The Essential Role of Saccharomyces
cerevisiae CDC6 Nucleotide-binding Site in
Cell Growth, DNA Synthesis, and Orc1 Association*
Bin
Wang
§,
Lou
Feng
§,
Yu
Hu
§,
Sheng He
Huang
,
C.
Patrick
Reynolds
¶,
Lingtao
Wu
, and
Ambrose Y.
Jong
§
From the Departments of
Pediatrics,
§ Microbiology, and ¶ Pathology, University of Southern
California, Los Angeles, California 90027
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ABSTRACT |
Saccharomyces cerevisiae
Cdc6 is a protein required for the initiation of DNA replication. The
biochemical function of the protein is unknown, but the primary
sequence contains motifs characteristic of nucleotide-binding sites. To
study the requirement of the nucleotide-binding site for the
essential function of Cdc6, we have changed the conserved Lys114 at the nucleotide-binding site to five other amino
acid residues. We have used these mutants to investigate in
vivo roles of the conserved lysine in the growth rate of
transformant cells and the complementation of cdc6
temperature-sensitive mutant cells. Our results suggest that
replacement of Lys with Glu (K114E) and Pro (K114P) leads to
loss-of-function in supporting cell growth, replacement of the Lys with
Gln (K114Q) or Leu (K114L) yields partially functional proteins, and
replacement with Arg yields a phenotype equivalent to wild-type, a
silent mutation. To investigate what leads to the growth defects
derived from the mutations at the nucleotide-binding site, we evaluated
its gene functions in DNA replication by the assays of the plasmid
stability and chromosomal DNA synthesis. Indeed, the K114P and K114E
mutants showed the complete retraction of DNA synthesis. In order to
test its effect on the G1/S transition of the cell cycle,
we have carried out the temporal and spatial studies of yeast
replication complex. To do this, yeast chromatin fractions from
synchronized culture were prepared to detect the Mcm5 loading
onto the chromatin in the presence of the wild-type Cdc6 or mutant
cdc6(K114E) proteins. We found that cdc6(K114E) is defective in the
association with chromatin and in the loading of Mcm5 onto chromatin
origins. To further investigate the molecular mechanism of
nucleotide-binding function, we have demonstrated that the Cdc6
protein associates with Orc1 in vitro and in
vivo. Intriguingly, the interaction between Orc1 and Cdc6 is
disrupted when the cdc6(K114E) protein is used. Our results suggest
that a proper molecular interaction between Orc1 and Cdc6 depends on
the functional ATP-binding of Cdc6, which may be a prerequisite step to
assemble the operational replicative complex at the G1/S transition.
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INTRODUCTION |
Cell cycle regulation is a complicated but highly coordinated
process. It has a conserved mechanism among eukaryotes from yeast to
human. The primary control of the eukaryotic cell cycle is provided by
a family of cyclin-dependent kinases
(CDKs)1 and their associated
cyclins, which regulate kinase activity. In unicellular yeast cells, a
single prototype CDK gene, CDC28 in the budding yeast
Saccharomyces cerevisiae or cdc2+ in
the fission yeast Schizosaccharomyces pombe functions at
different cell cycle stages. Different cyclins activate the same kinase as different points in the cycle. It is now known that the central control of cell cycle progression by CDK complexes is regulated positively and negatively to monitor each step of the progression (1,
2). This regulatory control, associated with checkpoints, orchestrates
various types of cell cycle genes throughout the cell cycle.
In S. cerevisiae, over 70 temperature-sensitive cell
division cycle (cdc) mutations have been isolated that
control events throughout the cell cycle (3, 4). One of them,
CDC6, is required in the late G1 and S phases of
the cell cycle. At the non-permissive temperature, the cdc6
mutant cells show a DNA synthesis defect (5). The cdc6
mutants undergo increased chromosomal loss and hyper-recombination,
suggesting a fairly direct role in DNA replication (6). Tandem copies
of different ARSs added to the mini-chromosome can suppress mitotic
loss in cdc6, but not in cdc7, cdc9,
cdc16, or cdc17 mutants (7), suggesting a role at
replication origins. The CDC6 mRNA fluctuates
periodically throughout the cell cycle (8-10), and its nuclear entry
is cell cycle-dependent (11). The 5'-untranslated region of
the CDC6 gene is very similar to a group of cell cycle genes
that are either precursor enzymes for DNA synthesis or are directly
involved in DNA replication (12). The involvement of Cdc6 in DNA
replication has also been supported by direct analysis of origin
function in cdc6-1 mutants using two-dimensional gel method
(13). The studies suggest that the Cdc6 and Orc5 protein interact and
determine the frequency of initiation of DNA replication in yeast.
Genetic interaction between CDC6 and ORC6 has
also been reported (14). The S. pombe cdc18+ is
homologous to S. cerevisiae CDC6 (15, 16). It has been proposed that the cdc18+ gene plays two roles in the cell
cycle, mediating the initiation of DNA replication and preventing
inappropriate mitosis. In S. cerevisiae, over-expression of
CDC6 induces G2 delay of the cell cycle (17).
Both results suggest a possible second function of CDC6 at
the G2/M phase boundary. Homologs of yeast Cdc6 have also
been found in human (18) and Xenopus (19). The conservation of Cdc6 among species implies an essential role for this protein.
Genomic footprinting has defined two cell cycle stages regarding the
formation of replication complexes (RC): pre-RC in G1 phase
and post-RC after S phase to M/G1 border (20-22). The
post-RC footprint closely resembles that produced in vitro
with purified ORC and Abf1, which protect A/B1 and B3 elements,
respectively. The pre-RC is defined by its further protecting the B2
element of ARS1 in addition to the A/B1 and B3 elements. The
data suggest that the initiation of DNA replication is not controlled
by the binding of ORC and Abf1 to the origins; instead, modification of
origins has to occur with the involvement of some additional factors.
The Cdc6 protein is required for establishment and maintenance of
pre-RC, because the arrested cdc6-1 mutant produces a
post-replication footprint (23-25). The Cdc6 protein is also required
for the loading of MCM proteins (26-30). In addition, the Cdc6 protein
interacts with Cdc28 protein complex after
p40sic1 is degraded at late G1 phase
(31). We have recently found that the Cdc6 can stimulate Abf1 binding
to the B3 domain of ARS1 DNA fragment (32). Currently, it is
believed that interactions of Cdc6 with a variety of other protein
complexes, such as the ORC, the MCM complex, and Cdc28 kinase are
important in coordinating chromosome replication and cell cycle control
(33). However, the detailed molecular mechanism of the CDC6
gene action is still largely unknown.
There is a conserved nucleotide-binding site in Cdc6 (34). It has been
shown that the yeast Cdc6 is able to bind ATP and GTP (10). At present,
the physiological roles of the nucleotide-binding site are still
obscure. One effective way to study the function of the
nucleotide-binding site is to carry out site-specific mutagenesis experiments to alter the indispensable Lys residue, and examine its
phenotypic consequence. In these studies, we have followed a series of
steps to unravel the functions of the nucleotide-binding site. First,
we changed the conserved Lys114 at the nucleotide-binding
site to five other amino acid residues. We show that replacement of Lys
with Glu (K114E) and Pro (K114P) leads to loss of function in
supporting cell growth, replacement of the Lys with Gln (K114Q) or Leu
(K114L) yields partially functional proteins, and replacement with Arg
yields a phenotype equivalent to wild-type. Second, based on the
plasmid stability assay and chromosomal DNA synthesis assay, the roles
of the conserved Lys is most likely required for DNA replication. This
guides us to investigate the formation of a functional replication
complex at G1/S transition. Third, we have prepared
synchronous chromatin fractions of both mutant and wild-type proteins
to examine the effect of the cdc6(K114E) mutation. We found that the
loading of the cdc6(K114E) and Mcm5 proteins is drastically altered in the chromatin. Finally, at the molecular level, we demonstrated that
Cdc6 interacts with Orc1 in vitro and in vivo.
Interestingly, the interaction between Orc1 and Cdc6 is impaired when
the cdc6(K114E) protein is used. Our results suggest that a proper
molecular interaction between Orc1 and Cdc6 depends on the functional
ATP binding of Cdc6. This may lead to assemble the operational
replicative complex at the G1/S transition. The biological
significance of the ATP binding in this process is discussed in this report.
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EXPERIMENTAL PROCEDURES |
Strains, Plasmids, and Media--
The yeast
cdc6-1 strain, 611, has been described (31).
Strain K4055 was
CDC6::hisGURA3hisG,trp1-1::TRP1
MET-CDC6 with URA3 looped out (9). Strain
BJ2168 (a prc1-407 prb1-1122 pep4-3 leu2 trpl
ura3-52) is a protease-deficient strain. Plasmid carrying alleles
mutated at lysine 114 were generated by cloning a 1.8-kb
HindIII-EcoRI fragment encompassing the
CDC6 ORF into M13mp19, mutagenizing as described
below, and cloning the resulting alleles into YEp352 (35). Lysine 114 has been changed to Glu (K114E), Gln (K114Q), Arg (K114R), Pro (K114P),
and Leu (K114L) (see Fig. 1). The YEp352 subclones are designated:
YEp352-K114E, YEp352-K114L, YEp352-K114R, YEp352-K114P, and
YEp352-K114Q (Table I). Plasmid YGp12 for the expression of untagged
proteins were prepared by cloning the wild-type and K114E allele under
the control of the GAL1,10 promoter in a 2-µm yeast vector
with URA3 as selectable marker. Plasmid YGp102 is the same
construct except that LEU2 is the selection marker (Fig. 2).
YCp5N is derived from a single-copy shuttle vector YCplac111 and
contains a 0.7-kb 5' upstream sequence of the CDC6 gene
(34). A T7 tag was inserted into 3' end of the CDC6 open
reading frame in which the TAG codon of CDC6 gene had been
removed, and the fusion was subcloned into YCp5N. YEp-HA-Mcm5 is
generated by inserting a MCM5 fragment into a high copy
vector, YEp352, in which Mcm5 is HA-tagged at its C-terminal 751 amino acids and the expression of the MCM5 gene is driven by its
own 444-bp promoter sequence (36). A full-length of ORC1
gene was subcloned into bacterial expression vector pET28b,
pET28a-ORC1, for the expression of 6xHis-Orc1 protein
YGp181-His-T7-ORC1 contains 6xHis and T7 tag at the N
terminus of the ORC1 gene driven by the Gal
promoter with Leu2 selection marker (Fig. 7C).
YGp123 is the vector with the insertion of 0.8-kb Gal1-Gal10
promoter on the vector YEplac112 with Trp1 as the selection
marker. The wild-type CDC6-T7 tag, cdc6(K114E)-T7
tag and temperature-sensitive cdc6-1-T7 tag genes were
subcloned into YGp123, individually, for co-expression experiments
(Fig. 7C). Yeast media YPD and CSM medium SD-ura were
purchased from Bio101, Inc. All chemicals were ordered from Sigma; and
restriction enzymes were purchased from Life Technologies, Inc. or New
England Biolabs.
In Vitro Mutagenesis--
Muta-Gene in vitro
mutagenesis kit (Bio-Rad) was used to carry out the experiments as
described below. The mutagenic (GGT CCG CCT GGC ACT GGC (G/C)(G/C/T)G
ACT) and universal primers were synthesized (12, 37); T7 DNA polymerase
instead of the Klenow fragment of DNA polymerase I was used in the
reaction (38). The mutagenized DNA is shown in Fig. 1.
Plasmid Stability Assay--
Yeast cdc6-1 shows
elevated chromosomal loss. The wild-type and mutant cdc6
genes were transformed into strain cdc6-1 to examine their
plasmid stability. A colony grown on selective medium was resuspended
in 0.2 ml of water. A 0.1-ml sample was used to inoculate 5 ml of
nonselective media (either YPD or SD plus uracil), and cultures were
grown at 30 °C with aeration for 5-10 generations. Dilutions of the
initial suspension were plated on YPD plates, and colonies were counted
to determine the initial concentration of cells. These plates were then
replica-plated to SD-ura to determine the percentage of plasmid-bearing cells.
PFGE Labeling--
The PFGE labeling method was used to
investigate yeast chromosomal DNA synthesis (39). In this method, yeast
cells are first labeled with 32Pi in
vivo and chromosomal DNA is then resolved by pulsed field gel
electrophoresis. Briefly, 20 ml of yeast cell culture was grown at room
temperature to the early log phase (~107 cells/ml) and
arrested by
-factor for 90 min. The cells were then washed with 20 ml of low phosphate medium (LPM) three times, and resuspended into the
same volume of LPM. After heat treatment (37 °C), 50 µCi of
radioactive 32Pi was added. The culture was
grown for another 1 h, and uptake was quenched with 50 mM cold phosphate. The labeled cells were harvested and
then washed with phosphate-buffered saline buffer, and molecules were
separated using the Bio-Rad CHEF-DR II Megabase DNA pulse field
electrophoresis system. The resulting gel was dried and autoradiographed.
Preparation of Synchronous Chromatin Fractions--
Yeast
chromatin was prepared according to Lue and Kornberg (40) with slight
modifications. Briefly, yeast strain K4055 cells harboring
plasmid fusions with co-transformation of YEp-Mcm5-HA and
YCp5N-Cdc6-T7, or YCp5N-cdc6(K114E)-T7. The transformant cells were
grown on the CSM medium in the presence of methionine
(MET+) to shut off the endogenous CDC6. The
cells were synchronized at G1 with
-factor at final
concentration of 10 µg/ml at room temperature for 4 h. The cells
were then released from
-factor block, grown on the fresh
MET+ medium, and collected at the indicated time points.
Aliquots of cells were harvested at different time intervals to monitor the degree of synchrony by measurement of percentage of budded cells
and FACS analysis (38). Yeast cells were washed once with 50 mM ice-cold EDTA and incubated in a solution containing 20 mM EDTA/2%
-mercaptoethanol at 30 °C for 30 min.
After incubation, the cells were centrifuged and washed once with 1 M sorbitol. Spheroplasts were generated by digesting the
cells with 100 µg/ml yeast lytic enzyme (ICN) in a solution
containing 1 M sorbitol, 5 mM
-mercaptoethanol at 37 °C for 60 min. The spheroplasts were cooled on ice for 10 min, centrifuged at 3000 × g for
10 min at 4 °C, and washed once with the 1 M ice-cold
sorbitol. The spheroplasts were collected by centrifugation and lysed
in a buffer containing 20% (w/v) Ficoll 400 (Sigma), 20 mM
HEPES-KOH, pH 7.5, 20 mM KCl, 5 mM
MgCl2, 3 mM dithiothreitol, 1 mM
phenylmethylsulfonyl fluoride (PMSF), and 1 mM EDTA with a
Teflon/glass homogenizer. Cell debris were removed by four spins at
3000 × g for 5 min at 4 °C. Uniform white
supernatants were recovered and were spun at 36,000 rpm and 4 °C for
30 min. Nuclear pellets were resuspended in 500 µl of the spheroplast
lysis buffer described above and subjected to Ficoll 400 gradient
centrifugation (20-45%) at 36,000 rpm and 4 °C for 30 min.
Chromatin pellets were resuspended in a buffer H/0.1 (50 mM
HEPES-KOH, pH 7.5, 100 mM KCl, 5 mM magnesium
acetate, 1 mM EDTA, 0.02% Nonidet P-40, 1 mM
dithiothreitol, 1 mM PMSF, and 10% glycerol) and stored at
80 °C.
Western Blots to Detect Mcm5 and Cdc6 Proteins--
10 µg of
protein from each chromatin sample were mixed with 4× SDS loading
buffer and boiled in a water bath for 5 min. The samples were resolved
by SDS-polyacrylamide gel (8-10%) electrophoresis at 4 °C for
4 h, and proteins were blotted onto an Immobilon-P polyvinylidene
difluoride transfer membrane (Millipore). The protein blots were probed
either with 12CA5 monoclonal antibody (Boehringer Mannheim) for
detecting HA-tagged proteins, or with anti-T7 tag monoclonal antibody
(Novagen) for detecting T7-tagged proteins, followed by alkaline
phosphatase-conjugated secondary antibody. The protein samples were
detected with the Tropix chemiluminescent system.
In Vitro and in Vivo Interaction between Orc1 and Cdc6--
500
ml of bacterial culture containing expressed 6xHis-T7-Orc1 tagged
protein was harvested and resuspended into 15 ml of Buffer I (20 mM Tris-HCl, pH 8, 500 mM NaCl, 20 mM imidazole, 0.1% Triton X-100) in the presence of 20 mM PMSF and 20 mM benzamidine. 10 mg of
lysozyme and 2 mg of DNase were added and incubated at 4 °C for 30 min. The mixture was centrifuged at 13,000 rpm for 20 min. The
supernatant was collected (~15 ml) and mixed with 0.2 ml of Ni-NTA
(Qiagene) at 4 °C for 60 min. The resulting slurry was packed onto a
column and washed with 20 ml of Buffer II (20 mM Tris-HCl,
pH 8, 150 mM NaCl, 50 mM imidazole, 0.1%
Triton X-100). The Orc1-charged matrix was ready for Cdc6 interaction
assay. GST-Cdc6 fusions were prepared from 250 ml of culture. Crude
extracts were made in Buffer II with 20 mM PMSF and 20 mM benzamidine. Aliquot was used for SDS-polyacrylamide gel
electrophoresis to analyze their expression (Fig. 7B,
lanes 4-6). The rest (~15 ml) was passed
through Orc1-charged matrix 10 times for the interaction studies.
For Orc1 and Cdc6 co-precipitation experiments (Fig. 7C),
yeast strain BJ5459 was first transformed with
YGp181-His-T7-ORC1 (selection marker Leu2). The
resulting strain was then transformed separately with either YGp123-
CDC6-T7, or YGp-K114E-T7, or YGp1233-cdc6-1-T7 with
selection marker Trp1. 500 ml of culture was induced with 2% galactose for 4 h. The cell pellets were disrupted by
Bead-BeaterTM in Buffer II, supplemented with 20 mM benzamidine, 20 mM PMSF, 1 mg of DNase.
Tagged Orc1 was purified by Ni-NTA matrix, concentrated 10-fold, and
probed with anti-T7 antibody on protein blots. Monoclonal antibody
against T7 tag can detect both His-T7-Orc1 and Cdc6-T7 on the same blot
if Orc1 can bring down Cdc6-T7 proteins in the experiment shown in Fig.
7C.
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RESULTS |
Site-directed Mutagenesis of the Nucleotide-binding Site of
the CDC6 Gene--
In vitro site-specific mutagenesis is a
powerful tool to probe the relationship between the structure and
activity of proteins, because the amino acid residues responsible for a
particular function can be identified directly (41). Previous analysis
of the primary sequence (34) has shown that CDC6
contains a motif conserved among known ATPases,
GXXGXGKT, ranging from residues 108-115 in CDC6 (Fig. 1A).
This element plays a role in binding the pyrophosphate moiety of
nucleotides, and the Lys residue is essential for electrostatic interaction between the proteins and the nucleotides (42). In order to
examine whether the conserved Lys residue represents an essential amino
acid residue as in other nucleotide-binding proteins, we have mutated
the Lys residue to five other residues. The mutagenic primers were
similar to the wild-type sequence except for one or two altered
nucleotides at the first two bases in the lysine codon (Fig. 1,
A and B). The following substitutions were made
at the conserved lysine: to glutamate to test the effect of introducing
a negative charge, to proline to test the effect of a general
disruption, to glutamine to test the effect of substituting an amide,
to leucine to test the effect of introducing a neutral amino acid, and
to arginine to test the effect of changing the configuration of the
positive charge. Although both Lys and Arg are basic amino acids, none
of the nucleotide-binding sites known so far contain Arg. Substitution
of arginine for lysine results in a protein with partial function in
some cases. For example, this substitution in yeast RAD3 was
found to abolish its ATPase and DNA helicase activities, but not to
change the ability to bind ATP (43). Mutagenesis was confirmed by DNA
sequencing (Fig. 1C).

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Fig. 1.
Site-directed mutagenesis of the
CDC6 gene at its nucleotide-binding site.
A, the consensus sequence of a nucleotide-binding domain is
shown on the top. The Cdc6 protein sequence from position
108 to 115 is presented in the middle, and the corresponding
CDC6 DNA sequence is lined up on the bottom. The
mutagenic primer is identical to the wild-type CDC6 DNA
sequence depicted above except that the nucleotide A is changed to G,
C, and/or T, resulting in the Lys residue altering to other amino acid
residues. B, alternations of the codon and the amino acid
residue are summarized. C, the DNA sequences of mutagenized
cdc6 genes are shown on the bottom.
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Functional Complementation of cdc6-1 Mutant Cells--
To test
the in vivo function of the nucleotide-binding site, we
examined whether the mutant genes can complement the cdc6-1 temperature-sensitive growth defect. We transformed the
cdc6-1 mutant strain with plasmids expressing the mutant
genes. Transformants were selected at the nonpermissive temperature. As
shown in Table I, the wild-type and
K114L, K114Q, and K114R are able to complement the defective mutant
cells at nonpermissive temperature. Mutant K114E, K114P, and vector
alone, however, fail to transform. The few colonies observed for K114E
and K114P are presumably due to the recombination between the episomal
and chromosomal copies. These results imply that the Lys to Glu
mutation may perturb the interaction between Cdc6 and nucleotides; the
Lys to Pro mutation may alter steric structure resulting in the loss of
CDC6 gene function. Thus, the result supports that the Lys
residue is essential for CDC6 gene function.
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Table I
Transformation frequency of the wild-type and mutated CDC6 gene into
cdc6-1 cells at the permissive (23 °C) and nonpermissive
temperature (37 °C)
Transformations are reported as colonies/ µg of transforming DNA.
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A trivial explanation of the above findings is that the mutant proteins
are not efficiently present. To ensure that failure of the mutant
alleles to complement cdc6-1 was due to a defective Cdc6
protein, and not merely due to absence of protein, we compared the
steady-state levels of wild-type Cdc6 and the cdc6(K114E) proteins. The
cdc6(K114E) protein was chosen, because it had the most drastic defect
in the complementation assay. Since Cdc6 is present at very low levels
in yeast and is barely detectable with the polyclonal Cdc6 antibody, we
cloned the wild-type and mutant alleles under the control of the
GAL1,10 promoter and introduced this construct into yeast
vectors marked with URA3 or LEU2 (YGp12 and
YGp102, respectively). Protein blots of extracts of cells carrying the
plasmids were probed with Cdc6 antibody. As shown in Fig.
2, both mutant and wild-type proteins
were expressed at the same level, demonstrating that expression levels
were not the cause of the lack of in vivo function of
cdc6(K114E) protein.

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Fig. 2.
Expression of mutant and wild-type Cdc6
proteins in yeast. On the left margin, the
protein markers from Life Technologies, Inc. are indicated: myosin
(205,000), phosphorylase b (97,400), bovine serum albumin
(68,000), ovalbumin (43,000), carbonic anhydrase (29,600), and lysozyme
(18,000). CDC6 was expressed under the control of the
GAL1,10 promoter as described under "Experimental
Procedures." We created strains carrying either one or two copies of
the wild-type or mutant allele by transforming strain BJ2168
with YGp12 alone or YGp12 and YGp102 constructs together. Cells were
grown in raffinose, and then 2% galactose was added for 3 h;
cells were then harvested. Fifty µg of each extract was
electrophoresed and blotted, and the blot was probed with polyclonal
Cdc6 antibody described previously. Lanes 1 and
4, strain BJ2168 carrying YGp12 and YGp102
(vectors only, no CDC6 insert); lane
2, strain BJ2168 carrying YGp12-CDC6;
lane 3, strain BJ2168 carrying both
YGp12-CDC6 and YGp102-CDC6; lane
5, strain BJ2168 carrying YGp12-K114E;
lane 6, strain BJ2168 carrying
YGp12-K114E and YGp102-K114E.
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Effects on the Growth Rate from cdc6-1 Transformant Cells--
To
further evaluate the effect of mutant alleles in the cdc6-1
background, cdc6-1 was transformed with the mutant
plasmids, the wild-type plasmid and vector alone, and transformants
were selected at room temperature. Transformant cells were grown to log
phase at the permissive temperature (25 °C), then transferred to the
nonpermissive temperature (37 °C) and incubated for another 8 h, during which growth rates were determined (Fig.
3A). Cells harboring the
wild-type plasmid grew at a rate similar to wild-type strains in this
medium (td = 120 min). For mutants K114E, K114P, and vector alone, cells essentially ceased to divide after 2 h and
totally arrested at 37 °C. The remaining strains grew more slowly
than wild-type, with doubling times of 125-130 min for K114R, and
150-160 min for K114L and K114Q at 37 °C. Thus, all of the mutants
generated show at least some impairment in growth rate, indicating that
an intact lysine residue is critical for optimal CDC6
function. Similar results can be observed from the plates incubated at
25 °C (permissive temperature) and 37 °C (nonpermissive temperature). No colonies were formed in the K114E and K114P
transformants (Fig. 3B). As the growth curves of the K114E
and K114P transformants are similar to cdc6-1 cells
transformed with vector only, it is clear that the K114E and K114P are
loss-of-function proteins that cannot rescue the cdc6-1
mutation.

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Fig. 3.
Growth properties of the transformants
carrying mutant cdc6 genes at the nonpermissive
temperature (37 °C). A, yeast carrying the indicated
plasmids were grown at SD-ura at room temperature until early log
phase. The cultures were shifted to 37 °C (nonpermissive
temperature) and harvested at different time points, and cells were
counted using a hemacytometer. The value given represents an average
number of several independent determinations. and solid
line, YEp352-CDC6; and thick
dashed line, YEp352-K114R; and
thin dashed line, YEp352-K114Q; + and
dotted line, YEp352-K114L; and
dashed/dotted line, YEp352-K114P; and
dashed line, YEp352-K114E; and
dashed line, YEp352 only. B, the
wild-type CDC6 (WT) and various mutant
transformant cells were streaked onto the plates and incubated at 25 and 37 °C, respectively. Picture was taken after 24 h of
incubation.
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Plasmid Stabilities in cdc6-1 Cells Expressing Mutant cdc6
Genes--
Temperature-sensitive mutation cdc6-1 shows
enhanced plasmid loss at the nonpermissive temperature, but plasmids
may be stabilized by the inclusion of a functional copy of
CDC6 on the plasmid. We tested the above mutated alleles of
cdc6 for their ability to confer stability on plasmids. As
shown in Fig. 4, cdc6-1 cells bearing a plasmid with the wild-type CDC6 gene maintains
~85% of plasmid after 10 generations under nonselective conditions, a loss rate similar to wild-type backgrounds. The K114R plasmid confers
slightly lower stability (~70%), and K114L and K114Q plasmids result
in 45-50% plasmid-containing cells after 10 generations. K114E and
K114P transformants, however, show dramatic plasmid loss, similar to
that of YEp352 transformants. The loss of plasmid stability is most
likely due to the defect(s) of DNA synthesis. To explore what mechanism
affects the growth rate and plasmid DNA stability, we investigated the
chromosomal DNA synthesis.

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Fig. 4.
Plasmid stability in cdc6-1
and its transformant cells. Strain cdc6-1 was
transformed with the mutant plasmids described in Fig. 1.
YEp352-CDC6 (wild-type gene) and YEp352 (vector only) were
used as positive and negative controls, respectively. Transformants
were grown in nonselective media for 5-10 generations and plated in
supplemented minimal medium or on YPD. The percentage of colonies
containing plasmids was determined by replica plating on to medium
lacking uracil. The value given represents an average number of several
independent determinations. and solid line,
YEp352-CDC6; and thick dashed
line, YEp352-K114R; and thin
dashed line, YEp352-K114Q; + and
dotted line, YEp352-K114L; and
dashed/dotted line, YEp352-K114P; and
solid line, YEp352-K114E; and
dashed line, YEp352 only.
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Yeast Chromosomal DNA Synthesis in cdc6-1 and Transformant
Cells--
The PFGE labeling technique is a novel method, which is
highly specific to chromosomal DNA synthesis in yeast. In this method, yeast cells are first labeled by 32P in vivo and
chromosomal DNA molecules are then resolved by pulsed field gel
electrophoresis. DNA synthesis can be measured by the 32P
incorporation into individual chromosomal DNA molecules. We have
previously used the PFGE labeling technique to verify that cdc6-1 cells fail to synthesize chromosomal DNA molecules
(39). In Fig. 5, we examined DNA
synthesis by the PFGE method in cdc6-1 cells transformed
with different cdc6 Lys114 mutants. The cultures
were synchronized with
-factor (at START), 32P label was
added, and cells were incubated at the non-permissive temperature. PFGE
was used to resolve the labeled chromosomal DNA molecules. Cells
transformed with wild-type CDC6 and vector YEp352 only, are
used as the positive and negative controls, respectively. The DNA
pattern (Fig. 5A, lanes 1 and
7) and 32P incorporation (Fig. 5B,
lanes 1 and 7) for the controls are as
expected. In the K114E transformant (lane 6) and
K114P transformant (lane 4), ethidium bromide
staining revealed a similar chromosomal pattern, yet little
radioisotope incorporation into chromosomal DNA was observed. However,
in cells transformed with K114R, K114Q, and K114L plasmids, a nearly
wild-type amount of 32P incorporation was observed at the
nonpermissive temperature (lanes 2, 3,
and 5). Taken together (Figs. 3-5), we conclude that the
K114E and K114P are loss-of-function mutations, K114Q and K114L are
partially functional proteins, and K114R functions equivalently to
wild-type. Since the only fully functional mutant represents a
conservative change in the conserved ATP-binding site, we argue that a
functional ATP-binding site in CDC6 is required for DNA synthesis in vivo.

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Fig. 5.
Chromosomal DNA synthesis in
cdc6-1 and transformants. Yeast
cdc6-1 transformants containing the wild-type
CDC6 and mutant cdc6 genes were subjected to the
PFGE-labeling protocol (see "Experimental Procedures"). The
cultures (20 ml) were grown in the SD-ura at room temp to the early log
phase (~107 cells/ml). Cells were washed with prewarmed
LPM three times. They continued growing in the LPM for 3 h at the
nonpermissive temperature (37 °C). The cultures were then
synchronized by adding -factor (10 units/ml) and incubating for 120 min (approximately one doubling time). The cultures were again washed
three times with LPM to remove pheromone. The washed cells were
resuspended into 20 ml of fresh LPM supplemented with 0.2 mCi of
radioactive 32P and incubated at 37 °C for 30 min.
Labeling was quenched with 50 mM cold phosphate buffer
before harvesting. Chromosomal DNA molecules were resolved by PFGE, and
DNA was visualized by ethidium bromide staining (A).
Incorporation of radioisotope was visualized by autoradiography
(B). All samples are aligned. Lane 1,
YEp352-CDC6; lane 2, YEp352-K114R;
lane 3, YEp352-K114Q; lane
4, YEp352-K114P; lane 5, YEp352-K114L;
lane 6, YEp352-K114E; lane
7, YEp352 only.
|
|
Effect of Mutant K114E on the Assembly of the Replicative
Complex--
It has been shown that the Cdc6 is essential for
establishment and maintenance of pre-replicative complexes (pre-RCs)
and the loading of MCM proteins at G1 phase on chromatin is
Cdc6-dependent. Since the initiation of DNA replication
requires the prior assembly of pre-RCs, we further tested the
hypothesis that the mutation of ATP-binding domain of Cdc6 would lead
to the failure of the pre-RC assembly. We selected the mutant
cdc6(K114E) for these studies, because it showed the most drastic
effect on the CDC6 gene functions. To do this, we
transformed wild-type CDC6 and mutant cdc6(K114E)
genes with its native promoter bearing on the single copy vector into
strain K4055, respectively. In this K4055 strain,
the chromosomal CDC6 gene is under the control of the MET3 repressive promoter. In the absence of methionine in
the medium, the chromosomal wild-type CDC6 gene is expressed
to support the cell growth. In the presence of methionine, the
chromosomal wild-type CDC6 gene is shut-off and endogenous
Cdc6 protein is rapidly degraded, resulting in a null mutant phenotype.
In the cdc6(K114E) transformant, the wild-type protein is basically
replaced by the mutant one. The impaired function of the mutant cdc6
protein, if any, can be examined. We have investigated the temporal and spatial regulation of the Cdc6 nucleotide-binding function(s) as shown
in Fig. 6.

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Fig. 6.
Association of Cdc6 and Mcm5 proteins in the
synchronized chromatin fractions. Yeast strain K4055
was transformed with either T7-tagged wild-type CDC6 gene
(A) or mutated cdc6(K114E) gene (B),
respectively. Both strains also contained the HA-tagged MCM5
gene. The cells were synchronized with -factor
arrest-andrelease method. In the presence of methionine, the
chromosomal CDC6 is shut off, and the plasmid carried
T7-tagged Cdc6 was expressed. Aliquot of cultures were collected every
20 min, and subjected to FACS analysis, counting percentage of budded
cell (upper panel), and protein blotting analyses
(bottom panel). Based on the percentage of budded
cells and FACS analysis, the first S phase is around 60 min in the
wild-type Cdc6 cells. Panel A shows the
expression of the wild-type T7-tagged Cdc6 and panel
B shows the expression of the mutated cdc6(K114E) protein.
The same blot was used to detect HA-tagged Mcm5 by the monoclonal
antibody 12CA5.
|
|
We determined the association of the cdc6(K114E) mutant protein with
chromatin and the loading of Mcm5 onto chromatin origin in
chromatin-binding assay. Chromatin was prepared from the yeast cell
released from G1-phase block at the indicated time points and probed either with anti-T7 tag (for detecting the association of
Cdc6 with chromatin) or with monoclonal antibody 12CA5 (for detecting
HA-tagged Mcm5 associated with chromatin). In the presence of
methionine, the endogenous Cdc6 was shut off, so growth of the cell was
totally dependent on Cdc6 produced from the plasmids harboring in the
cell. The cultures were synchronized by the
-factor arrest-and-release method. Based on the percent of budded cells and
FACS analysis, the peak of the S phase is around ~60 min (Fig. 6A, upper panel) in the wild-type
cells. Orc2, which was used as an internal control showed a constant
level throughout the cell cycle (data not shown). The association of
wild-type Cdc6 with chromatin was in a cell cycle-dependent
manner, i.e. Cdc6 was detected when the cell was released
from G1 phase block. It remained on chromatin during
G1 phase, decreased at the start of S phase and disappeared
from chromatin during G2 and M phases, and reappeared after
M phase (Fig. 6A, bottom panel). The
association of Mcm5 with chromatin showed a quite similar pattern to
that of the Cdc6 protein. i.e. Mcm5 was loaded onto
chromatin during G1 phase, replaced from chromatin during S
phase and was not detected during G2 and M phases. The
association of cdc6(K114E) mutant proteins with chromatin, however, was
dramatically different from that the wild-type Cdc6 (Fig.
6B). The cdc6(K114E) mutated proteins can associate with
chromatin around 20 min, but then faded quickly from the chromatin.
Under the same condition, the association of Mcm5 with chromatin was
determined in the presence of the mutant cdc6(K114E) proteins. The
loading of the Mcm5 protein onto chromatin was delayed for at least 60 min after the cell was released from the G1 phase block
(Fig. 6B, lower panel). There was no
second time associating or re-loading for Cdc6 and Mcm5, suggesting
that the cell cycle was arrested and the cell ceased to grow.
Consistent with these observations, atypical FACS pattern in the mutant
transformants was observed. Presumably, this is due to defective DNA
synthesis and/or abnormal morphology. Thus, the data suggested the
improper chromatin assembly and the perturbation of the cell cycle
progression in the cdc6(K114E) background.
Functional Orc1 and Cdc6 Interaction Depends on the Normal
ATP-binding Site of Cdc6--
During the process of Cdc6 purification,
we consistently observed the co-fractionation of Orc1 with Cdc6. In the
glycerol gradient centrifugation, both Orc1 and Cdc6 proteins
co-sediment in a large 450-660-kDa protein
complex.2 Due to their
structural similarity, it has been proposed that the Cdc6 and Orc1 may
be in a single gene family (44). This information led us to investigate
the relationship between Cdc6 and Orc1 in vitro and in
vivo.
In the in vitro interaction studies, bacterially expressed
Orc1-His-tagged protein retained on the nickel-chelating matrix was
used as a protein affinity column. The same vector without the Orc1
insert was used as negative control. Yeast extracts, as the Cdc6
protein source, were then passed through the Orc1-charged matrix. After
extensive washing, the interacting components were analyzed by protein
blots. The presence of Cdc6 protein can be detected by anti-Cdc6
polyclonal antibodies, indicating an interaction between Orc1 and Cdc6
proteins (Fig. 7A). To test
whether this is a direct interaction between Cdc6 and Orc1, we used
bacterially expressed wild-type GST-Cdc6 and mutant GST-cdc6 proteins
for interaction studies. In the Escherichia coli background,
one would assume that yeast adaptor protein(s), if any, can be avoided. In Fig. 7B (lane 1), the wild-type
GST-Cdc6 was able to interact with Orc1, suggesting a direct
interaction. Intriguingly, there was no detectable GST-cdc6(K114E) band
(Fig. 7B, lane 2). This result
suggests that Orc1 could not interact with mutated cdc6(K114E) protein.
To avoid artifact, we used another mutant protein GST-cdc6-1 as
control. The mutation point of the cdc6-1 protein has been determined
as Gly260 to Glu mutation.2 This mutation leads
to a temperature-sensitive mutation. However, there is no defect in
Orc1/Cdc6 interaction (Fig. 7B, lane
3). The failure of Orc1/cdc6(K114E) interaction is not due
to the absence of protein, as the same level of GST-Cdc6
(lane 4), cdc6(K114E) (lane
5), and cdc6-1 (lane 6) were
expressed and used in this experiment.

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Fig. 7.
Interaction between Orc1 and Cdc6.
A, bacterially expressed 6xHis-T7-Orc1 tagged protein was
retained on the nickel-chelating matrix used as a protein affinity
column (lane 1). The same vector without the Orc1
insert was used as negative control (lane 2).
Yeast extracts were prepared from strain BJ5459/
YGp123-CDC6 and used as the Cdc6 protein source. The
extracts were then passed through the Orc1-charged matrix, washed
extensively, and subjected to protein blotting experiments. The
retention of Cdc6 protein, if any, was detected by anti-Cdc6 polyclonal
antibodies. B, E. coli extracts containing
GST-Cdc6 (lane 1), GST-cdc6(K114E)
(lane 2), and GST-cdc6-1 (lane
3), individually, were used for Orc1-matrix interaction as
described under "Experimental Procedures." As controls, aliquot of
bacterial expressed GST-Cdc6 (lane 4),
GST-cdc6(K114E) (lane 5), and GST-cdc6-1
(lane 6) was analyzed by anti-Cdc6 polyclonal
antibodies, indicating that they were expressed about the same level.
C, yeast strain BJ5459 was transformed with
YGp181-His-T7-ORC1 (selection marker Leu2) first.
The resulting strain was then transformed separately with either
YGp123-Cdc6-T7 (lane 1), or YGp123-K114E-T7
(lane 2), or YGp123-cdc6-1-T7 (lane
3) with selection marker Trp1. 500 ml of culture
was induced with 2% galactose for 4 h. The tagged Orc1 was
purified by Ni-NTA matrix, concentrated 10-fold, and probed with
anti-T7 antibody on the protein blots. The T7 monoclonal antibody can
detect not only 6xHis-T7-Orc1 tag protein but also Cdc6-T7, if there is
an interaction between Orc1 and Cdc6 proteins. The upper
arrow indicates the predicted size of Orc1 and the
lower arrow indicates the predicted size of
Cdc6.
|
|
In in vivo interaction studies, 6xHis-T7-Orc1-tagged protein
was expressed in yeast. This strain co-expressed wild-type Cdc6-T7 tag,
mutant cdc6(K114E)-T7 tag, or cdc6-1-T7 tag proteins, separately. The
6xHis-T7-Orc1-tagged protein was isolated by nickel-chelating matrix,
then concentrated, and probed with anti-T7 monoclonal antibody. In Fig.
7C, there is an obvious co-precipitation between Orc1 and
Cdc6 (lane 1), but the interaction is
insignificant in our observation of Orc1 and cdc6(K114E) mutant protein
(lane 2). Again, normal interaction between Orc1
and temperature-sensitive cdc6-1 protein was observed (lane
3), suggesting that the temperature sensitivity of
cdc6-1 follows a defective mechanism different from the one
in ATP-binding mutation. Taken together, the results suggest that ATP
binding may induce or maintain a proper conformation of Cdc6, which is
important for Orc1 interaction. The failure of the proper interaction
may lead to the impairment of replicative complex assembly.
 |
DISCUSSION |
Cdc6 plays an essential role in the initiation of DNA replication.
Little, however, is known about structure/function relationships in the
CDC6 gene. All eukaryotic Cdc6 contains a conserved region known as Walker A-motif, GXXGXGKT (18). This
element may play a role in binding the pyrophosphate moiety of
nucleotides. The Lys residue is essential for electrostatic interaction
between the proteins and the nucleotides. Secondary structural
predictions show that the structure around residues 108-115 in yeast
Cdc6 are consistent with the model for an ATP-binding protein (data not
shown). In order to examine whether the conserved Lys residue represents an essential amino acid residue as in other
nucleotide-binding proteins, we have mutated the Lys residue to five
other amino acids. In general, many proteins with the
GXXGXGKT nucleotide-binding site consensus
sequence possess ATPase activity, including the helicase,
RAD3 (43). In other ATP- and GTP-binding proteins, this
sequence has been suggested to mediate functional conformational change
(45). It has recently been demonstrated that the bGST-Cdc6 can bind ATP
and GTP in vitro. It has also been proposed that Cdc6 is
active as an ATPase (10). The in vivo function(s) of nucleotide binding in Cdc6 is still unclear. Site-directed mutagenesis of the conserved Lys may be useful to delineate its biological function
in vivo. Indeed, we generated five point-mutated cdc6 at
Lys114 of the nucleotide-binding site. We have used four
approaches to explore the effect on the cell functions of various
point-mutants of cdc6: (i) complementation of the temperature-sensitive
strain, (ii) effect on the growth rate, (iii) determination of plasmid stability in the transformant mutants, and (iv) determination of DNA
synthesis in these mutants. The PFGE labeling method to examine DNA
synthesis in cdc6-1 and transformant cells was particularly informative (Fig. 5). Since synthesis on individual chromosomes giving
chromosomal size DNA products is observed directly, it may be that this
technique creates the fewest experimental artifacts among all current
available methods for yeast chromosomal DNA synthesis. Our data clearly
demonstrated that a functional nucleotide binding activity is required
for the CDC6 in DNA synthesis. Our results also demonstrate
that the change of Lys to Glu (K114E) and to Pro (K114P) lead to
loss-of-function in all of these respects; change of the Lys to Gln
(K114Q) and to Leu (K114L) leads to partially functional proteins.
Interestingly, Cdc6(K114R) shows a behavior equivalent to wild-type. No
functional ATPase to date contains an Arg in place of Lys at the same
position (46), implying that nucleotide hydrolysis may not be a major
function of this domain in Cdc6. This leads to the model that
nucleotide binding may be involved in conformational or allosteric
changes in the protein, resulting in sequential events at chromatin in
the G1/S transition of the cell cycle.
How do these observations reconcile with the in vivo
function of initiation of DNA replication? The Cdc6 protein is required for establishment and maintenance of pre-RC, and the Cdc6 protein is
also required for the loading of MCM proteins. In addition, the Cdc6
protein interacts with Cdc28 protein complex after
p40sic1 is degraded at late G1phase
(31). The Cdc28 protein kinase may then activate the initiation of DNA
replication (47). It is generally accepted that a stepwise assembly of
the functional replication complex is required for the coordination of
the DNA replication and cell cycle control (for review, see Ref. 48). It is well established that ATP is essential for prokaryotic DNA replication, but is much less understood in eukaryotic DNA replication. We have systematically investigated the temporal and spatial regulation of the wild-type CDC6 and mutated cdc6(K114E) gene
function(s) to explore the possible involvement of ATP in this process
(Fig. 6). This system is dynamic and useful for understanding the
molecular events from late G1 phase (a stable
protein-origin complex) into the S phase (a mobile DNA machinery).
Indeed, we demonstrated that the impaired function of cdc6(K114E) leads
to an abnormal assembly of the replicative complex. At this stage, the
preparation of replicative complexes was still quite crude, preventing
us from drawing a conclusive picture. Nevertheless, two major findings allowed us to unravel the possible molecular mechanism action of ATP
binding. First, we have demonstrated that there is a direct interaction
between yeast Orc1 and Cdc6 proteins. The information is consistent
with observations from many other laboratories regarding the structure
and functions of the ORC-origin complex, and the interaction between
Cdc6 and Orc1 is conserved in eukaryotes (50, 51). Moreover, we
demonstrated that the interaction between Orc1 and Cdc6 is defective in
the cdc6(K114E) mutation. This observation is original. The simplest
model is that the ATP-binding mutation fails to form Orc1/Cdc6 complex
properly, which results in a deficiency in the formation of an
operational pre-replicative complex. Thus, the requirement of ATP for
Cdc6/Orc1 interaction is involved in a quite early stage of DNA
replication. This possibility is supported by our chromatin studies
(Fig. 6).
In summary, with the availability of these mutants, we have clearly
demonstrated that the in vivo function of the conserved Lys114 in the nucleotide-binding site of CDC6 is
required for cell growth and DNA synthesis. We have also demonstrated
that, at the molecular level, the mutant cdc6 at its nucleotide binding
fails to interact with Orc1. The fact that the ATP-binding domain is
highly conserved among Cdc6 and Orc1 protein families suggests their
similar role in the initiation of DNA replication (18, 19, 47). Perhaps by sequentially contributing ATPase activity or ATP-governed
conformational change to the ATP-dependent ORC/MCM
functions result in the ordered events during initiation of replication
(18, 30, 44). The highly conserved central domain of the MCM and RF-C
family proteins also contains a predicted consensus motif for
DNA-dependent ATPase (49). It is tempting to speculate that
the MCM proteins of the pre-replicative complex, driven by the Cdc6 and
Orc1 ATPase or ATP-governed conformational change, start to unwind the
origin sequence during initiation of replication. ATP binding in the MCM and RF-C families may follow a similar mechanism. Further studies
of the roles of ATP in Cdc6/Orc1, MCM, and RF-C families may reveal
this complicated, yet extremely important regulation in DNA replication
and cell cycle progression.
 |
ACKNOWLEDGEMENT |
We thank Dr. Zhou Chen for help with the
mutagenesis study.
 |
FOOTNOTES |
*
This work was supported by National Institutes of Health
Grant GM48492 and the Neil Bogart Memorial Laboratories of the T. J. Martell Foundation for Leukemia, Cancer and AIDS Research.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Childrens Hospital
Los Angeles, 4650 Sunset Blvd., Mail Stop 57, Los Angeles, CA 90027. Tel.: 323-669-5647; Fax: 323-953-9940; E-mail:
ajong{at}chla.usc.edu.
2
L. Feng and A. Y. Jong, unpublished data.
 |
ABBREVIATIONS |
The abbreviations used are:
CDK, cyclin-dependent kinase;
ARS, autonomously replicating
sequence;
cdc, cell division cycle;
GST, glutathione
S-transferase;
PFGE, pulsed field gel electrophoresis;
PMSF, phenylmethylsulfonyl fluoride;
RC, replication complex;
kb, kilobase pair(s);
HA, hemagglutinin;
LPM, low phosphate medium;
FACS, fluorescence-activated cell sorting.
 |
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