analyzed on a 10%
polyacrylamide gel. B, each synthesized MCM protein was
incubated with histone-Sepharose beads in 0.3 M NaCl. After
centrifugation, unbound proteins were saved, and bound proteins were
eluted twice with 2 M NaCl. Unbound (5 µl) (lane
2), bound proteins (10 µl) (lanes 3 and
4), and total synthesized proteins (lane 1) were
analyzed on a 10% SDS-polyacrylamide gel, and the radioactivity was
visualized by Bio-Image Analyzer.
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To determine the region of MCM2 responsible for binding to histone, the
histone binding activity of the truncated forms of MCM2 was examined.
First, MCM2 proteins that were deleted externally from the amino
terminus were synthesized, and histone binding was examined (Fig.
2). A large portion (76%) of the
recovered native MCM2 was detected in the bound fraction. Deleting the
amino-terminal region (amino acid residues 1-62) did not affect the
binding with histone. Smaller proteins were also synthesized using this
MCM2 construct (
1-62), which may result from the initiation of
protein synthesis from methionines at a downstream region in this
system. These smaller proteins did not bind the histone-Sepharose.
Deleting amino acid residues 1-76 decreased the binding, since almost
equal amounts of MCM2 were recovered in the unbound and bound
fractions. Almost no binding was detected in MCM2 deleted amino acid
residues 1-92, 1-116, and 1-166.

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Fig. 2.
Histone binding activity of MCM2 deleted from
the amino terminus. DNA constructs that delete the amino-terminal
region of MCM2 were prepared as described under "Experimental
Procedures." Native and truncated forms of MCM2 were synthesized
in vitro and examined for binding to histone-Sepharose, as
described in Fig. 1. Unbound (lane 2), bound (lanes
3 and 4) proteins, and total proteins (lane
1) were analyzed in 10% SDS-polyacrylamide gels. The largest
protein bands, indicated with arrows, were probably
full-size products of the constructs. The number of amino acid residues
deleted are indicated at the top.
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Next, the MCM2 proteins, deleted externally from the carboxyl terminus,
were synthesized, and histone binding was examined (Fig.
3). A large portion (approximately 75%)
of the MCM2 proteins recovered from the bound fractions contained the
amino-terminal region amino acid residues 1-153, 1-227, and 1-282.
However, the MCM2 protein containing amino acid residues 1-134 showed
a decrease in binding activity (55% was recovered in the bound
fraction), and the MCM2 protein containing amino acid residues 1-92
and 1-116 lost the binding activity. These results suggest that the
region of amino acid residues 63-153, which contains a high
concentration of charged amino acids, is required for binding to
histone (Fig. 4). Consistently, the MCM2
fragment from amino acid residues 63 to 153 bound to histone (Fig. 3).
MCM2 is the only one MCM protein containing such a highly charged
region, and this feature is conserved from S. cerevisiae to
mammals.

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Fig. 3.
Histone binding activity of MCM2 deleted from
the carboxyl terminus. MCM2 proteins that were deleted from the
carboxyl terminus and a fragment of 63-153 residues were synthesized
in vitro and examined for binding to histone-Sepharose.
Unbound (lane 2), bound (lanes 3 and
4), and total proteins (lane 1) were analyzed by
10% (for 1-282), 15% (for 1-227), or 20% (for others)
SDS-polyacrylamide gel electrophoresis. The number of remaining amino
acid residues in the truncated MCM2 are indicated at the
top.
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Fig. 4.
The region of MCM2 required for binding
to histone. The results in Figs. 2 and 3 are summarized. The
structures of truncated MCM2 proteins are shown; the
DNA-dependent ATPase domain and two candidates of nuclear
localization signals (NLS) are indicated. The histone
binding activity of the MCM2 protein is indicated at the
right by +/ , and binding of intermediate strength is
indicated by (+). The region required for binding to histone is
enlarged at the bottom, where basic amino acid residues are
underlined and acidic residues are
overlined.
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A Region of H3 Histone Required for the Binding with MCM2
Protein--
Our group reported that MCM protein complexes containing
MCM2, -4, -6, and -7 specifically bind to a histone H3-Sepharose column
(16). To identify the region(s) of H3 histone responsible for the
binding to MCM2, various deletion mutants of H3 histone were produced
as fusion proteins, and the binding of these H3 histones to MCM2 was
examined by far-Western blotting analysis (Fig.
5). An H3 histone fragment of amino acid
residues 1-67 (lane 3) was bound with MCM2 with efficiency
relatively similar to that of the fusion protein containing full-size
H3 histone (lane 5). Several smaller proteins were also
detected in all fusion proteins, which are probably nonspecifically
recognized in this analysis. The fusion proteins containing H3
fragments amino acid residues 1-45 (lane 1) and 1-55
(lane 2) lost the binding activity. A fragment of amino acid
residues 26-90 (lane 8) retained the binding activity, but
a fragment from 36 to 90 (lane 9) lost almost all of the
binding ability. Two fragments that contained the carboxyl-terminal
region but lacked the amino-terminal end (lanes 6 and
7) did not bind to MCM2. These results suggest that the
region of amino acid residues 26-67 is required for binding to MCM2.
Consistent with these results, a fragment from 26 to 67 was capable of
binding to MCM2 (lane 10).

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Fig. 5.
MCM2 binds to an amino-terminal region of
histone H3. A, native and truncated forms of histone H3 were
synthesized as thioredoxin fusion proteins and electrophoresed on 15%
polyacrylamide gels. Lane 1, amino acid residues 1-45;
lane 2, 1-55; lane 3, 1-67; lane 4,
1-90; lane 5, 1-136; lane 6, 46-136;
lane 7, 91-136; lane 8, 26-90; lane
9, 36-90; lane 10, 26-67. Proteins were transferred
to a membrane and stained with Ponceau S. B, the membrane
was incubated with an amino-terminal portion of MCM2 (amino acid
residues 1-282), and the radioactivity on the membrane was measured.
C, the structures of H3 histone in the fusion proteins are
shown; the amino acid residue numbers are indicated, and the binding
activity of the fusion proteins with MCM2 is also indicated on the
right by +/ .
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Requirement of an Amino-terminal Region of MCM2 Protein for Nuclear
Localization--
Mouse MCM2 proteins are localized in nuclei when
they are expressed in COS cells (11), suggesting that MCM2 contains a
nuclear localization signal. Two regions that may be required for the nuclear localization of MCM2 were found in the amino-terminal region;
one region is amino acid residues 18-34 where a bipartite-type nuclear
localization signal (36) is present, and another region is amino acid
residues 118-152 where positively charged amino acid residues are
highly concentrated (Fig. 4). To determine the region required for
nuclear localization of MCM2, two DNA constructs producing deletion
mutants of MCM2 proteins as GFP fusion proteins were prepared and
transfected into HeLa cells (Fig. 6).
Full size MCM2 (Fig. 6A) as well as the MCM2 that was
deleted from the amino terminus to amino acid residue 92 (Fig.
6C) were localized to the nucleus. However, the mutant MCM2
deleted from the amino terminus to amino acid residue 153 was not
concentrated in the nucleus; they were localized uniformly throughout
the cells (Fig. 6E). A nuclear localization signal from SV40
T antigen was ligated to the mutant MCM2-GFP fusion protein
(
1-153). This fusion protein was localized to the nucleus (Fig.
6G). These results suggest that the second region from amino
acid residues 118 to 152 is capable of localizing MCM2 to nucleus.

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Fig. 6.
An amino-terminal region is required for the
nuclear localization of MCM2 protein. Genes encoding the MCM2
proteins fused to GFP were transfected into HeLa cells, and the cells
were visualized by fluorescence microscopy. Full size MCM2 protein
(A and B) as well as those deleted the amino
terminus ( 1-92 (C and D) and 1-153
(E and F)) were fused with GFP. A nuclear
localization signal from SV40 T antigen was fused to the mutant MCM2
( 1-153), and the fluorescence of this fusion protein was observed
(G and H). The MCM2-GFP fluorescence is shown on
the left (A, C, E, and
G) and nuclear staining with 4',6'-diamidino-2-phenylindole
is shown on the right (B, D,
F, and H). The bar in H
indicates 20 µm.
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Inhibition of the DNA Helicase Activity by MCM2--
We reported
the results suggesting that the human MCM protein complex containing
MCM4, -6, and -7 has both DNA helicase and DNA-dependent
ATPase activities (20). When MCM protein complexes were fractionated by
glycerol gradient centrifugation, these activities were detected in the
350-kDa fraction, where MCM2 protein was almost absent, but a main peak
of the four MCM proteins was detected at a position of 230 kDa (Fig.
7, A and C) (20).
When MCM proteins in the gradient fraction were electrophoresed in
native agarose gel, two major complexes of approximately 450 and 600 kDa were detected (Fig. 7B). The amounts of the 600-kDa
complex in the gradient fractions were almost proportional to the DNA
helicase activity (Fig. 7, B and C). These
results suggest that MCM4, -6, and -7 form a hexamer and function as a
DNA helicase, as previously suggested (20). Two complexes of similar
sizes were also detected on SDS-polyacrylamide gel after protein
cross-linking (20). Correlation between the amounts of the 600-kDa
complex and DNA helicase activity was more clearly observed when the
complex was detected on SDS-polyacrylamide gel after protein
cross-linking, which is probably due to the fact that composition of
MCM complexes is slightly changed during native gel electrophoresis.
The results in Fig. 7 also indicated that DNA helicase activity was
mainly detected in the fractions lacking MCM2. Therefore, it was
possible that MCM2 protein can inhibit the DNA helicase activity by the MCM4, -6, and -7 protein complex.

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Fig. 7.
Complex formation of MCM proteins.
A, MCM2, -4, -6, and -7 protein complexes purified by
histone-Sepharose column chromatography were separated by glycerol
gradient centrifugation (20). Proteins in the fractions (fractions
2-11) were electrophoresed on a 10% polyacrylamide gel containing SDS
and stained with silver. Under these conditions, ferritin (440 kDa) and
catalase (232 kDa) sedimented at fractions 3 and 6, respectively.
B, proteins in the gradient fractions were electrophoresed
on a 5% polyacrylamide gel under non-denaturing conditions and were
stained with silver. As markers, thyroglobulin (669 kDa) and ferritin
(440 kDa) were electrophoresed. C, DNA helicase activity
that displaces 17-mer oligonucleotides annealed to M13 DNA was
measured. In the left-end lane, DNA helicase activity in the
absence of the gradient fraction was measured. The position of the
displaced oligomers is indicated.
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Histidine-tagged mouse MCM2, purified from baculovirus-infected
Sf9 cells, was added to a DNA helicase reaction containing purified MCM4, -6, and -7 complex (fractions near 350 kDa were pooled).
By adding an almost stoichiometrical amount of MCM2, the DNA helicase
activity was severely inhibited (only 12% of the activity was
detected) (Fig. 8A). In
contrast, His-MCM2 had no inhibitory effect on the DNA helicase
activity of SV40 T antigen and mouse DNA helicase B (37) (data not
shown). His-MCM2 itself did not exhibit DNA helicase activity. As shown
in Fig. 8B, a 600-kDa complex was mainly detected in the
purified MCM4, -6, and -7 complex, although complexes of smaller
molecular mass were also detected, which is probably due to the
presence of a small amount of human MCM2 in this fraction. The addition
of His-MCM2 prevented the formation of the 600-kDa complex and
increased the number of smaller molecular mass complexes. A weak band
of about 400 kDa was detected in the presence of His-MCM2 only (data
not shown). Therefore, the inhibitory effect of MCM2 on the DNA
helicase activity was correlated with the ability of MCM2 to
disassemble the 600-kDa complex. To examine this correlation further,
the fraction that mainly contains the MCM4, -6, and -7 complex was incubated with or without MCM2 and then the mixture was separated by
glycerol gradient centrifugation (Fig.
9). Without adding MCM2, the DNA helicase
activity was detected in the fractions 3-5 (near 350 kDa) (Fig.
9C, left) where MCM4, -6, and -7 co-sedimented (Fig.
9A, left), and a 600-kDa complex was mainly detected in native gel (Fig. 9B, left). After incubation with MCM2,
however, MCM2, -4, -6, and -7 were mainly detected at the position of
230 kDa (Fig. 9A, right) where a 450-kDa complex was mainly
detected in native gel (Fig. 9B, right). The DNA helicase
activity was not detected in these fractions (Fig. 9C,
right). When MCM2 was incubated in the absence of the MCM4, -6, and -7 complex and then centrifuged, it was detected in a broad region
of the gradient fractions, but the peak was detected at fractions 2-4
(data not shown). These results suggest that MCM2 specifically inhibits the DNA helicase activity of MCM4, -6, and -7 by preventing the formation of the hexamer.

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Fig. 8.
Mouse MCM2 inhibits the DNA helicase activity
of the human MCM4, -6, and -7 complex. A, increasing amounts
of His-MCM2 proteins, purified from baculovirus-infected Sf9
cells, were added to the DNA helicase reaction mixture which contained
a purified human MCM4, -6, and -7 complex. The amount of MCM2 and the
presence of MCM4, -6, and -7 complex are indicated at the
top of the figure. The position of the displaced oligomers
is indicated. At the bottom, displaced oligomers were
expressed as the percentage of the oligomers that were displaced in the
reaction containing only MCM4, -6, and -7 complex. B, the
purified MCM4, -6, and -7 complex was incubated with or without
His-MCM2 under the same conditions as those used for the DNA helicase
assay, except that bovine serum albumin was omitted. The proteins were
electrophoresed on a 4% polyacrylamide gel under non-denaturing
conditions and were stained with silver. Thyroglobulin (669 kDa) and
ferritin (440 kDa) markers migrated to the positions indicated.
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Fig. 9.
Disassembly of MCM4, -6, and -7 hexamers with
MCM2. A, MCM4, -6, and -7 complex (fractions 3-5
in Fig. 7) containing approximately 7 µg of total proteins was
incubated with or without His-MCM2 (4.2 µg) in 50 µl of 50 mM Tris-HCl, pH 7.9, 20 mM 2-mercaptoethanol, 5 mM MgCl2, 5 mM ATP, and 0.01%
Triton X-100 for 30 min at 37 °C, and then the mixture was separated
by glycerol gradient centrifugation. Proteins in fractions
2-8 were electrophoresed on a 10% polyacrylamide gel containing
SDS and were stained with silver. Ferritin and catalase sedimented at
fractions 2 and 5, respectively. Presence or absence of MCM2 during
incubation is indicated by +/ at the top. B,
fractions were concentrated to approximately 10-fold. Protein complexes
of the concentrated sample were analyzed by 5% polyacrylamide gel
electrophoresis under non-denaturing conditions. As markers,
thyroglobulin and ferritin were electrophoresed, and proteins were
stained with silver. C, DNA helicase activity of the
concentrated sample was examined. In the left-end lane, DNA
helicase activity in the absence of the sample was measured. Presence
or absence of MCM2 is indicated at the bottom.
|
|
 |
DISCUSSION |
The results indicate that a highly charged region near the amino
terminus of MCM2, which is unique among the six MCM proteins, has an
affinity for histone, and this region interacts with an amino-terminal
half region of H3 histone in vitro. This highly charged
region of MCM2 is present from yeast to mammals. We showed that MCM2
can inhibit the DNA helicase activity of the human MCM4, -6, and -7 complex. Thus, MCM2 may play a different role from that of the other
MCMs in the initiation of DNA replication.
A region required for the nuclear localization of MCM2 was mapped near
the histone-binding domain. These findings seem to be correlated with
the finding that the DNA-binding domains of transcription factors are
located near their nuclear localization signal (38). MCM4, -6, and -7 do not have a typical nuclear localization signal. Consistently, Kimura
et al. (11) have shown that mouse MCM2 proteins, which are
expressed in COS cells, are localized to nucleus, but MCM4, -6, and -7 proteins are not. In the same experiments, it has also been shown that
co-expressing MCM6 with MCM2 localizes MCM6 to the nucleus. MCM2 can be
isolated from cell extracts in a complex with MCM4, -6, and -7 (13-16). Thus, MCM2 may carry MCM4, -6, and -7 to nucleus in
vivo.
MCM protein complexes containing MCM2, -4, -6, and -7 have been
purified by histone-Sepharose column chromatography (16). The results
of this study suggest that the MCM protein complexes containing these
four MCM proteins bind to histone-Sepharose through the interaction of
MCM2 protein with histone H3. A major peak of MCM2, -4, -6, and -7 was
detected at the position of 230 kDa in glycerol gradient centrifugation
(Fig. 7A and Ref. 20), suggesting that they form dimers.
However, a 450-kDa complex was mainly detected in the same position,
which was analyzed by native agarose gel electrophoresis (Fig.
7B) and also in SDS-polyacrylamide gel electrophoresis after
cross-linking (20). When the MCM proteins in the position of 230 kDa
were chemically cross-linked and then separated by glycerol gradient
centrifugation, the resultant 450-kDa complex, which was detected on an
SDS-polyacrylamide gel, sedimented at the position of 230 kDa (data not
shown). Therefore, it is probable that MCM2, -4, -6, and -7 detected at
the position of 230 kDa form a heterotetramer. A portion of MCM4, -6, and -7 may be separated from MCM2 during elution from the
histone-Sepharose column, and the separated MCM4, -6, and -7 mainly
form a hexamer of 600 kDa, which sedimented at the position of 350 kDa
in glycerol gradient centrifugation (Fig. 7B and Ref. 20).
It is possible that two of the separated MCM4, -6, and -7 trimers are
assembled into a hexamer during these processes. We previously
suggested that the hexamer of MCM4, -6, and -7 exhibits both DNA
helicase and ATPase activities (20). Here, we show that MCM2 inhibits
both the DNA helicase activity and the formation of MCM4, -6, and -7 hexamers (Figs. 8 and 9). These results support the notion that the
MCM4, -6, and -7 hexamer of 600 kDa exhibits DNA helicase activity and also suggest a possibility that MCM2 protein plays a regulatory role in
the DNA helicase activity of the MCM4, -6, and -7 complex. By adding
MCM2, the 600-kDa hexamer of MCM4, -6, and -7 was converted to the
450-kDa complex containing MCM2, and DNA helicase activity was not
detected in this complex (Fig. 9). These results are consistent with
the notion that the 450-kDa complex is a heterotetramer consisting of
MCM2, -4, -6, and -7. Other smaller complexes of approximately 400 kDa
slightly increased in the presence of MCM2 (Fig. 8B), and
one of these complexes may be a trimer of MCM4, -6, and -7, which was
separated from the hexamer. The system where MCM2 regulates DNA
helicase activity of the MCM4, -6, and -7 complex seems to be similar
to the
replication system (39). In this system,
P proteins bring
dnaB helicase to the
replication origin which is occupied by
O.
Since
P proteins inhibit the dnaB helicase activity, they must be
removed from the origin with the assistance of the heat-shock proteins
DnaJ, DnaK, and GrpE.
MCM2 may be released from the MCM4, -6, and -7 complex to activate its
DNA helicase activity at the onset of DNA replication. Cdk2/cyclin
and/or Cdc7/Dbf4 protein kinases, both of which are required for the
initiation of DNA replication (reviewed in Ref. 40) may phosphorylate
MCM2 to remove it from the MCM4, -6, and -7 protein complex. The
finding that a mutant of MCM5 (bob-1) bypasses the requirement for Cdc7
kinase in S. cerevisiae (41) seems to be consistent with
this notion. The region containing the bob-1 mutation is evolutionarily
conserved in MCM2 and MCM4 as well as MCM5. Recently, Lei et
al. (42) have reported that MCM2 in addition to MCM3, -4, and -6 can be phosphorylated by Cdc7/Dbf4 in vitro. Since the
affinity of MCM2 for histone H3 is high, a structural change of the
nucleosome may cause MCM2 to bind H3 histone in the chromatin, which
may concomitantly result in the release of the MCM4, -6, and -7 protein
complex. A third possibility is that newly synthesized H3 histone may
remove MCM2 from the chromatin. This correlates with the findings that
histone synthesis precedes DNA synthesis during the cell cycle
(43).
The region of H3 histone necessary for binding to MCM2 is amino acid
residues 26-67 near the amino terminus. This region, which forms an
-helical structure (44) and is involved in histone-DNA binding in
the nucleosome (45), is required for cell growth in S. cerevisiae (46). It has been reported that the regions of amino
acid residues 1-30 and 50-70 of H3 histone are exposed at the surface
of the nucleosome in solution, which was determined by binding of
site-specific antibodies of H3 histone to the nucleosome and the
chromatin (47). The interaction between MCM2 protein and histone H3 is
strong and specific, but the physiological significance of the
interaction remains to be clarified. This interaction may be involved
in the chromatin binding of MCM protein complexes before the initiation
of DNA replication or in a structural change of the nucleosome at the
initiation of DNA replication; alternatively, it may be required for
the chromatin assembly process during DNA replication.
We thank Masato Tanaka for providing plasmid
DNA containing a nuclear localizing signal of SV40 T antigen; Takemi
Enomoto for providing mouse helicase B; Sachiko Ohta for help in the
nucleotide sequencing; and Emma Jones for revision of the manuscript.