In contrast, heliquinomycin at an IC50 value of 5.2 lm inhibited the ATPase activity of the MCM4/6/7 complex in the pres-ence of single-stranded DNA.. To elucidate its cellular targets f
Trang 1minichromosome maintenance 4/6/7 helicase
Yukio Ishimi1,2, Takafumi Sugiyama1, Ryou Nakaya1, Makoto Kanamori3, Toshiyuki Kohno2,
Takemi Enomoto3 and Makoto Chino4
1 College of Science*, Ibaraki University, Japan
2 Macromolecular Structure Research Group*, Mitsubishi Kagaku Institute of Life Sciences, Tokyo, Japan
3 Molecular Cell Biology Laboratory, Graduate School of Pharmaceutical Sciences, Tohoku University, Miyagi, Japan
4 Pharmaceuticals Group, Nippon Kayaku Co Ltd., Tokyo, Japan
Minichromosome maintenance (MCM) proteins are
essential factors for the prevention of the loss of
extra-chromosomal DNA in Saccharomyces cerevisiae [1–3]
A heterohexameric MCM2–7 protein complex has been
identified as a component of the DNA replication
licensing system that ensures a single round of DNA
replication per cell cycle [4–7] This complex functions
as a replicative DNA helicase that drives the unwinding
of the DNA duplex prior to semiconservative DNA
synthesis at the replication forks This notion is
sup-ported by the following findings First, all of the
MCM2–7 proteins possess DNA-dependent ATPase
motifs that are common features of DNA helicases [8]
Second, the MCM4/6/7 subcomplex forms the core of the MCM2–7 hexamer and exhibits intrinsic DNA helicase activity in vitro [9–12] Third, in S cerevisiae, MCM2–7 proteins play an essential role in both the ini-tiation and elongation of DNA replication [13], and these proteins migrate on the genome together with the replication forks [14,15] One of the intricacies related
to the function of the MCM2–7 complex is that an iso-lated MCM2–7 complex does not exhibit definite DNA helicase activity in vitro, but the MCM4/6/7 hexamer does Further, the interaction between the MCM2 protein and the MCM4/6/7 hexamer, or between the MCM3/5 proteins and the MCM4/6/7 hexamer,
Keywords
anticancer drug; DNA replication; MCM
4/6/7 helicase
Correspondence
Y Ishimi, Ibaraki University, 2-1-1 Bunkyo,
Mito, Ibaraki 310-8512, Japan
Fax: +81 29 228 8439
Tel: +81 29 228 8439
E-mail: ishimi@mx.ibaraki.ac.jp
(Received 9 March 2009, revised 13 April
2009, accepted 16 April 2009)
doi:10.1111/j.1742-4658.2009.07064.x
The antibiotic heliquinomycin, which inhibits cellular DNA replication at a half-maximal inhibitory concentration (IC50) of 1.4–4 lm, was found to inhi-bit the DNA helicase activity of the human minichromosome maintenance (MCM) 4/6/7 complex at an IC50value of 2.4 lm In contrast, 14 lm heliqui-nomycin did not inhibit significantly either the DNA helicase activity of the SV40 T antigen and Werner protein or the oligonucleotide displacement activity of human replication protein A At IC50values of 25 and 6.5 lm, heliquinomycin inhibited the RNA priming and DNA polymerization activi-ties, respectively, of human DNA polymerase-a/primase Thus, of the enzymes studied, the MCM4/6/7 complex was the most sensitive to heliqui-nomycin; this suggests that MCM helicase is one of the main targets of heliquinomycin in vivo It was observed that heliquinomycin did not inhibit the ATPase activity of the MCM4/6/7 complex to a great extent in the absence
of single-stranded DNA In contrast, heliquinomycin at an IC50 value of 5.2 lm inhibited the ATPase activity of the MCM4/6/7 complex in the pres-ence of single-stranded DNA This suggests that heliquinomycin interferes with the interaction of the MCM4/6/7 complex with single-stranded DNA
Abbreviations
BrdU, bromodeoxyuridine; FITC, fluorescein isothiocyanate; IC 50, half-maximal inhibitory concentration; MCM, minichromosome
maintenance; RPA, replication protein A.
*[Corrections added on 18 May 2009 after first online publication: in affiliation 1, ‘Macromolecular Structure Research Group’ has been replaced by ‘College of Science’, and in affiliation 2 ‘Macromolecular Structure Research Group’ has been inserted.]
Trang 2inhibits helicase activity [16,17] On the basis of these
previous reports, we propose that a structural change
in the MCM2–7 complex may generate the MCM4/6/7
hexamer, which, in turn, exhibits helicase activity
Another possibility is that the DNA helicase activity of
the MCM2–7 complex may be attributed to the
inter-action of this complex with other proteins It has been
reported that the CMG complex, which consists of the
Cdc45 protein, MCM2–7 hexamer and the GINS
com-plex purified from Drosophila embryo extracts, exhibits
DNA helicase activity in vitro [18] Furthermore, it has
been demonstrated recently that the MCM2–7 complex
prepared from S cerevisiae exhibits DNA helicase
activity in the presence of potassium acetate or
gluta-mate These results suggest that the MCM2–7 complex
functions as a replicative DNA helicase in vivo [19]
Heliquinomycin, which is an antibiotic [20,21],
inhib-its cellular DNA replication and RNA synthesis To
elucidate its cellular targets for the inhibition of DNA
synthesis, we examined the effects of heliquinomycin
on the DNA helicase activities of the MCM4/6/7
com-plex, SV40 T antigen and Werner protein, on the
oligo-nucleotide displacement activity of replication protein
A (RPA), and on the RNA priming and DNA
poly-merization activities of the DNA polymerase-a/primase
complex The results indicated that, among all the
enzymes examined, the MCM4/6/7 helicase was the
most sensitive to heliquinomycin It was observed that,
in the absence of single-stranded DNA, heliquinomycin
did not inhibit the ATPase activity of the MCM4/6/7
complex to a great extent; in contrast, in the presence
of DNA, this antibiotic inhibited the ATPase activity
This result suggests that heliquinomycin inhibits the
helicase activity of MCM4/6/7 by interfering with the
interaction of this complex with single-stranded DNA
Results
Sensitivity of cellular DNA replication to
heliquinomycin
Heliquinomycin with a relative molecular mass of
698 Da was isolated from Streptomyces sp as an
anti-biotic (Fig S1) [20] It has been shown that
heliquino-mycin inhibits DNA replication in various transformed
cells at a half-maximal inhibitory concentration (IC50)
of 1.4–4 lm [21] In these experiments, DNA synthesis
was measured by the incorporation of labelled
thymi-dine into DNA To confirm this, human HeLa cells
were pulse labelled with bromodeoxyuridine (BrdU) in
the presence of increasing concentrations of
heliquino-mycin (Fig 1A) BrdU incorporated into DNA was
detected by staining the cells with anti-BrdU Ig and
A
B
Fig 1 Effect of heliquinomycin (HQ) on the incorporation of BrdU into DNA in HeLa cells (A) Logarithmically growing HeLa cells were incubated with the indicated concentrations of heliquinomycin for
1 h and then pulse labelled with BrdU for 20 min The incorporated BrdU in the cells was detected by incubation of the cells with anti-BrdU Ig, followed by FITC-labelled anti-rat Ig MCM7 was detected
by incubation of the cells with anti-MCM7 Ig, followed by Cy3-labelled anti-mouse Ig (B) One hundred Cy3-stained cells were selected, and the fluorescence intensity of FITC in the cells was quantified The average level of intensity in the cells cultured in the presence of heliquinomycin was expressed in comparison with that
in the cells cultured in the absence of heliquinomycin.
Trang 3then with fluorescein isothiocyanate (FITC)-labelled
second Ig The cells were also stained with specific Ig
to detect MCM7 protein in the nucleus In the absence
of heliquinomycin, approximately 27% of the cells
were stained with anti-BrdU Ig As the concentration
of heliquinomycin was increased, the proportion of BrdU-positive cells and the intensity of the signal grad-ually decreased Almost no BrdU-positive cells were detected in the presence of 14 lm heliquinomycin In contrast, staining with anti-MCM7 Ig was not changed
by the presence of heliquinomycin The fluorescence derived from the incorporated BrdU was quantified in these experiments, and the IC50 value was determined
to be 2.3 lm (Fig 1B), which is similar to the value reported previously [21]
Sensitivity of MCM4/6/7 helicase to heliquinomycin
Heliquinomycin at an IC50 value of 7–14 lm inhibits the activity of the cellular DNA helicase called DNA helicase I [22], but scarcely affects the activities of topoisomerases and the replication of the SV40 chromosome in vitro [21] To understand the cellular targets of this antibiotic during DNA replication, the effects of this antibiotic on the helicase activities of the human MCM4/6/7 complex, SV40 T antigen and Werner protein were examined In addition to these three proteins, the human DNA polymerase-a/primase complex and the human RPA complex were purified
to near homogeneity (Fig S2) Some unidentified pro-teins were found in the purified DNA polymerase-a/ primase complex and the human RPA complex It has been reported that MCM3 interacts with DNA polymerase-a/primase [23] We examined the presence
of MCM4, 5 and 6 proteins in the purified DNA
+ MCM4/6/7
A
B
+ Tag
00.431.44.31443 (μ M ) HQ 0
0.431.44.31443
17-mer 17-mer/M13
0
(μ M ) HQ
Tag
MCM
0
(μ M ) HQ 0.43
120
100
80
60
40
20
0
1.4 4.3 14 43
+ Werner
17-mer
17-mer/M13
Fig 2 Effect of heliquinomycin on DNA helicase activity (A) Top: effects of increasing concentrations of heliquinomycin (HQ) on the DNA helicase activities of the MCM4/6/7 complex and the SV40 T antigen Dimethyl sulfoxide solution (0.4 lL) containing or lacking heliquinomycin was added to the reaction mixture The final con-centrations of heliquinomycin added to the reaction mixture are indicated at the top The DNA helicase activity was measured as the activity that displaces 17-mer oligonucleotides annealed to M13mp18 single-stranded DNA Bottom: the proportion of dis-placed 17-mer oligonucleotides in total DNA was considered to be 100% in the control reaction mixture lacking heliquinomycin, and the proportions in the mixtures containing heliquinomycin were cal-culated in relation to the control value The horizontal line is dis-played on a logarithmic scale Four independent experiments were performed for the MCM4/6/7 complex, and an average of the val-ues was plotted together with the standard deviations Two inde-pendent experiments were performed for the T antigen and an average of the values was plotted (B) Top: effects of increasing concentrations of heliquinomycin on the DNA helicase activity of the Werner protein Bottom: proportion of displaced 17-mer oligo-nucleotides Two independent experiments were performed, and
an average of the values was plotted together with the error bars.
Trang 4polymerase-a/primase complex (Fig S3) Only small
amounts of MCM4 (0.1% of total protein) and
MCM5 (0.9%) proteins were detected, and MCM6
was not found
DNA helicases were added to the DNA helicase
reaction mixtures at the minimum amounts required to
displace almost all of the 17-mer oligonucleotides
Heliquinomycin at a concentration of 14 lm did not
inhibit the helicase activity of the SV40 T antigen to a
great extent, but inhibited that of the MCM4/6/7
com-plex at an IC50 value of 2.4 lm (Fig 2A)
Heliquino-mycin (14 lm) did not inhibit the helicase activity of
the Werner protein to a great extent (Fig 2B) It
should be noted that the mobility of displaced
frag-ments in the absence or presence of heliquinomycin
was different The RPA complex displaces
oligonucleo-tides annealed to M13 single-stranded DNA without triggering ATP hydrolysis [24] We found that heliqui-nomycin scarcely affected the oligonucleotide displace-ment activity of RPA (Fig S4) We also examined the effect of heliquinomycin on the reactions of RNA priming (Fig 3A) and DNA polymerization activity (Fig 3B) of the DNA polymerase-a/primase complex When dT50was used as a template, an RNA primer of approximately 10 nucleotides was synthesized only in the presence of the above complex The synthesis
of the RNA primer was inhibited in the presence of heliquinomycin at an IC50 value of 25 lm The DNA polymerization activity of the DNA polymerase-a/ primase complex was measured using activated DNA
as a template and a primer The observed reduction in the level of the incorporated nucleotides indicates that heliquinomycin at an IC50value of 6.5 lm inhibits the DNA polymerization activity of the complex These results indicate that, among the enzymes studied, MCM4/6/7 is the most sensitive to heliquinomycin and the DNA polymerase-a/primase complex is also rela-tively sensitive to this antibiotic (Table 1)
Sensitivity of MCM4/6/7 helicase to heliquinomycin
To understand the mechanism by which heliquino-mycin inhibits the activity of MCM4/6/7 helicase, we
0 0.43 1.4 4.3 14 43
HQ (μ M )
0 0.431.44.3 1443 (μ M ) HQ
17 10 50
nt
+ Pol α-primase
A
B
0
(μ M ) HQ 0.43 1.4 4.3 14 43
120
100
80
60
40
20
0
100
80
60
40
20
0
Fig 3 Effect of heliquinomycin on the RNA priming activity of the DNA polymerase-a/primase complex (A) Top: effect of increasing concentrations of heliquinomycin (HQ) on the RNA priming action
of the DNA polymerase-a/primase RNA priming activity was mea-sured by the analysis of oligoA synthesis when dT 50 was used as the template The products were electrophoresed under denaturing conditions The three oligonucleotides of A10, 17-mer and dT50 were labelled at their 5¢ ends and electrophoresed to determine the size of the synthesized oligoA fragment The arrow indicates the position of the RNA primer synthesized by DNA polymerase-a/prim-ase Bottom: radioactivity of the synthesized RNA primer The radioactivity recorded for RNA in the control reaction mixture lack-ing heliquinomycin was considered to be 100%, and that recorded for the reaction mixtures containing heliquinomycin was presented
in relation to this control value Two independent experiments were performed, and an average of the values was plotted together with error bars (B) Effect of increasing concentrations of heliquinomycin
on the DNA polymerization activity of the DNA polymerase-a/prim-ase The reaction was performed using activated DNA as a primer and template The acid-insoluble radioactive material trapped on the glass fibre filter was measured The radioactivity recorded in the case of the control reaction mixture lacking heliquinomycin was considered to be 100%, and that recorded for the reaction mixture containing heliquinomycin was presented in relation to this control value Two independent experiments were performed, and an aver-age of the data was plotted together with the error bars.
Trang 5examined the effect of heliquinomycin on the formation
of the MCM4/6/7 complex (Fig 4) In the absence of
heliquinomycin, the MCM4/6/7 complex, as detected
using anti-MCM4 IgG, exhibits a trimeric or hexameric
structure, depending on its mobility in the gel We
observed that the hexamer–trimer proportion increased
slightly with an increase in the heliquinomycin
concen-tration The hexameric form of the MCM4/6/7 complex
was dominant in the presence of 14 lm of heliquino-mycin, and larger complexes were detected in the pres-ence of 43 lm of heliquinomycin Thus, it appears that higher concentrations of heliquinomycin affect signifi-cantly the formation of the MCM4/6/7 complex We also examined the sensitivity of the ATPase activities
of the MCM4/6/7 complex and the SV40 T antigen
to heliquinomycin in the absence of single-stranded DNA (Fig 5) Heliquinomycin inhibited the ATPase
0 0.43 1.4 4.3 14 43 (μM) HQ
669
440 kDa
(4/6/7)2
(4/6/7)
Fig 4 Effect of increasing concentrations of heliquinomycin (HQ)
on the formation of the MCM4/6/7 complex The MCM4/6/7
com-plex was incubated in the presence or absence of heliquinomycin
and subsequently electrophoresed on a native polyacrylamide gel.
The proteins in the gel were transferred onto a filter and the
MCM4 protein was detected by incubating the filter with rabbit
anti-MCM4 IgG, followed by horseradish peroxidase-conjugated
anti-rabbit IgG Finally, the bound antibodies were examined for
chemiluminescence using West Pico chemiluminescent substrate
(Thermo Scientific, Rockford, IL, USA) The positions to which
thyroglobulin (669 kDa) and ferritin (440 kDa) migrated in the gel
are indicated.
Tag
MCM
0
0
ATP
120 100 80 60
40 20 0
Fig 5 Top: effect of increasing concentrations of heliquinomycin (HQ) on the ATPase activities of the MCM4/6/7 complex (340 ng) and the SV40 T antigen (200 ng) in the absence of single-stranded DNA After incubation under these conditions, an aliquot of the mix-ture was subjected to thin layer chromatography The radioactivity at the sites to which Piand ATP migrated was measured, and the ratio
of the released P i to ATP was calculated The ratio obtained in the case of the reaction performed without the enzymes was subtracted from that obtained in the reactions performed with the enzymes Bottom: the ratio obtained in the case of the control reaction mixture which lacked heliquinomycin was considered to be 100%, and that recorded for the reaction mixture that contained heliquinomycin was presented in relation to this control value Two independent experi-ments were performed for the MCM4/6/7 complex, and an average
of the values was plotted together with error bars.
Table 1 Sensitivity of the enzymes to heliquinomycin The IC50
values indicated are those calculated in the present study as well
as in a previous investigation [21] Those determined in the
pre-vious investigation are marked by an asterisk.
IC50(l M )
SV40 chromosome replication in vitro* > 72
Trang 6activities of these two helicases only slightly We also
examined its effect on these activities in the presence of
heat-denatured, single-stranded DNA (Fig 6); under
these conditions, the ATPase activity of the MCM4/6/7
complex increased manifold [9] Heliquinomycin
scar-cely inhibited the ATPase activity of the T antigen, but
inhibited that of the MCM4/6/7 helicase at an IC50
value of 5.2 lm The ATPase activity of MCM4/6/7
was also stimulated in the presence of M13mp18
single-stranded DNA in place of heat-denatured DNA, and
the stimulated activity was also inhibited in the
pres-ence of heliquinomycin (Fig S5) These results suggest
that heliquinomycin interferes with the interaction of
the MCM4/6/7 complex with single-stranded DNA, which is required to increase the ATPase activity of this complex
Discussion
Heliquinomycin was first characterized as a compound that inhibits bacterial cell growth, and was found to inhibit DNA synthesis in several cancer cells at an
IC50 value of 1.4–4 lm [21] In the present study, we found that heliquinomycin inhibits BrdU incorporation into DNA at an IC50 value of 2.3 lm; this result is consistent with the previous findings The reported study also indicated that the cell cycle progression of HeLa cells was retarded during the S phase and the cells were arrested in the G2 phase in the presence of heliquinomycin [21] Heliquinomycin inhibits the cellular DNA helicase, helicase I, at an IC50 value of 7–14 lm, but does not inhibit the activity of topoi-somerases Our study indicates that heliquinomycin inhibits the activity of human MCM4/6/7 helicase at
an IC50value of 2.4 lm, but scarcely inhibits the DNA helicase activity of the SV40 T antigen and the Werner protein, or the oligonucleotide displacement activity of human RPA Further, it inhibits the RNA priming and DNA polymerization activities of the human DNA polymerase-a/primase at IC50values of 25 and 6.5 lm, respectively We also examined the effect of heliquino-mycin on the DNA helicase activity of human REC-QL4 protein Heliquinomycin inhibited this activity at
an IC50 value of 14 lm (data not presented) Thus, among the enzymes studied, MCM4/6/7 helicase was found to be the most sensitive to heliquinomycin These results suggest that MCM helicase and DNA polymerases may be the critical targets of heliquinomy-cin during cellular DNA replication Further, we observed that the checkpoint system that is induced by the inhibition of DNA polymerases during DNA repli-cation is not induced in HeLa cells treated with 4.3 lm heliquinomycin (data not presented) This suggests that the MCM helicase, rather than the DNA polymerases,
is the main target of heliquinomycin in vivo
Heliquinomycin not only inhibited the helicase activ-ity of the MCM4/6/7 complex, but also inhibited the single-stranded DNA-dependent ATPase activity of the complex Heliquinomycin suppressed the ATPase activity of the complex in the absence of single-stranded DNA, but the enzymatic activity was significantly less sensitive to heliquinomycin Thus, heliquinomycin may inhibit the ATPase activity and DNA helicase activity of the MCM4/6/7 complex by affecting the ability of this complex to interact with single-stranded DNA The finding that the activities
0
(μ M ) 0.431.44.31443 00.431.44.31443 HQ
+ MCM4/6/7
A
B
+ Tag
Pi
ATP
Tag
MCM
0
(μ M ) HQ
120
100
80
60
40
20
0
Fig 6 (A) Effect of increasing concentrations of heliquinomycin
(HQ) on the ATPase activities of the MCM4/6/7 complex (120 ng)
and SV40 T antigen (200 ng) in the presence of single-stranded
DNA The radioactivity at the sites to which Piand ATP migrated
was measured, and the ratio of the released Pito ATP was
calcu-lated The ratio in the case of the reaction performed without the
enzyme was subtracted from that in the case of the reactions
per-formed with these enzymes (B) The ratio obtained in the case of
the control reaction mixture which lacked heliquinomycin was
con-sidered to be 100%, and that recorded for the reaction mixture that
contained heliquinomycin was presented in relation to this control
value Two independent experiments were performed for the
MCM4/6/7 complex, and an average of the values was plotted
together with error bars.
Trang 7of DNA polymerase-a/primase are also inhibited at
higher concentrations of heliquinomycin may suggest
that heliquinomycin interacts with single-stranded
DNA to interfere with the activities However, there is
no evidence for the interaction of heliquinomycin with
single-stranded DNA In contrast, the formation of the
MCM4/6/7 complex was inhibited by heliquinomycin
at higher concentrations Although these
concentra-tions are higher than those that inhibit MCM4/6/7
helicase activity, it is possible that heliquinomycin
interacts directly with the MCM4/6/7 complex to
inhi-bit the interaction of this complex with single-stranded
DNA, even at low concentrations
MCM proteins are considered to be one of the
most sensitive diagnostic markers for the detection
of cancer cells in human tissues [25] The expression
of MCM proteins appears to be critical for the
development of cancer cells, as this expression shows
a strong correlation with the malignant
transforma-tion of cells The finding that MCM2–7 proteins are
overexpressed in transformed cancer cells [26]
suggests that the upregulation of MCM protein
expression may play a role in the development of
cancer cells Consistent with this notion, it has
recently been reported that deregulated expression of
the MCM7 protein accelerates the transformation of
cells [27] Thus, MCM proteins are among the most
critical targets for achieving the inhibition of cancer
cell growth Furthermore, heliquinomycin may
have useful applications in the development of
MCM-specific anticancer drugs
Materials and methods
BrdU labelling of HeLa cells
HeLa cells were cultured in Dulbecco’s modified Eagle’s
medium supplemented with 7% fetal calf serum Cells
cul-tured on coverslips were incubated with dimethyl sulfoxide
or increasing concentrations of heliquinomycin for 1 h and
then pulse labelled with 20 lm BrdU for 20 min After being
permeabilized and blocked by incubation with 0.1% Triton
anti-MCM7 mouse Ig (sc-9966; Santa Cruz Biotechnology,
blocking solution The cells were washed with the same
solution and then incubated with cyanine-3
(Cy3)-conju-gated anti-rabbit IgG (Jackson ImmunoResearch, West
solu-tion They were then re-fixed, treated with 4 m HCl for
30 min at room temperature and incubated with rat anti-BrdU Ig (clone BU1/75; Harlan Sera Laboratory, Belton, Leicestershire, UK), followed by incubation with FITC-conjugated anti-rat IgG (Cappel, Organon Teknika Corpo-ration, Durham, NC, USA) Positive immunoreactivities were detected with fluorescence microscopy (BX-9000; KEYENCE, Osaka, Japan)
DNA helicase and ATPase activities of the DNA helicases
A human MCM4/6/7 complex was prepared, and its DNA helicase activity was measured, as reported previously, except for some minor modifications [9] The standard reac-tion mixture (20 lL) contained 50 mm Tris/HCl (pH 7.9),
20 mm 2-mercaptoethanol, 10 mm ATP, 10 mm magnesium
a 17-mer oligonucleotide annealed to M13mp18 DNA and
an approximately100 ng sample of human MCM4/6/7 com-plex, a 25 ng sample of SV40 T antigen or a 1.25 ng sample
of Werner protein, in the presence or absence of heliquino-mycin at the indicated concentrations This mixture was
anal-ysed using 12% PAGE The ATPase activity was measured
by incubating either the MCM proteins (120–340 ng) or the
bovine serum albumin, 10 mm magnesium acetate, 10 mm ATP and heliquinomycin at the indicated concentrations in the presence or absence of 5 lg of single-stranded DNA (heat-denatured) Further, 0.5 lL of the reaction mixture was spotted onto a poly (ethyleneimine)-cellulose thin layer chromatography plate (Cellulose F; Merck, Darmstadt,
period of 2 h using a solution of 0.8 m acetic acid and 0.8 m LiCl The radioactivity on the plate was detected using a Bio-Image Analyser (FLA3000; Fuji, Tokyo, Japan)
Formation of the MCM4/6/7 complex The reaction mixture (10 lL) containing 50 mm Tris/HCl
presence or absence of heliquinomycin The resulting solu-tion was analysed on a 5% acrylamide gel in 50 mm Tris/ HCl (pH 8.0) and 50 mm glycine Subsequently, the gel was immersed in a solution containing 49 mm Tris, 38 mm
order to achieve protein denaturation The proteins in the gel were then transferred onto a membrane filter (Immobilon;
Trang 8Millipore, Billerica, MA, USA) and anti-MCM4 IgG were
used to detect MCM4 on the filter [9]
RNA priming and DNA synthesis with DNA
polymerase-a/primase
The DNA polymerase-a/primase complex was purified from
HeLa cells by immunoadsorption, followed by elution from
a column coated with a monoclonal antibody (SJK237), as
reported previously [28] DNA polymerase activity was
measured using a reaction mixture (20 lL) containing
20 mm Tris/HCl (pH 7.9), 3.3 mm 2-mercaptoethanol,
and 85 ng DNA polymerase-a/primase complex in the
pres-ence of heliquinomycin at the indicated concentrations The
reaction was terminated by the addition of 30 lL of sodium
added, and the acid-insoluble radioactive material trapped
on a glass fibre filter was measured in a liquid scintillation
cocktail The reaction mixture (10 lL) used for the
mea-surement of the RNA priming activity contained 40 mm
Tris/HCl (pH 7.5), 10 mm magnesium acetate, 1 mm
heliquinomycin at the indicated concentrations This
(0.6 units) was added, and the mixture was further
(0.1% bromophenol blue, 0.1% xylene cyanol, 10 mm
EDTA and 98% formamide), and the products were
analy-sed on a 25% polyacrylamide gel containing 7 m urea The
at their 5¢ ends and used as markers The gel was dried and
the radioactivity was detected using a Bio-Image Analyser
Preparation of the RPA complex
cDNAs for human RPA1, RPA2 and RPA3 were
synthe-sized from mRNA extracted from HeLa cells by the reverse
transcription-polymerase chain reaction (RT-PCR) method
(Invitrogen, Carlsbad, CA, USA), and were cloned into the
baculovirus vectors pVL1393, pAcUW31 and pVL1393,
-RPA1 fusion protein, and RPA2 as a flag-RPA2 fusion
protein High-5 cells were co-infected with the three viruses
expressing the RPA1, RPA2 and RPA3 proteins for 2 days
The recombinant RPA proteins in the lysates of the
infected cells were purified by performing
nickel-nitrilotri-acetic acid (Qiagen, Hilden, Germany) affinity column
chromatography as follows The purification involved the
suspension of the infected cells in lysis buffer consisting
of 10 mm Tris/HCl (pH 7.5), 130 mm NaCl, 1% Triton X-100, 10 mm NaF, 10 mm sodium phosphate buffer,
San Jose, CA, USA) The mixture was incubated for
40 min on ice, and insoluble components were separated by centrifugation at 137 000 g (TLS55; Beckman, Fullerton,
lysate, 1/10 vol of nickel-nitrilotriacetic acid-agarose was
rocking platform Agarose beads were then collected by centrifugation and thoroughly washed with buffer A [50 mm sodium phosphate buffer (pH 6.0), 300 mm NaCl and 10% glycerol] containing 20 mm imidazole Next, the beads were washed once with buffer B [50 mm sodium phosphate buffer (pH 8.0), 300 mm NaCl and 10% glyc-erol] containing 20 mm imidazole, and the proteins bound
to the beads were eluted by adding buffer B containing
300 mm imidazole at a volume equivalent to 1 bed This
platform and separation of the beads by centrifugation The proteins were eluted twice more The eluates were pooled and diluted to decrease the NaCl concentration to
50 mm, and the solution thus obtained was concentrated using Centricon 30 (Millipore) The concentrated proteins were loaded onto a MonoQ column (GE Healthcare, Pis-cataway, NJ, USA), and the bound proteins were eluted using a linear NaCl gradient (0.1–0.6 m) The RPA1 (70 kDa), RPA2 (34 kDa) and RPA3 (14 kDa) proteins were co-eluted with approximately 0.3 m NaCl, and were concentrated using Microcon 30 after the salt concentration had decreased to 0.1 m The oligonucleotide displacement activity of RPA was measured using the same reaction mix-ture as that employed to assess the DNA helicase activity, except that the reaction mixture contained 200 ng of RPA complex
Purification of Werner helicase High-5 cells infected with recombinant virus encoding
centrifugation The cells were lysed with 0.5% Nonidet
P-40 in buffer C [50 mm Tris/HCl (pH 7.9), 150 mm NaCl, 10% glycerol, 1 mm phenylmethylsulfonyl fluoride and
added to the lysate at a final concentration of 0.5 m After incubation for 30 min on ice, the cell lysate was centrifuged
The supernatant was passed through a DE52 (Whatman, Maidstone, Kent, UK) column equilibrated with 0.5 m NaCl in buffer C to remove nucleic acids Flow-through fractions were loaded on to a nickel-nitrilotriacetic acid
Trang 9onto the nickel-nitrilotriacetic acid column, the column was
washed with buffer D containing 0.2 m NaCl and 25 mm
imidazole, and eluted with buffer D containing 0.2 m NaCl
and 200 mm imidazole The fractions containing Werner
Pooled fractions were loaded onto a MonoS column (GE
Healthcare) After the column had been washed with buffer
H [25 mm Hepes/NaOH (pH 7.8), 1 mm EDTA, 10%
glyc-erol, 0.01% Nonidet P-40, 1 mm phenylmethylsulfonyl
bound proteins were eluted with buffer H containing 0.5 m
NaCl Fractions around the main peak were pooled,
Germany) and then fractionated on Superdex 200 HR in
buffer H containing 0.1 m NaCl The purified protein was
concentrated with Vivaspin and dialysed against buffer H
containing 0.1 m NaCl
Other materials
The SV40 T antigen was prepared as reported previously
[28] Heliquinomycin was purified from Streptomyces sp
MJ929-SF2, as reported previously [29], and 1 mg of the
To prepare activated DNA, calf thymus DNA (30 mg) was
mix-ture (10 mL) containing 50 mm Tris/HCl (pH 7.5), 5 mm
then dialysed against 50 mm Tris/HCl (pH 8.1) and 5 mm
Acknowledgements
This study was supported in part by a Grant-in-Aid
for Scientific Research from the Ministry of Education,
Science, Sports and Culture of Japan
References
1 Tye BK (1999) MCM proteins in DNA replication
Annu Rev Biochem 68, 649–686
2 Bell SP & Dutta A (2002) DNA replication in
eukary-otic cells Annu Rev Biochem 71, 333–374
3 Forsburg SL (2004) Eukaryotic MCM proteins: beyond
replication initiation Microbiol Mol Biol Rev 68, 109–
131
4 Kubota Y, Mimura S, Nishimoto S, Takisawa H &
Nojima H (1995) Identification of the yeast
MCM3-related protein as a component of Xenopus
DNA replication licensing factor Cell 81, 601–609
5 Chong JPJ, Mahbubani HM, Khoo C-Y & Blow JJ
(1995) Purification of an MCM-containing complex as a
component of the DNA replication licensing system Nature 375, 418–421
6 Madine MA, Khoo C-Y, Mills AD & Laskey RA (1995) MCM3 complex required for cell cycle regulation
of DNA replication in vertebrate cells Nature 375, 421–424
7 Prokhorova TA & Blow JJ (2000) Sequential MCM/P1 subcomplex assembly is required to form a heterohex-amer with replication licensing activity J Biol Chem
275, 2491–2498
8 Koonin EV (1993) A common set of conserved motifs
in a vast variety of putative nucleic acid-dependent ATPases including MCM proteins involved in the initia-tion of eukaryotic DNA replicainitia-tion Nucleic Acids Res
21, 2541–2547
9 Ishimi Y (1997) A DNA helicase activity is associated with an MCM4, -6, and -7 protein complex J Biol Chem 272, 24508–24513
10 Lee J-K & Hurwitz J (2000) Isolation and characteriza-tion of various complexes of the minichromosome maintenance protein of Schizosaccharomyces pombe
J Biol Chem 275, 18871–18878
11 Kaplan DL, Davey MJ & O’Donnell M (2003) Mcm4,6,7 uses a ‘pump in ring’ mechanism to unwind DNA by steric exclusion and actively translocate along
a duplex J Biol Chem 278, 49171–49182
12 Bochman M & Schwacha A (2007) Differences in the single-stranded DNA binding activities of MCM2-7 and MCM467: MCM2 and 5 define a slow ATP-dependent step J Biol Chem 282, 33759–33804
13 Labib K, Tercero JA & Diffley JF (2000) Uninterrupted MCM2–7 function required for DNA replication fork progression Science 288, 1643–1647
14 Aparicio OM, Weinstein DM & Bell SP (1997) Compo-nents and dynamics of DNA replication complexes in
S cerevisiae: redistribution of MCM proteins and Cdc45p during S phase Cell 91, 59–69
15 Katou Y, Kanoh Y, Bando M, Noguchi H, Tanaka H, Ashikari T, Sugimoto K & Shirahige K (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a
stable replication-pausing complex Nature 424, 1078–1083
16 Ishimi Y, Komamura Y, You Z & Kimura H (1998) Biochemical function of mouse minichromosome main-tenance 2 protein J Biol Chem 273, 8369–8375
17 Sato M, Gotow T, You Z, Komamura-Kohno Y, Uchiyama Y, Yabuta N, Nojima H & Ishimi Y (2000) Electron microscopic observation and single-stranded DNA binding activity of Mcm4,6,7 complex J Mol Biol
300, 421–431
18 Moyer SE, Lewis PW & Botchan MR (2006) Isolation
of the Cdc45–MCM2–7–GINS (CMG) complex, a can-didate for the eukaryotic DNA replication fork helicase Proc Natl Acad Sci USA 103, 10236–10241
Trang 1019 Bochman ML & Schwacha A (2008) The Mcm2–7
complex has in vitro helicase activity Mol Cell 31,
287–293
20 Chino M, Nishikawa K, Tsuchida T, Sawa R,
Nakamura H, Nakamura KT, Muraoka Y, Ikeda D,
Naganawa H, Sawa T et al (1997) Heliquinomycin, a
new inhibitor of DNA helicase, produced by
of heliquinomycin J Antibiot 50, 143–146
21 Chino M, Nishikawa K, Yamada A, Ohsono M, Sawa
T, Hanaoka F, Ishizuka M & Takeuchi T (1998) Effect
of a novel antibiotic, heliquinomycin, on DNA helicase
and cell growth J Antibiot 51, 480–486
22 Tuteja N, Tuteja R, Rahman K, Kang L-Y & Falaschi
A (1990) A DNA helicase from human cells Nucleic
Acids Res 18, 6785–6792
23 Tho¨mmes P, Fett R, Schray B, Burkhart R, Barnes M,
Kennedy C, Brown NC & Knippers R (1992) Properties
of the nuclear P1 protein, a mammalian homologue of
the yeast Mcm3 protein Nucleic Acids Res 20, 1069–
1074
24 Georgaki A, Strack B, Podust V & Hu¨bscher U (1992)
DNA unwinding activity of replication protein A FEBS
Lett 308, 240–244
25 Laskey R (2005) The Croonian lecture 2001 hunting the
antisocial cancer cell: MCM proteins and their
exploita-tion Philos Trans R Soc Lond B Biol Sci 360, 1119–
1132
26 Ishimi Y, Okayasu I, Kato C, Kwon H-J, Kimura H,
Yamada K & Song S-Y (2003) Enhanced expression
of MCM proteins in cancer cells derived from uterine
cervix Eur J Biochem 270, 1089–1101
27 Honeycutt KA, Chen Z, Koster MI, Miers M,
Nuchtern J, Hicks J, Roop DR & Shohet JM (2006)
Deregulated minichromosomal maintenance protein MCM7 contributes to oncogene driven tumorigenesis Oncogene 25, 4027–4032
28 Ishimi Y, Claude A, Bullock P & Hurwitz J (1988) Com-plete enzymatic synthesis of DNA containing the SV40 origin of replication J Biol Chem 263, 19723–19733
29 Chino M, Nishikawa K, Umekita M, Hayashi C, Yamazaki T, Tsuchida T, Sawa T, Hamada M & Takeuchi T (1996) Heliquinomycin, a new inhibitor of DNA helicase, produced by Streptomyces sp MJ929-SF2 I Taxonomy, production, isolation, physico-chemi-cal properties and biologiphysico-chemi-cal activities J Antibiot 49, 752–757
Supporting information
The following supplementary material is available: Fig S1 Structure of heliquinomycin
Fig S2 SDS–PAGE of purified proteins
Fig S3 Detection of MCM proteins in purified DNA polymerase-a/primase complex
Fig S4 Effect of heliquinomycin on the oligonucleo-tide displacement activity of RPA
Fig S5 Effect of heliquinomycin on the ATPase activities of MCM4/6/7 in the presence of M13mp18 single-stranded DNA
This supplementary material can be found in the online version of this article
Please note: Wiley-Blackwell is not responsible for the content or functionality of any supplementary materials supplied by the authors Any queries (other than missing material) should be directed to the corre-sponding author for the article