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INTRODUCTION |
Initiation of chromosomal DNA replication and cell cycle
progression are tightly regulated in eukaryotes. In the yeast
Saccharomyces cerevisiae, several cdc
(cell division cycle) mutants that
block initiation of chromosomal DNA replication have been isolated and characterized (1, 2), for example, cdc28, cdc4,
cdc6, and cdc7. The stepwise assembly of proteins
at origins of DNA replication is a crucial part of regulating entry
into S phase. Two key factors that mediate such cell cycle regulation
are Cdc6 protein level and availability and the presence of an active
cyclin-dependent kinase (Cdk) (see Ref. 3 for review). The
origin recognition complex is bound to origins of DNA replication at
all stages of the cell cycle in S. cerevisiae (4-6).
However, Cdc6 is not recruited to origins until late in M phase and is
required for the association of the Mcm2-7 family proteins at
origins to form a prereplicative complex (6-8).
The Cdc6 protein-dependent stage of the assembly reaction
is inhibited by active Clb-Cdks (6, 9, 10). Because Cdc6 protein is
synthesized only from late M phase until late G1 (11), prereplicative
complexes can only be assembled during this period of the cell cycle. S
phase cyclin-Cdk (Cdc28p/Clb5p or Cdc28p/Clb6p) activity is required
for the chromatin association of Cdc45p just before the initiation of
chromosomal DNA replication (12). The previous results demonstrated
that Cdc28 protein-Clb kinase is required throughout S phase to
activate origins when they are scheduled to fire (13).
The Cdc7/Dbf4 complex is a Cdk-like protein kinase (see Refs. 14 and 15
for review) that is also required for entry into S phase at a very late
stage. CDC7 transcript levels are constant throughout the
cell cycle, whereas DBF4 transcription is cell cycle
regulated and the transcript abundance peaks near the G1 to
S phase transition. The DBF4 gene was first identified as a gene that could suppress cdc7 mutants when present in
multiple copies (16), and Dbf4p is now known to be required for
Cdc7p-mediated kinase activity. It has been shown that a
Cdc7p-associated H1 kinase activity is cell cycle regulated (17). It is
not clear, however, whether this H1 kinase activity reflects the normal
activity of the Cdc7p kinase. Recently, homologues of Cdc7p have been
identified in Schizosaccharomyces pombe (18), mouse (19),
Xenopus, and humans (20-22), and a Dbf4p homologue has also
been found in S. pombe (23, 24) and human (25). The presence
of these homologues in evolutionarily distant species suggests that
Cdc7p/Dbf4p plays a conserved role in the initiation of DNA replication
in eukaryotes.
Several lines of evidence suggest that Cdc7p/Dbf4p acts directly at
origins of DNA replication and that the Mcm proteins may be one of the
targets of the kinase. DBF4 was isolated in a one-hybrid screen for autonomously replicating sequence
(ARS)1-interacting factors
(26). Interestingly, its interaction with origins was found to be
independent of its ability to bind to Cdc7p. Two recent reports (27,
28) suggest that Cdc7p activity is required to activate individual
origins rather than for a global activation of S phase, because
temperature-sensitive cdc7 mutants demonstrate an inability
to activate late rather than early replication origins at a
semi-permissive temperature. Potential target(s) of Cdc7p that are
required for the initiation of DNA replication were revealed through
genetic analysis of a mutant in S. cerevisiae that could
bypass the requirement for CDC7 or DBF4 (17). The mutation was found to reside in MCM5/CDC46 (29), a member of the Mcm family of six sequence-related proteins (30). Since then, a
number of reports have suggested that some Mcm proteins such as Mcm2
and Mcm3 are in vitro substrates of Cdc7p kinases from
various sources (21, 23, 31). Another study recently reported that all
yeast Mcm proteins, except for Mcm5 expressed and prepared from insect
cells, were substrates for the Cdc7/Dbf4p protein kinase complex
(32).
RAD53 encodes a dual specificity protein kinase that is
required for all three DNA damage checkpoints at G1, S
phase, and G2 (33). It is thought to be part of the
transducer class (34). Several lines of circumstantial evidence suggest
that in addition to its checkpoint function, Rad53p could be also
involved in the regulation of temporal order origin firing during
chromosomal DNA replication (35).
In this report, we describe the expression and the purification of
recombinant yeast Cdc7p/Dbf4p to near homogeneity and biochemical analysis of the Cdc7p/Dbf4p complex-associated protein kinase activity.
We show that both subunits of the recombinant enzyme are
auto-phosphorylated and that this reaction requires active Dbf4p and
Cdc7p. The recombinant protein efficiently phosphorylates Mcm2 and also
phosphorylates with somewhat lower efficiency the remaining Mcm
proteins as well as the largest subunit of DNA polymerase 
-primase
and RPA. However, when Mcm2 protein was pretreated with an
alkaline phosphatase, it was not phosphorylated by the Cdc7p/Dbf4p kinase, suggesting that Mcm2 protein must be prephosphorylated by an
unknown kinase(s) to be a substrate for Cdc7/Dbf4 protein kinase.
Analysis of mutant Cdc7/Dbf4 protein kinases suggests that ARS-binding
and Cdc7 protein kinase activation domains of Dbf4p collaborate to form
an active Cdc7p/Dbf4p complex that functions in chromosomal DNA
replication. Finally, inhibition of the Cdc7p/Dbf4p complex kinase
activity by Rad53p-mediated phosphorylation is demonstrated. Based on
these results, it is proposed that Rad53p regulates the temporal
activation of chromosomal DNA replication origins in yeast by
regulating the activity of Cdc7p/Dbf4p protein kinase.
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MATERIALS AND METHODS |
Yeast Strains--
The following yeast strains were used in this
study: S. cerevisiae 208 (MATa cdc7-1
ura3-52 leu2-3, 112), L128-2D (MATa dbf4-1 ura3
trp1), L202-1A (MATa dbf4-2 ura3 leu2 trp1),
KKY743 (MAT dbf4-3 ura3 leu2 trp1), KKY744
(MAT dbf4-4 ura3 leu2 trp1), and KKY745
(MAT dbf4-5 ura3 leu2 trp1) were previously
described (16). YKY9 (MATa leu2 trp1-298 ura3-52 prb
PEP4::TRP1), and YKY30 (MAT
ura3 leu2 trp1 bob1 CDC7::LEU2
PEP4::TRP1) were used for expression and
purification of GST-Mcm 2-7 proteins. CB001-2HA-Rad53
(MAT leu2 trp1 ura3 prb pep4::URA3
RAD53::[YCpRAD53-HA]) was constructed by
transforming S. cerevisiae CB001 (36) with 2HA-tagged
RAD53 plasmid (Ycp-RAD53-HA) (37) digested with restriction enzyme BamHI. The dbf4-1 sup1 was isolated from
spontaneous revertants of temperature-sensitive L128-2D strain.
Primer DNA and Plasmid DNA--
Standard DNA manipulations were
carried out according to Sambrook et al. (38).
Oligonucleotides
5'-GGCCAGATCTCATAATGACAAGCAAAACGA-3', 5'-GGCCAGATCTTTGCTATTCAGATATTAGG-3',
5'-GGCCGGATCCAAGAAAATGGTTTCTCCAACGAAA-3', and
5'-GGCCGGATCCCTATATTTGAAATCTGAGATT-3'
(underlined nucleotides represent either BglII or
BamHI sites, and bold nucleotides are the initiation
or termination codons) were synthesized on an automated DNA synthesizer
(Beckman) and were used for polymerase chain reaction amplification of
the DNA fragments containing CDC7 and DBF4 on the
plasmid pKK709 and pKK616 DNA (16). Amplified DNA was digested with
either BglII (for CDC7) or BamHI
enzyme (for DBF4) and inserted into either BglII-
or BamHI-digested baculovirus vector pBacPAK9. The resulting
constructs, pBac-Cdc7p and pBac-Dbf4p, were co-transfected with
Bsu36I-linealized wild type virus BacPAK6 viral DNA into Sf9 cells using cationic liposome-mediated transfection
according to the Invitrogen protocol, and recombinant virus was plaque
purified. The presence of an insert in a putative recombinant virus was verified by polymerase chain reaction using primers complementary to
the polyhedron locus. Recombinant virus Bac-Cdc7p 17 and Bac-Dbf4p 32
were amplified in Sf9 cells to a titer of 108
plaque-forming units/ml. Recombinant virus containing mutant cdc7-1 or dbf4-1~5 were constructed as wild
type CDC7 and DBF4 genes.
Expression of Recombinant Protein--
Sf9 cells were
grown to confluence in T-75 Falcon flasks at 27 °C in Grace's
medium supplemented with 10% fetal bovine serum, 50 µg/ml
gentamycin, and 0.1% pleuronic F-68 and infected with recombinant
virus pBac-Cdc7 17 or pBac-Dbf4 32 at a multiplicity of infection of
10. Cells were harvested at 72 h post-infection by centrifugation
at 1,200 × g and washed twice with serum-free Grace's medium.
Purification of the Recombinant Cdc7p-Dbf4p Protein Kinase
Complex--
Recombinant virus pBac-Cdc7 17 and pBac-Dbf4 32 were
used to infect 2 L of Sf9 cells (2 × 106
cells/ml) at a multiplicity of infection of 10. After 72 h the cells were harvested, washed with serum-free Grace's medium,
resuspended in 60 ml of ice-cold HSL buffer (50 mM
Tris-HCl, pH 7.4, 1% (v/v) Nonidet P-40, and 500 mM NaCl)
containing a mixture of protease inhibitors (10 µg/ml aprotinin, 5 µg/ml leupeptin, 100 µg/ml bacitracin, 250 µg/ml soybean trypsin
inhibitor, 1 mM phenylmethylsulfonyl fluoride, and 10 mM benzamidine), and subjected to sonication by Ultrosonic
Disruptor UD-201 (Tomy, Japan). To the cell extract (52 ml), obtained
by centrifugation at 27,000 × g for 20 min, 16.3 g of solid (NH4)2SO4 (50%
saturation) was gradually added with stirring. After 30 min stirring,
the extract was centrifuged at 27,000 × g for 20 min.
The precipitated protein was dissolved in 30 ml of buffer A (50 mM Tris-HCl, pH 7.5, 10 mM 2-mercaptoethanol, 1 mM EDTA, 10% glycerol (v/v), and 1 mM
phenylmethylsulfonyl fluoride) and dialyzed against 2 liters of buffer
A containing 50 mM NaCl for 4 h at 0 °C. After
centrifugation at 27,000 × g for 20 min, the
suspension was loaded onto an S-Sepharose column (80 ml) equilibrated in buffer A containing 100 mM NaCl. The column was washed
with three bed volumes of the same buffer, and the protein was eluted with a 320-ml linear gradient of 100-600 mM NaCl in buffer
A. Fractions containing Cdc7p and Dbf4p, as identified by activity assays and Western blot analysis (0.2-0.3 M NaCl), were
pooled and loaded onto a Hi Trap Heparin column (5 ml) equilibrated
with buffer A containing 200 mM NaCl. After washing with
three bed volumes of equilibration buffer, protein was eluted with a
120-ml linear gradient of 0.2-1 M NaCl in buffer A. The
peak fractions (0.5-0.65 M NaCl) were pooled, dialyzed
twice against 1 liter of buffer A containing 100 mM NaCl
for 3 h, and loaded onto a Mono Q column (HR5/5, 1 ml)
pre-equilibrated with buffer A containing 100 mM NaCl.
Protein was eluted with a 15-ml linear gradient from 100 to 700 mM NaCl in buffer A. Active fractions (0.3-0.4
M NaCl) were pooled together, dialyzed against 50%
glycerol, 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 1 mM
phenylmethylsulfonyl fluoride for 2 h and stored at
80 °C.
Purification of Yeast 2HA-tagged Rad53p Protein
Kinase--
S. cerevisiae CB001 cells expressing 2HA-tagged
Rad53p were grown at 30 °C in 24L YPD medium to
A600 = 1-2 and harvested by centrifugation at 8,000 rpm for 20 min. Cells were resuspended in
buffer A (36) to 1 g/ml and disrupted by passage through a Gaulin
homogenizer at 10,000 psi. To cell extracts, NaCl was added to a final
concentration of 0.5 M. After stirring at 0 °C for 30 min, cell debris was removed by centrifugation in a Beckman JLA10.5
rotor at 8,000 rpm for 20 min, and the supernatant was saved. To the
supernatant, 10%(w/v) Polymin P (pH 8) solution was slowly added to
0.5% (final concentration), mixed for 15 min, and centrifuged in a
Beckman JLA 10.5 rotor at 8000 rpm for 20 min. 0.313 g of ammonium
sulfate was added per ml of supernatant, and the mixture was stirred
for 30 min. The protein precipitate was collected by centrifugation in
a Beckman JLA 10.5 rotor at 10,000 rpm for 30 min. The pellet was
stored at 80 °C. The precipitate was resuspended in 20 ml of
buffer A and dialyzed against 2 liters of buffer A for 2 h and 2 liters of 0.1 M NaCl in buffer A for 2 h. Insoluble
material was removed from the dialysate by centrifugation in a Beckman
JA20.0 rotor at 10,000 rpm for 10 min. The supernatant was applied on
an S-Sepharose column (75 ml) equilibrated with 0.1 M NaCl
in buffer A, the column was washed with three column volumes of 0.1 M NaCl in buffer A, and protein was eluted with one column
volume of 0.5 M NaCl in buffer A. The protein was eluted at
0.5 M NaCl. Fractions were checked by Western blot analysis followed by immunostaining. The fractions containing the protein was
pooled together (Fraction I). Fraction I was dialyzed twice against 2 liters of 0.05 M NaCl in buffer A for 2 h and
centrifuged in a Beckman JA20.0 rotor at 10,000 rpm for 10 min to
remove any precipitates. The supernatant was applied on a Mono Q column
(HR 10/10) equilibrated with 0.05 M NaCl in buffer A. The
column was washed with three column volumes of 0.05 M NaCl
in buffer A, and then protein was eluted with 20 column volumes of
linear gradient from 0.05 to 0.5 M NaCl in buffer A. Fractions containing the Rad53 protein were identified by Western blot
analysis and pooled (Fraction II). Fraction II was dialyzed twice
against 2 liters of 0.1 M NaCl in buffer A for 2 h and
applied on a Mono S column (HR 5/5) equilibrated with 0.1 M
NaCl in buffer A. The column was washed with three column volumes of
0.1 M NaCl in buffer A and then eluted with a 30-ml linear
gradient of 0.1-0.5 M NaCl in buffer A. Fractions
containing Rad53p were identified by protein kinase activity assay
using Histone H1 and Western blot analysis, and the active fractions
were pooled (Fraction III). Fraction III was dialyzed against 1 liter
of 0.1 M NaCl in buffer A for 3 h and applied on a
Hi-Trap Heparin column (HR5/5) equilibrated with 0.1 M NaCl
in buffer A. The column was washed with three column volumes of 0.1 M NaCl in buffer A and then eluted with 20 ml of linear
gradient of 0.1-0.5 M NaCl in buffer A. Fractions containing Rad53 protein were identified by protein kinase activity assays and Western blot analysis, and the active fractions were pooled
(Fraction IV).
HU-activated Rad53p was also purified as described above from
CB001 cells expressing 2HA-tagged Rad53p after treating cells with 0.2 M hydroxyurea for 3 h at 30 °C. More detailed
characterization of the purified 2HA-tagged Rad53p will be published elsewhere.
SDS Polyacrylamide Gel Electrophoresis and Protein
Determination--
4-20% gradient SDS-PAGE was performed by the
method of Laemmli (38). Protein concentration was determined
by the method of Bradford with bovine serum albumin as standard as
described previously (31, 36).
Immunoblot Analysis--
Following SDS-PAGE, proteins were
electroblotted onto an Immobilon-P (Millipore) membrane that was
incubated for 30 min with blocking buffer, Tris-buffered saline (50 mM Tris-HCl, pH 7.4, and 100 mM NaCl)
containing 2% nonfat dry milk, and incubated overnight with rabbit
polyclonal antipeptide antiserum to Dbf4p (provided by J. Diffley) or
with rabbit polyclonal antiserum to Cdc7p expressed in
Escherichia coli (provided by H. Masai). After washing three
times with blocking buffer, the membrane was incubated with alkaline
phosphatase-conjugated goat anti-rabbit IgG for 1.5 h at room
temperature, washed three times with blocking buffer, and washed twice
with Tris-buffered saline. Color was developed as described
previously (31).
Protein Kinase Assay--
Protein kinase activity was assayed
with GST-Mcm2p purified from S. cerevisiae as a substrate.
Reaction mixtures (20 µl) contained 20 mM Hepes, pH 7.5, 10 mM MgCl2, 1 mM dithiothreitol,
10% (v/v) glycerol, 300 pmol of [
-32P]ATP (specific
activity, 10,000 cpm/pmol), 10-30 pmol of GST-Mcm2p, and Cdc7p/Dbf4p
protein kinase complex or cell extracts. After 5 min at 30 °C,
reactions were stopped by the addition of 5 µl of stop solution,
heated at 95 °C for 3 min, and subjected to 4-20% SDS-PAGE. After
electrophoresis, the gel was stained with Coomassie Brilliant Blue,
destained, dried and autoradiographed (31). To measure radioactivity
incorporated into the protein, the corresponding protein bands were
excised from the gel and quantitated in the presence of scintillation
fluid using a scintillation spectrometer.
Other Materials and Methods--
The purification of yeast
GST-Mcm2-Mcm7 from S. cerevisiae was described previously
(31), and their purity was more than 95% (data not shown). Calf thymus
histone H1 (more than 95% pure) was obtained from Nacalai Tesque,
Japan. Other methods and materials used in this study were
previously described (31, 36, 39).
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RESULTS |
Expression of Cdc7p/Dbf4p Protein Kinase Complex in Insect
Cells--
The goal of this study was to characterize the Cdc7p/Dbf4p
protein kinase and its role in chromosomal DNA replication initiation. Initially, attempts were made to purify the Cdc7p/Dbf4p complex from
S. cerevisiae cell extracts. However, because of its low abundance and the cell cycle-dependent expression of its
activity, it was not possible to purify the complex to
homogeneity.2 Thus, Cdc7p or
Dbf4p were expressed in insect cells using the baculovirus
system. Although insect cells expressed each protein, the majority of
the protein was insoluble (data not shown). Attempts to reconstitute a
soluble Cdc7/Dbf4 complex by mixing two cell extracts containing Cdc7p
and Dbf4p did not produce any significant amount of soluble, active
Cdc7p/Dbf4p complex. Therefore, Cdc7p and Dbf4p were co-expressed in
insect cells by mixed infection using the baculovirus system. When
soluble extracts were prepared from these cells and precipitated with
rabbit antiserum against Cdc7p, both Cdc7p and Dbf4p were precipitated
efficiently (see below). Furthermore, the immunoprecipitates
incorporated a significant amount of 32P into a GST-Mcm2p
substrate when co-incubated with the substrate and
[-32P]ATP.
Purification of Cdc7p/Dbf4p Protein Kinase Complex--
The
Cdc7p/Dbf4p protein kinase complex was purified to near homogeneity
from insect cells expressing both Cdc7p and Dbf4p as described under
"Materials and Methods." Fig. 1 shows
the elution profile of the partially purified protein complex when
applied to a Hi Trap Heparin column, eluted from the column with a
0.2-1 M NaCl linear gradient, and assayed for protein
kinase activity using GST-Mcm2p as a substrate. Two peaks of kinase
activity, which were not detected in uninfected insect cells (data not
shown), are observed; the first peak coincides with peaks of both Cdc7p and Dbf4p (as detected by immunoblotting), suggesting that this peak
represent the Cdc7p/Dbf4p protein kinase complex. On the other hand,
the second activity peak did not include a significant amount of Dbf4p
polypeptide, although it did include some Cdc7p polypeptide (Fig. 1).
One explanation for this result is that the second activity peak might
coincide with the elution position of an insect cell Dbf4 homologue.
Alternatively, Cdc7p might be activated by unknown factor(s).
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Fig. 1.
Purification of Cdc7p/Dbf4p protein kinase
complex. The S-Sepharose fractions of the lysate prepared from
cells expressing Cdc7p/Dbf4p were applied to a Hi-Trap Heparin column
(HR5/5) equilibrated with 0.2 M NaCl in buffer A, the
column was washed with the same buffer, and proteins were eluted with a
30-ml 0.2-1.0 M NaCl linear gradient in buffer A. A, column fractions were analyzed for total protein
(A280) and the concentration of NaCl.
Quantification of protein kinase activity (see B) for
GST-Mcm2p (closed circles) and Dbf4p autophosphorylation
(open circles) is also shown. B, protein kinase
activity in the column fractions was measured using GST-Mcm2p as a
substrate. C, immunoblotting with antisera to Cdc7p and
Dbf4p. In B and C, Ori is the pool of S-Sepharose
fractions that was applied to the Hi-Trap Heparin column.
Arrows indicate the positions of GST-Mcm2p, Cdc7p, or Dbf4p.
D, the pooled fractions from the Hi-Trap Heparin column were
further purified by MonoS and MonoQ chromatography, and the most
purified sample (about 0.5 µg) was subject to SDS-PAGE. The protein
was visualized by staining with Coomassie Brilliant Blue (D,
lane A) or transferred to an Immobilon filter and
immunoblotted with Cdc7p- (D, lane B) or
Dbf4p-antibody (D, lane C).
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For the purpose of this study, the fractions in the first peak of
kinase activity were pooled and further purified by chromatography using several columns (see "Materials and Methods"). The product of
the purification is nearly homogeneous and includes two major polypeptides (85 and 58 kDa, respectively) in nearly equimolar quantities. These polypeptides match the expected sizes of Dbf4p and
Cdc7p and react appropriately with Cdc7p and Dbf4p antibodies (Fig.
1D). Therefore, we concluded that these two polypeptides are
Dbf4p and Cdc7p, respectively. We designate here the Cdc7p/Dbf4p complex purified from insect cells as Cdc7p/Dbf4p* until it will be
found that the Cdc7p/Dbf4p protein kinase complex from insect cells is
the same as the complex from yeast cells.
Characterization of the Purified Cdc7p-Dbf4p* Protein
Kinase--
The untagged and soluble form of the Cdc7p/Dbf4p* protein
kinase (Fig. 1) was used for extensive biochemical characterization. Previous work (31) demonstrated that the best substrate for the
Cdc7p/Dbf4p* protein kinase is GST-Mcm2p purified from yeast cells, so
this protein was used as a substrate in many of the following
experiments. As shown in Fig. 2, the
Cdc7p/Dbf4p* protein kinase quickly phosphorylated GST-Mcm2p (Fig. 2,
closed circles) and autophosphorylated both Dbf4p (Fig. 2,
open squares) and Cdc7p (data not shown) in 10 min at
30 °C. These data were used to determine that the
Vmax of the kinase activity is 14.5 mol of
phosphate/mol of Cdc7/Dbf4p* complex/min at 30 °C. However, the
Km for the substrate could not be determined because
only 0.2 mol of phosphate was incorporated per mole of substrate, which
is most likely because the substrate is not 100% active.
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Fig. 2.
Kinetics of the protein kinase activity
associated with the Cdc7p/Dbf4p* complex. The standard protein
kinase reaction containing 2.5 µg (about 20 pmol) GST-Mcm2p and 10 ng
Cdc7p/Dbf4p* was incubated at 30 °C for the indicated time. An
aliquot was withdrawn and analyzed by SDS-PAGE. After staining with
Coomassie Brilliant Blue, the gel was dried, the bands corresponding to
GST-Mcm2p and Dbf4p were excised, and incorporated radioactivity was
measured in a scintillation spectrometer. Closed circles,
GST-Mcm2p; open square, Dbf4p.
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As found for most other protein kinases, the activity requires 5-10
mM Mg2+; 1-5 mM Zn2+
or Ca2+ partially substitutes for Mg2+ giving
40 and 55% maximal activity, respectively. As shown in Fig.
3A, an addition of 0.2 M NaCl severely inhibited phosphorylation of the GST-Mcm2p
substrate. In contrast, NaCl up to 0.8 M did not
inhibit the autophosphorylation of Dbf4p. Similar results were observed
with KCl (data not shown). Therefore, these data strongly suggest that
the Cdc7p/Dbf4p* kinase complex is very stable in vitro and
may not dissociate even at a high monovalent salt concentration.
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Fig. 3.
Biochemical properties of the Cdc7p/Dbf4p*
complex. A, salt sensitivity of the Cdc7p/Dbf4p*
complex. The purified Cdc7p/Dbf4p* complex was incubated in the
presence of [-32P]ATP, GST-Mcm2p, and various
concentrations of NaCl for 5 min at 30 °C. Kinase activity toward
each substrate was analyzed and quantified as described.
Autoradiography of the dried gel is shown in panel a, and
quantification of activity is shown in panel b.
B, autophosphorylation of Dbf4p in the Cdc7p/Dbf4p* complex.
The purified Cdc7p/Dbf4p* complex was preincubated at 30 °C with
unlabeled ATP in the absence of exogenously added substrate; 20-µl
aliquots were withdrawn at the indicated times and used for Western
blot of Dbf4 (panel a) or quantitative kinase assay for 5 min at 30 °C in the presence of Mcm2p and [-32P]ATP
(panel b). Solid bars, phosphorylation of
GST-Mcm2p; shaded bars, phosphorylation of Dbf4p.
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As described above and shown in Figs. 2 and 3A, the
Cdc7p/Dbf4p* protein kinase complex, especially Dbf4p, undergoes
autophosphorylation and phosphorylates exogenously added GST-Mcm2p.
When the complex was preincubated with unlabeled ATP, the mobility of
Dbf4p during SDS-PAGE decreased significantly as the reaction proceeded
(Fig. 3B). Similar results were observed with Cdc7p (data
not shown), although its autophosphorylation was to a lesser extent
than that of Dbf4p. Nevertheless, preincubation with unlabeled ATP did
not change the activity of the protein kinase toward the GST-Mcm2p substrate (Fig. 3B). This result suggests that
autophosphorylation of Dbf4p/Cdc7p* does not influence its kinase activity.
Many proteins are required for the initiation of DNA
replication in S. cerevisiae, and these proteins are
potential substrates for the Cdc7p/Dbf4p protein kinase complex.
Therefore, several replication proteins were tested for their ability
to act as a substrate for Cdc7p/Dbf4p* in vitro. The
proteins tested included origin recognition complex (six subunits)
(provided by John Diffley) (40), Cdc6p (provided by Ambrose Jong) (41),
Pol2* (four subunits, Pol2p, Dpb2, Dpb3, and Dpb4) (36), Pol3 complex
(three subunits, Cdc2p, Hys2p, and Pol32) (42),3
Pol32 (provided by Naomi Nakashima), and proliferating cell nuclear antigen (42). None of those proteins were efficiently
phosphorylated in vitro by the kinase (data not shown).
However, as shown in Fig. 4, members of
the Mcm protein family purified from yeast cells were phosphorylated
in vitro by the Cdc7p/Dbf4p* kinase complex. Mcm2p was the
best substrate, as reported previously (31). However, these data are
different from those obtained with yeast Mcm proteins expressed and
purified from insect cells (32). Although other proteins such as RFA
(RPA) complex, the 180-kDa catalytic subunit of DNA polymerase
-primase complex (36), and Mcm10 were also phosphorylated by the
kinase, they were phosphorylated less efficiently than GST-Mcm2p.
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Fig. 4.
Substrate specificity of Cdc7p/Dbf4p* protein
kinase complex. 1 µg of the indicated proteins were incubated
with the Cdc7p/Dbf4p* complex in the presence of
[-32P]ATP at 30 °C for 5 min, and the
phosphorylated proteins were analyzed by SDS-PAGE. After protein
staining with Coomassie Brilliant Blue, the radioactivity incorporated
into the protein of interest was quantified as described. Values were
normalized to the activity level with GST-Mcm2Nt (GST-fused to amino
acids 1-504 of Mcm2p), for which 100% activity represents
incorporation of 2.4 pmol of phosphate into 12 pmol of substrate.
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The amino acid residues of GST-Mcm2p phosphorylated by the Cdc7p/Dbf4p*
protein kinase complex were analyzed, demonstrating that more than 95%
of the incorporated 32P was in serine and less than 5% was
in threonine (data not shown). This confirms that Cdc7p/Dbf4p is a
serine/threonine kinase (17).
Prephosphorylated Mcm2p Is a Substrate for the Cdc7p/Dbf4p* Protein
Kinase Complex--
It was previously shown that Mcm2p is
phosphorylated by other protein kinases during the cell cycle in
addition to Cdc7p/Dbf4p (31). Because the GST-Mcm2p substrate was
prepared from unsynchronized yeast, it was likely that it was isolated
in a phosphorylated form. Therefore, Mcm2p was dephosphorylated using
E. coli alkaline phosphatase (BAP), repurified by MonoQ
column chromatography, and tested as a substrate for Cdc7p/Dbf4p*
protein kinase. As shown in Fig.
5A, BAP treatment increased
the mobility of GST-Mcm2p during SDS-PAGE, suggesting that GST-Mcm2p
purified from yeast cells is already phosphorylated. Unexpectedly, the
dephosphorylated GST-Mcm2p was a poor substrate for Cdc7p/Dbf4p*;
virtually no 32P was incorporated into the dephosphorylated
protein (Fig. 5, B and C). It is unlikely that
this is due to an inhibition of the kinase, because efficient
autophosphorylation of Cdc7p/Dbf4p* is observed in the presence of
dephosphorylated GST-Mcm2p. Furthermore, if dephosphorylated GST-Mcm2p
was mixed with native GST-Mcm2p, the same amount of 32P as
without dephosphorylated GST-Mcm2p was incorporated into GST-Mcm2p
(Fig. 5B, lanes m-p). Thus, GST-Mcm2p
phosphorylated in vivo is a substrate for Cdc7p/Dbf4p*
protein kinase complex in vitro, but dephosphorylated
GST-Mcm2p is not. These results are consistent with earlier results
indicating that Mcm2p is sequentially phosphorylated during the cell
cycle and that the last phosphorylation of Mcm2p is blocked at the
restrictive temperature in cdc7 mutant cells (31).
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Fig. 5.
Effect of dephosphorylation on GST-Mcm2p as a
substrate for Cdc7p/Dbf4p* protein kinase. GST-Mcm2p was treated
with E. coli BAP, repurified by MonoQ column chromatography
and used as a substrate for Cdc7p/Dbf4p* protein kinase complex.
A, Coomassie Brilliant Blue-stained gel after SDS-PAGE of
GST-Mcm2p with and without BAP treatment. Lanes a,
c, e, and f were GST-Mcm2p that was
purified from wild type, bob1-CDC7,
bob1-CDC7, and wild type yeast cells,
respectively. Lanes b and d were the same as
lanes a and c, except for BAP treatment.
B, Cdc7p/Dbf4p* protein kinase assay was carried out as
described above in the presence of various amounts (0-2.4 µg
indicated in C) of GST-Mcm2p for 5 min at 30 °C.
Autoradiograph of the dried gel is shown. Lane a, without
GST-Mcm2p; lanes b-g, 0.1, 0.2, 0.4, 0.8, 1.6, and 2.4 µg
of GST-Mcm2p without BAP treatment, respectively; lanes
h-l, 0.1, 0.2, 0.4, 0.8, and 1.6 µg of GST-Mcm2p with BAP
treatment, respectively; lane m, without GST-Mcm2p
pretreated with BAP; lane o, 0.4 µg of GST-Mcm2p
pretreated with BAP; lane p, 0.4 µg of GST-Mcm2p without
BAP treatment; lane q, 0.4 µg of GST-Mcm2p without BAP
treatment and 0.4 µg of GST-Mcm2p pretreated with BAP. C,
protein kinase activity of lanes a-l in B was
quantified as described.
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Previous studies indicate that bob1 mutants bypass the
requirement for CDC7/DBF4 in the yeast cell cycle (29). This
suggests that GST-Mcm2p purified from
bob1-CDC7 yeast cells should not be
phosphorylated at the Cdc7p/Dbf4p sites and could therefore be a better
substrate for the Cdc7p/Dbf4p* protein kinase in vitro. This
prediction was tested, and the results are shown in Fig. 6. Contrary to our prediction, GST-Mcm2p
purified from bob1-CDC7 cells was two to three
times less active as a protein kinase substrate than GST-Mcm2p from
wild type cells (Fig. 6). There are several possible explanations for
this result. For example, there may be another protein kinase in
bob1-CDC7 yeast that partially phosphorylates GST-Mcm2p at the same sites phosphorylated by Cdc7p/Dbf4p.
Alternatively, in bob1-CDC7 cells,
GST-Mcm2p may undergo a post-translational modification that causes a
conformational change inhibiting its subsequent phosphorylation by the
Cdc7p/Dbf4p* protein kinase complex. Another possibility is that the
bob1 mutation blocks an upstream modification of GST-Mcm2p
that is required prior to its phosphorylation by Cdc7p/Dbf4p*.
Nonetheless, GST-Mcm2p from bob1-CDC7 cells
could be phosphorylated by Cdc7p/Dbf4p* (Fig. 6B) to a
greater extent than dephosphorylated GST-Mcm2p (Fig. 5C).
S. cerevisiae Mcm2p was also expressed in and purified from insect cells (provided by Y. Ishimi) and was tested as a substrate for
the Cdc7p/Dbf4p* protein kinase; the activity was two to three times
less than that of GST-Mcm2p from yeast cells (data not shown). These
results are also consistent with the conclusion that an upstream
phosphorylation of Mcm2p is required prior to its phosphorylation by
the Cdc7p/Dbf4p* protein kinase.
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Fig. 6.
GST-Mcm2p from
bob1-CDC7 strain as a substrate for
Cdc7p/Dbf4p* protein kinase. Various amounts (0-10 µg) of
GST-Mcm2p from either wild type or bob1-CDC7
cells were added into the kinase assay reactions as described.
A, autoradiograph of a dried gel is shown. Lanes
1 and 8, without GST-Mcm2p. Lanes 2-7, with
0.01, 0.1, 0.2, 0.3, 0.5 and 1.0 µg of GST-Mcm2p from wild type
yeast, respectively. Lanes 9-12, with 0.1, 0.1, 0.3, 0.5, and 1.0 µg of GST-Mcm2p from bob1-CDC7
yeast, respectively. B, protein kinase activity was
quantified as described above.
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Molecular Characterization of Dbf4 Mutants--
Several
temperature-sensitive dbf4 mutants were previously isolated
and characterized (16, 43, 44). This work extends those studies and
determines the nucleotide sequence changes in several mutant alleles of
the DBF4 gene. In dbf4-1 and dbf4-2, there is a single-base pair change in the coding region of
DBF4 resulting in a single amino acid change from
Pro277 to Leu and from Pro308 to Leu,
respectively (Fig. 7A). In
dbf4-3, there are two base changes resulting in a single
amino acid change from Pro308 to Ser in the coding region.
On the other hand, in dbf4-4, there is a single-base change
in the coding region that creates a stop codon at amino acid 596 (from
Gln596 to term (TAG)). Therefore, dbf4-4 mutant
may produce a truncated Dbf4p. Finally, there are three base pair
changes in dbf4-5. Two of those base pair changes result in
a stop codon at amino acid 112 (from TGG (Trp112) to TAA
(Term)) and the remaining base pair change creates a GAA
(Glu414) to AAA (Lys) substitution. Thus, the
dbf4-5 mutant may generate two polypeptides, one with 111 amino acid residues and another with 584 amino acids (assuming that
Met120 is used as an initiation codon). This expectation
was confirmed by the experiments described below (Fig. 7C).
Interestingly, all mutations reside in the regions previously
identified as the Cdc7-binding region (amino acids 241-416 and
573-695) and ARS-binding region (amino acids 81-278) by two-hybrid
and one-hybrid assays (26, 45), respectively.
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Fig. 7.
Expression of mutant Cdc7p/Dbf4p
complexes. A, the schematic diagram indicates the
identity and location of the amino acid changes or deletions in
dbf4-1-dbf4-5 (16, 43, 44). The Cdc7p- and ARS-binding
regions of Dbf4p (26, 45) are indicated, and the ability of each mutant
protein to complement dbf4 yeast when
expressed from a single-copy plasmid is indicated (+ if able to
complement; if unable to complement). B, one-hybrid assay
using mutants dbf4-1, dbf4-3, dbf4-4,
dbf4-5, and dbf4-5 derivatives and
ARS1 was performed at three different temperatures (25, 30, and 37 °C) as described (26). C, Sf9 cells
co-infected with baculovirus containing CDC7or
cdc7-1 and either dbf4-1, dbf4-3,
dbf4-4, dbf4-5 or its derivatives were grown at
28 °C and cell lysates were prepared as described under "Materials
and Methods." Cdc7p/Dbf4p complexes were immunoprecipitated with
rabbit antiserum to Cdc7p, and the precipitates were analyzed by
Western blot with rabbit antibody to Cdc7p or Dbf4p. Lysates were from
cells expressing proteins as follows. Lane a, baculovirus
vector only; lane b, Cdc7-1p; lane c, Cdc7p;
lane d, Dbf4p; lane e, Cdc7p/Dbf4p; lanes
f, Cdc7-1p/Dbf4p; lane g, Cdc7p/Dbf4-1p; lane
h, Cdc7p/Dbf4-3p; lane i, Cdc7p/Dbf4-4p; lane
j, Cdc7p/Dbf4-5'p; lane k, Cdc7p/Dbf4-5"p; lane
l, the purified Cdc7p/Dbf4p*. AbCdc7 and abDbf4 show rabbit
antiserum against Cdc7p and Dbf4p, respectively. Immunoprecipitation
was carried out with rabbit antiserum and rabbit IgG is also seen in
the figure (IgG indicated with arrow). Although the results
for Cdc7p/Dbf4-5p and Cdc7p/Dbf4-5p''' were not shown in this figure,
the results were the same as that of Cdc7p/Dbf4-5'p and of Cdc7p/Dbf4p,
respectively.
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To find which mutation site(s) in dbf4-5 is responsible for
temperature-sensitive cell growth phenotype, we generated three additional dbf4-5-derivative mutant genes. These were
dbf4-5' that contains the dbf4-5 gene, whose
first 112 amino acid residues were deleted, dbf4-5", which
is the same as dbf4-5', but the second mutation site (AAA
(Lys414)) in dbf4-5 was reversed to a wild type
sequence (GAA(Glu414)), and dbf4-5''', which
contains only the second mutation (AAA(Lys414) of
dbf4-5, respectively (Fig. 7A). Those genes were
tested to be able to complement lethality of
dbf4 at 25 and 37 °C. The results showed
that the dbf4-5 and dbf4-5' mutant genes
complemented lethality of dbf4 at 25 °C,
but not at 37 °C, while the dbf4-5''' gene complemented
lethality of dbf4 at 25 °C and 37 °C.
Surprisingly, the dbf4-5" gene did not complement lethality
of dbf4 mutation at 25 °C. These results
indicate that either the first mutation site (TGG (Trp112)
to TAA (Term)) or the second mutation site (GAA (Glu414) to
AAA (Lys) substitution) is not sufficient to reconstitute temperature
sensitivity in the dbf4-5 mutant. Therefore, it was concluded that the two mutation sites are required for making the
dbf4-5 mutant temperature-sensitive.
A yeast strain containing an ARS1-lacZ reporter
construct was tested for expressing 
-galactosidase after
transformation with Dbf4 fused to GAD (26). As shown in Fig.
7B, all mutant genes, except for dbf4-5 and its
derivatives, showed a more or less wild type level of ARS1
binding activity at all temperatures tested. Interestingly, the
dbf4" mutant had neither complementation activity of
dbf4 lethality at 25 °C (Fig.
7A) nor ARS1 binding activity at all
temperatures, whereas the dbf4-5 and other dbf4-5
derivatives, which complemented dbf4 lethality
at 25 °C, did not exhibit ARS1 binding activity (Fig.
7B). Therefore, if the ARS binding activity of Dbf4p is
essential for yeast cell growth as shown previously (26), Dbf4-5p and
Dbf4-5'p should retain the activity, which could not be detected under
our assay conditions. Alternatively, the ARS binding activity missing
in Dbf4-5p or Dbf4-5'p may be substituted with the conformational
change of the protein by the second mutation (GAA (Glu412)
to AAA (Lys412) change) in dbf4-5. From the
results with dbf4-5", the latter is more likely possibility.
The dbf4-1-dbf4-5 mutants described above are reportedly to
be temperature-sensitive in growth in vivo (16, 43, 44). Thus, temperature sensitivity of the kinase activity was tested by
co-expression of the mutant Dbf4p with wild type Cdc7p in insect cells
followed by partial purification and kinase assay. As shown in Fig.
7C, this analysis was carried out for six mutant Dbf4p complexes (Cdc7p/Dbf4-1p, Cdc7p/Dbf4-3p, Cdc7p/Dbf4-4p, Cdc7p/Dbf4-5p, Cdc7p/Dbf4-5'p, and Cdc7p/Dbf4-5" (the dbf4-5" mutant gene
did not complement dbf4 lethality)) as well as
for the mutant Cdc7-1p/Dbf4p complex. In all cases, the Cdc7p antiserum
precipitated both kinase subunits Cdc7p and Dbf4p (Fig. 7C
and data not shown for Cdc7p/Dbf4-2p and Cdc7p/Dbf4-5'p), indicating
that the mutant protein complexes were stably formed in insect cells
in vivo. Interestingly, the bands corresponding to Dbf4p,
Dbf4-4p, Dbf4-5, Dbf4-5', and Dbf4-5"p were diffuse in character when
detected on a Western blot. These diffuse bands were not observed when
cell extracts were pretreated with BAP (data not shown), suggesting
that the diffuse quality of these bands results from protein
phosphorylation. This phosphorylation could be due to
autophosphorylation of the complex, because in the mutant complexes
(Cdc7-1p/Dbf4p (Fig. 7C, lane f), Cdc7p/Dbf4-1p (Fig. 7C, lane g), and Cdc7p/Dbf4-3p (Fig.
7C, lane h)), Dbf4p migrated as a single band in
SDS-PAGE and the immunoprecipitates with Cdc7p antibodies did not have
any significant protein kinase (data not shown). These data suggest
that Cdc7p/Dbf4-4p, Cdc7p/Dbf4-5p, Cdc7p/Dbf4-5'p, and Cdc7p/Dbf4-5"p
complexes retain a significant amount of the protein kinase activity at
permissive temperatures, whereas other mutant complexes do not.
The temperature sensitivity of the protein kinase activity of the
Cdc7p/Dbf4p mutants was tested using partially purified protein
complexes. As shown in Fig.
8A, although the mutant
Cdc7-1p/Dbf4p protein complex could be partially purified, only a small
amount of protein kinase activity was detected at 25 °C. Therefore,
we could not convincingly demonstrate that this activity is
temperature-sensitive. Similar results were also obtained for
Cdc7p/Dbf4-1p and Cdc7p/Dbf4-3p (the kinase activity of Cdc7p/Dbf4-2p
was not tested but is expected to be the same as Cdc7p/Dbf4-3p). In the
cases of Cdc7p/Dbf4-4p and Cdc7p/Dbf4-5p, a significant amount of
protein kinase activity co-eluted with the mutant protein complex on Hi
Trap Heparin column chromatography (data not shown), thus letting us to
measure their temperature sensitivities. Those kinase activities were
more quickly inactivated at 35 °C than the wild type protein kinase
(Fig. 8, B and C, and data not shown), indicating
that those mutations confer temperature sensitivity on the protein
kinase of Cdc7p/Dbf4p complex. The dbf4-4 mutation generates
a termination in the second Cdc7p-binding domain (Fig. 7A).
This domain may not be essential for Cdc7 activation at 25 °C (26).
However, the cell growth of the mutant and the Cdc7p/Dbf4p kinase are
both temperature-sensitive, thus the domain also plays an important
role for the kinase activity.
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Fig. 8.
Partial purification and heat inactivation of
mutant Cdc7p/Dbf4p complexes. A, Sf9 cells
co-infected with baculovirus containing cdc7-1and
DBF4 or with baculovirus containing CDC7 and
DBF4 were lysed and applied to an S-Sepharose column. The
Cdc7-1p/Dbf4p or Cdc7p/Dbf4p complex was eluted from the column with
0.5 M NaCl in buffer A. The eluted fractions were applied
to a Hi-Trap Heparin-Sepharose column, eluted from the column by
0.2-1.0 M NaCl linear gradient in buffer A as Fig. 1, and
analyzed by SDS-PAGE followed by Western blotting with anti-Cdc7p or
Dbf4p serum. Protein kinase activity was assayed using GST-Mcm2p as a
substrate as described. B, partially purified wild type
Cdc7p/Dbf4p and Cdc7p/Dbf4-5p (MonoS fractions) were preincubated at
35 °C for the indicated times; aliquots were removed, and the
protein kinase activity was measured at 25 °C as described. The
figure shows activity remaining for the Cdc7p/Dbf4p and Cdc7p/Dbf4-5p
complexes; the mutant protein was more rapidly inactivated at 35 °C
than the wild type Cdc7p/Dbf4p complex. C, The remaining
GST-Mcm2 kinase activity was measured as for Fig. 2.
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Genetic Interaction between DBF4 and RAD53--
To understand
better the function of Dbf4p, mutations were identified that suppress
the temperature-sensitive growth phenotype of dbf4-1 after
treating dbf4-1 mutant cells with ethyl methane sulfonate as
published (16). More than 50 mutants were obtained that separated into
two complementation groups. One of those complementation groups,
dbf4-1 sup1, was further characterized in this study. Genetic mapping experiments indicated that dbf4-1 sup1
mutation resides at 17.0 cM from the CDC60 locus on
chromosome XVI, where RAD53 and PEP4 are closely
located (data not shown). Thus, a single copy plasmid of either
RAD53 or PEP4 was introduced into the
dbf4-1 sup1 mutant. Only the mutant cell harboring the
RAD53 gene on a single-copy plasmid became
temperature-sensitive. Thus, we concluded that dbf4-1 sup1
is an allele of rad53 mutations. This result suggests that
RAD53, which encodes a serine/threonine protein kinase and
plays a role in both S phase checkpoint and DNA damage checkpoint in
yeast (34), interacts with Dbf4p and regulates the function of Dbf4p
during S phase.
Rad53 Phosphorylates Cdc7p/Dbf4p and Inhibits the Mcm2p Protein
Kinase Activity--
Previous studies also indicate that
RAD53 interacts with DBF4 genetically and
physically (32, 46). These prompted us to test in vitro the
ability of Rad53p to directly phosphorylate the Cdc7p/Dbf4p* protein
kinase complex. Rad53p was purified to near homogeneity from S. cerevisiae cells expressing 2HA-tagged Rad53p (Fig.
9A, panel a).
Although cells were not treated with either HU or methyl methane
sulfonate to activate Rad53p, Rad53p had a significant
autophosphorylation activity as well as histone H1 kinase activity,
indicating that it was an active protein kinase (Fig. 9A,
panel c). It was noted that autophosphorylation of Rad53p is
stimulated in the presence of a substrate histone H1. Because the
Cdc7p/Dbf4p* complex also exhibits autophosphorylation (Figs. 2 and 3),
the Cdc7p/Dbf4p* complex was preincubated with a cold ATP to mask its
autophosphorylation. Then the Cdc7p/Dbf4p* complex was incubated with
the purified 2HA-Rad53p. As shown in Fig.
10A, 32P was
incorporated into Dbf4p, and the band of the protein were shifted
upward in SDS-PAGE in Rad53p-dependent manner. Similarly, 32P were incorporated into Cdc7p, but to a considerably
lesser extent (Fig. 10A). If lane 2 in Fig.
9B (1.2 pmol of 32P was incorporated into 60 pmol of histone H1) is compared with lane 6 in Fig.
10A (1.0 pmol of 32P was incorporated into 0.1 pmol of Cdc7p/Dbf4p*), the Cdc7p/Dbf4p* purified from insect cells was
at least 100 times more active as substrate for Rad53p protein kinase
than calf thymus histone H1. Thus, this result strongly suggests that
the Cdc7p/Dbf4p complex, particularly Dbf4p, may be one of in
vivo targets of the Rad53p protein kinase. In addition,
phosphorylation of the Cdc7p/Dbf4p* complex by Rad53p altered the
protein kinase activity of the complex; when 2HA-Rad53p was
co-incubated with Cdc7p/Dbf4p* and GST-Mcm2p, the phosphorylation of
GST-Mcm2p by Cdc7p/Dbf4p* was strongly inhibited (Fig. 10, A
and B). Furthermore, 2HA-Rad53p purified from yeast cells
treated with 0.2 M HU was more efficient in inhibiting the
Cdc7p/Dbf4p* complex than nonactivated 2HA-Rad53p; 10-fold less amount
of 2HA-Rad53p purified from HU-treated yeast cells inhibited the
Cdc7p/Dbf4p* kinase activity as much or more than 2HA-Rad53p from yeast
cells (Fig. 10). As shown in Fig. 10C, Cdc7p/Dbf4p* was
preincubated with either 2HA-Rad53p or 2HA-Rad53p from HU-treated yeast
cells, and the phosphorylation of GST-Mcm2p by Cdc7p/Dbf4p* was
inhibited by time-dependent manner. Taken together, these results and those of Dohrmann et al. (46) suggest that
Rad53p directly interacts with and regulates the Cdc7p/Dbf4p protein kinase activity in vivo during S phase.
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Fig. 9.
Purification of 2HA-tagged Rad53p. A. 2HA-tagged Rad53 protein was purified from S. cerevisiae
CB001 cells as described under "Materials and Methods." The
fractions from the hydroxylapatite column (the last step of the
purification) were analyzed by SDS-PAGE followed by staining with
Coomassie Brilliant Blue (panel a) or analyzed by Western
blot with rabbit polyclonal antibody against HA (panel b).
Fractions were assayed for protein kinase activity using calf thymus
histone H1 as a substrate (panel c). Left of the
gel indicates the molecular masses of marker proteins in kDa.
B, the purified 2HA-tagged Rad53p underwent
autophosphorylation as well as phosphorylating histone H1. Protein
kinase assay was carried out with 2HA-Rad53p in the absence or presence
of histone H1 or GST-Mcm2p. Lane 1, no Rad53p; lane
2, 30 ng of 2HA-Rad53p; lane 3, 10 ng 2HA-Rad53p and 2 µg of histone H1; lane 4, 20 ng of 2HA-Rad53p and 2 µg
of histone H1; lane 5, 30 ng of 2HA-Rad53p and 2 µg of
histone H1; lane 6, 10 ng of 2HA-Rad53p and 2.4 µg of
GST-Mcm2p; lane 7, 20 ng of 2HA-Rad53p and 2.4 µg of
GST-Mcm2p; lane 8, 30 ng of 2HA-Rad53p and 2.4 µg of
GST-Mcm2p; lane 9, 50 ng of 2HA-Rad53p and 2.4 µg of
Rad53p. The middle band located between 2HA-Rad53 and
histone H1 in A (panel c) and B was
phosphorylated by 2HA-Rad53p was a contaminant of calf thymus histone
H1. In the presence of histone H1, but not GST-Mcm2p, 2HA-Rad53 was
further autophosphorylated. This may be autoactivation of Rad53p.
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Fig. 10.
Rad53p inhibits GST-Mcm2p phosphorylation by
Cdc7p/Dbf4p* protein kinase. A kinase reaction containing
Cdc7p/Dbf4p* and unlabeled ATP was preincubated at 30 °C for 15 min
to mask the sites which are autophosphorylated. 2HA-tagged Rad53p
purified from either HU-treated (lanes 11-14 and
16-18) or untreated (lanes 4-9) S. cerevisiae cells and [-32P]ATP were added, and
the incubation was continued for 5 min with and without GST-Mcm2p.
After incubation, the phosphorylated proteins were analyzed as
described. A, autoradiography of the dried gel after
SDS-PAGE. Lane 1, 5 ng of Cdc7p/Dbf4*; lane 2, 5 ng of Cdc7p/Dbf4p* plus GST-Mcm2p; lane 3, 5 ng of
Cdc7p/Dbf4p* plus 2 µg of histone H1; lanes 4-6, the same
as lane 1 plus 10, 30, and 50 ng of 2HA-tagged Rad53p
purified from yeast cells, respectively; lanes 7-9, the
same as lanes 4-6, except that 0.2 µg of GST-Mcm2p was
added; lane 10, 5 ng of Cdc7p/Dbf4p* plus 0.2 µg of
GST-Mcm2p; lanes 11-14, the same as lane 10 plus
1, 3, 5, and 10 ng of HU-activated 2HA-Rad53p, respectively; lane
15, 5 ng of Cdc7p/Dbf4p*; lanes 16-18, the same as
lane 10, except for 1, 5, and 10 ng of HU-activated
2HA-Rad53p, respectively. B, kinase activity on GST-Mcm2p
shown in A was quantified as described above. C,
effect of preincubation with 2HA-Rad53p on the GST-Mcm2p
phosphorylation activity associated with Cdc7p/Dbf4p*. The purified
Cdc7p/Dbf4p* kinase complex (0.5 µg/ml) was preincubated with
2HA-Rad53p (0.5 µg/ml) or 2HA-Rad53p from HU-treated yeast cells (50 ng/ml) in the presence of cold ATP. At the indicated times, an aliquot
was withdrawn, and the remaining protein kinase activity was measured
after addition of GST-Mcm2p and [-32P]ATP as Fig. 8.
Because 2HA-Rad53p does not phosphorylate GST-Mcm2p, the
phosphorylation activity seen in this experiment represents the
phosphorylation catalyzed by Cdc7p/Dbf4p*.
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DISCUSSION |
This study reports the biochemical characterization of Cdc7p/Dbf4p
protein kinase that was purified to near homogeneity from insect cells
co-expressing Cdc7p and Dbf4p. Because an active, untagged, soluble and
pure Cdc7p/Dbf4p protein kinase from yeast cells has not been
characterized previously, this report becomes the first paper to
describe a thorough biochemical characterization of Cdc7p/Dbf4p
complex, which plays an important function during the initiation of
chromosomal DNA replication in yeast. To distinguish between
Cdc7p/Dbf4p complex from yeast cells and Cdc7p/Dbf4p complex purified
from insect cells, we designate the Cdc7p/Dbf4p complex purified from
insect cells as Cdc7p/Dbf4p* until it can be confirmed that the
Cdc7p/Dbf4p complex purified from insect cells is identical to that
from yeast cells. However, the results reported here are in general
agreement with previous genetic and biochemical studies of this enzyme,
except for temperature sensitivity of the Cdc7-1p/Dbf4p and
Cdc7p/Dbf4-1-Cdc7p/Dbf4-3p mutant complexes.
The substrate specificity of Cdc7p/Dbf4p* observed here is fairly
narrow; in the Mcm protein family, Mcm2p is the preferred phosphorylation target, with less efficient use of Mcm3p as a substrate
(Fig. 4). This result confirms previous reports that Mcm2p is a
substrate for Cdc7p/Dbf4p* (31) but differs in that Mcm6p was
previously reported as a substrate for Cdc7p/Dbf4p*. The reason for
this discrepancy is not clear at present, but it may reflect the higher
purity of the protein kinase complex used in this study.
Although GST-Mcm2p is a good in vitro substrate for
Cdc7p/Dbf4p*, less than 0.2 mol of phosphate/mol of GST-Mcm2p was
incorporated by the protein kinase (Fig. 2). When the GST-Mcm2p that
was phosphorylated with Cdc7p/Dbf4p* was completely digested with the
Lys-C endopeptidase, we could detected at least five different
32P-labeled
oligopeptides.4 Thus, it is
estimated that as few as 4% of the GST-Mcm2p molecules may be
phosphorylated at specific sites by Cdc7p/Dbf4p*. There are several
factors that may contribute to this result. First, data presented here
(Figs. 5 and 6) and elsewhere (31) indicate that sequential
phosphorylation events target Mcm2p during the cell cycle in S. cerevisiae, and the ability of Cdc7p/Dbf4p to utilize Mcm2p as a
substrate is dependent on proper prephosphorylation at specific protein
sites of Mcm2p. For example, BAP treatment of Mcm2p after purification
from yeast cells completely blocked its phosphorylation in
vitro by Cdc7p/Dbf4p* (Fig. 5), and Mcm2p from
bob1/CDC7 cells (Fig. 6), insect cells, or
E. coli (data not shown) were much poorer substrates for the
Cdc7p/Dbf4p* protein kinase than Mcm2p from yeast cells. Furthermore,
it was previously shown that only a portion of the phosphorylated Mcm2p
is further phosphorylated during S phase in vivo and that
various mcm2 mutations affect the Cdc7p/Dbf4p
phosphorylation activity (31). These results suggest that protein
conformation or post-translational modification plays a role in the
recognition of a substrate by Cdc7p/Dbf4p protein kinase.
Alternatively, it is still possible that Cdc7p/Dbf4p* purified from
insect cells may not be properly activated and is not active as
Cdc7p/Dbf4p from yeast cells.
It has been shown that the nuclear and subnuclear localization of the
Mcm2p and Mcm3p are temporally regulated with respect to the cell
cycle. The nuclear concentration of these proteins increases at the end
of mitosis, remains elevated throughout G1 phase, and
decreases at the beginning of S phase. Once inside the nucleus, a
fraction of the total Mcm2p and Mcm3p becomes tightly associated with
DNA (32, 47). The association of these proteins with chromatin
presumably leads to the initiation of DNA synthesis, and their
subsequent disappearance from the nucleus presumably prevents
reinitiation of DNA synthesis at replication origins. This temporally
and spatially restricted localization of Mcm2p and Mcm3p in the nucleus
may serve to ensure that DNA replication occurs once and only once per
cell. However, Mcm2p and Mcm3p are abundant in the cell (4 × 104/cell and 2 × 105/cell, respectively),
yet Mcm2 is limiting for DNA replication (48). If we assume that the
binding of Mcm2p at replication origins plays an important in
vivo function, and subsequently Cdc7p/Dbf4p protein kinase
specifically phosphorylates the origin-bound Mcm2p through an ARS
binding activity of Dbf4p, it can be argued that less than 1,000 molecules of Mcm2p (about 2-3% of the total pool of Mcm2p) might be a
substrate for the kinase (there are less than 1,000 replication origins
in yeast). Interestingly, this calculation roughly matches with the
calculation of the fraction of GST-Mcm2p phosphorylated by Cdc7p/Dbf4p*
in vitro (4%). Therefore, the difference between
chromatin-bound and unbound Mcm2p could be the phosphorylated state of
the protein, because the BAP-pretreated GST-Mcm2p was inert as a
substrate for Cdc7p/Dbf4p* protein kinase complex. Thus, we further
speculate that if chromatin-bound Mcm2p could be specifically purified
from the cell, it might be a better substrate for Cdc7p/Dbf4p* protein
kinase in vitro than the pool of total nuclear Mcm2p.
It has been recognized that Mcm2-7 family proteins form a complex and
play an essential role in the initiation of chromosomal DNA replication
in several eukaryotes (see Ref. 30 for review). Thus, it is reasonable
to suggest that a Mcm2-7 family protein complex might be the best
substrate for the Cdc7p/Dbf4p protein kinase. In S. cerevisiae, Mcm2-7 family proteins form a complex in
vivo and bind to chromatin during the cell cycle, as in other eukaryotes. However, unlike for other eukaryotes (49-51), a reasonable quantity of the Mcm2-7p complex from yeast has not yet been
successfully isolated; thus it has not been possible to directly test
its potential as a substrate for Cdc7p/Dbf4p* protein kinase.
Nonetheless, the substrate specificity that we observe in this study is
very similar to that of S. pombe Hsk1p-Dfb1p using the
Mcm2-7 complex as a substrate (23).
Mutant protein kinase complexes including Dbf4-1p,
Dbf4-2p, Dbf4-3p, Dbf4-4p, Dbf4-5p, and Dbf4-5p derivatives were also
characterized in this study. Complexes that included Cdc7-1p/Dbf4p,
Cdc7p/Dbf4-1p, and Cdc7p/Dbf4-3p had no significant protein kinase
activity in vitro, although these complexes could be
isolated and partially purified (Fig.
8A).2 Therefore, it is possible that
these mutant complexes might be extremely heat labile and readily
inactivated in insect cells during or prior to purification.
Alternatively, those mutant complexes were not properly formed in
insect cells; thus those might be artifacts of the protein expression
in insect cells. However, Cdc7p/Dbf4-4p and Cdc7p/Dbf4-5p complexes
expressed in insect cells exhibited a temperature-sensitive protein
kinase activity. Thus, we rather consider another possibility that a
small amount of the protein kinase activity detected in the mutant
Cdc7p/Dbf4p complexes may be directly related to the nature of their
mutations rather than protein expression artifact. This may explain why a multicopy plasmid harboring GLC8, which encodes the
regulatory subunit of Glc7 protein phosphatase, specifically suppresses
temperature sensitivity of mutants cdc7-1,
dbf4-1, dbf4-2, and dbf4-3 but not
dbf4-4 and dbf4-5.5
The characterization of complexes containing Dbf4-5p, Dbf4-5'p,
Dbf4-5", or Dbf4-5'''p was particularly informative and interesting. Previous studies showed that Dbf4p has separate ARS-binding and Cdc7p-binding domains (26, 46). Its interaction with ARSs was found to
be independent of its ability to bind to Cdc7p (26). The
dbf4-5 allele has sequence changes at two sites; one change is near or in the ARS-binding domain and the other change is in the
Cdc7p-binding region (Fig. 7A), and two distinct
polypeptides are produced from this dbf4 allele. As shown in
Fig. 7, Dbf4-5'p, the larger polypeptide produced by dbf4-5,
had no ARS binding activity, although it has activation of Cdc7p
protein kinase activity. More importantly, the Cdc7p/Dbf4-5p and
Cdc7p/Dbf4-5'p complexes exhibited a temperature-sensitive protein
kinase activity (Fig. 8C and data not shown), and the
dbf4-5 and dbf4-5' genes complement the lethality
of the dbf4 mutation and support
temperature-sensitive cell growth of dbf4
(Fig. 7A). These data clearly demonstrate that each single
mutation site of dbf4-5 (either TGG (Trp112) to
TAA (Term) or GAA (Glu412) to AAA (Lys412)
change) is not sufficient to reconstitute temperature sensitivity of
the dbf4-5 mutation. As published previously (26), deletion of a portion of the ARS-binding domain of DBF4
(i.e. the dbf4-5"allele) abrogated the ability of
the gene to complement dbf4 (Fig.
7A). However, the truncated gene product formed a complex
with Cdc7p and exhibited a protein kinase activity on GST-Mcm2p (data
not shown). Thus, these results suggest that a defect of the first mutation site (deletion of a portion of the ARS-binding domain) is
suppressed by the second mutation of dbf4-5 and therefore
suggest that both the ARS-binding domain and Cdc7p protein kinase
activation domains collaborate to form an active Cdc7p/Dbf4p complex
that functions in the mutant cells. Nevertheless, it should be
emphasized that these results are the first demonstration that the Mcm2
phosphorylation activity of the mutant Cdc7p/Dbf4p complex is
temperature-sensitive in vitro, because the previous studies
always used immunoprecipitates from cell extracts and histone H1 as a
substrate to assay the kinase activity (17).
In previous studies, Jackson et al. (17) showed that mutant
extracts from cdc7-1 and dbf4-1 demonstrate
temperature-sensitive Cdc7p/Dbf4p kinase activity but that
temperature-resistant kinase activity is restored when the two extracts
are mixed. In contrast, mixed extracts from insect cells expressing
Cdc7-1p and Dbf4p or Cdc7p and Dbf4-1-Dbf4-3p were inactive as protein
kinases, regardless of the temperature, when we performed a similar
experiment. This might be due to tight association of the mutant
complex in our samples. Furthermore, Jackson et al. (17)
detected a temperature-resistant protein kinase activity in the
immunoprecipitates from the mixture of dbf4-1 and
cdc7-1 mutant cell extracts; therefore, it is possible that
exchange of the polypeptides in their experiments is achieved in
immunoprecipitates. However, this possibility is considered less
likely, because temperature-resistant protein kinase activity was not
detected in our hands in similarly prepared immunoprecipitates from
yeast cells as well as insect cells expressing the mutant Cdc7p/Dbf4p
(data not shown). Therefore, the difference in these results may derive
from the fact that Jackson et al. (17) measured histone H1
kinase activity and our experiments measured Mcm2p kinase activity.
Nevertheless, it is equally possible that the mutant Cdc7p/Dbf4p is not
properly formed and/or phosphorylated in insect cells. Therefore,
further characterization of the wild type and mutant Cdc7p/Dbf4p
complexes expressed and purified from both yeast cells and other
eukaryotic cells is needed.
The purified Cdc7p/Dbf4p* complex showed an autophosphorylation of both
Cdc7p and Dbf4p. Especially, Dbf4p was highly phosphorylated. Apparently, this autophosphorylation did not alter its protein kinase
activity (Fig. 3B). Interestingly, Dbf4p is
hyperphosphorylated, presumably by Rad53p, upon treatment of cells with
HU or upon inactivation of DNA replication enzymes, and this
hyperphosphorylated kinase is less active than unmodified Cdc7p/Dbf4p
kinase (32). Because the checkpoint regulator Rad53p is required for
modification of Dbf4p in response to HU, Cdc7p/Dbf4p kinase may play a
role in the response of yeast cell to DNA replication inhibitors (32). Recently, Dohrmann et al. (46) showed that dbf4-1
is a synthetic lethal with rad53-31 and Dbf4p physically
interacts with Rad53p by a two-hybrid assay. Furthermore, we show here
that an extragenic suppressor screen of dbf4-1 yielded a new
temperature-sensitive allele of rad53. All these results
strongly suggest that Rad53p directly interacts with and regulates
Cdc7p/Dbf4p protein kinase activity in vivo. Thus, this
possibility was directly tested in vitro in this study using
the highly purified Cdc7p/Dbf4p* complex and 2HA-tagged Rad53p. As
shown in Fig. 10, a small amount of 2HA-tagged Rad53p significantly
phosphorylated both Dbf4p and Cdc7p. But they were not equally
phosphorylated. Although we do not know at the present time which
protein phosphorylation has an effect, these phosphorylations
consequently inhibited the GST-Mcm2p protein kinase activity of
Cdc7p/Dbf4p*. This result further strengthens the possibility that
Rad53p directly regulates the protein kinase activity of Cdc7p/Dbf4p by phosphorylation.
Weinreich and Stillman (32) recently showed that a Cdc7p monomer was
bound to chromatin at all times during the cell cycle, whereas Dbf4p
was only associated with chromatin at certain times. From these results
they proposed that the binding of Dbf4p to chromatin might determine
which origins become activated. In addition, the binding of Dbf4p to
the chromatin-bound Cdc7p would form an active Cdc7p/Dbf4p kinase
complex that could promote initiation at that origin. However, we argue
against this possibility, because we completely failed to reconstitute
Cdc7p/Dbf4p in vitro by mixing two extracts containing Cdc7p
and Dbf4p. Thus, if the binding of Dbf4p to the chromatin-bound Cdc7p
may form an active Cdc7p/Dbf4p kinase complex, we speculate that
another factor(s) might play a role for its association.
Two recent reports (27, 28) suggest that Cdc7p activity is required to
activate individual origins rather than being required for a global
activation of S phase. Also, it is well known that in the presence of
HU, which activates Rad53p, only early firing origins are activated in
S phase (35). How can these results be explained by phosphorylation and
inactivation of Cdc7/Dbf4p protein kinase by Rad53p protein kin
,
TOP
ABSTRACT
INTRODUCTION
