(Received for publication, August 21, 1995; and in revised form, December 4, 1995)
From the
hCDC47 is a putative human homologue of yeast CDC47 and a member
of the MCM protein family, which has been implicated in the regulatory
machinery causing DNA to replicate only once in the S phase. In the
present study, we performed an initial characterization of hCDC47. We
found that hCDC47 protein was present in the nucleus of cultured human
cells in two different forms; one extractable by a non-ionic detergent
and the other resistant to such extraction and tightly associated with
the nucleus. The levels of the nucleus-bound form gradually diminished
during S phase progression, although the total amount of nuclear hCDC47
protein remained relatively constant, suggesting that the nucleus-bound
form becomes dissociated from the nuclear structure during DNA
replication. This behavior of hCDC47 protein is very similar to that of
other mammalian MCM proteins reported recently. We also found that
expression of hCDC47 mRNA was repressed in quiescent cells but was
induced at the late G to S phase by growth factor
stimulation. Together, these findings indicate that hCDC47 protein
together with other MCM proteins participates in the regulation of
mammalian DNA replication.
Replication in eukaryotic cells is a precisely regulated event, which occurs only once in the S phase of the cell cycle to maintain the integrity of the genome. DNA replication proceeds in two stages: initiation of the replication fork and elongation of the new strands. Little is known so far about cellular factors that regulate the initiation of chromosomal DNA replication.
By using mutants that
fail to maintain minichromosomes with autonomously replicating
sequences (ARSs), ()several genes termed MCM have been
isolated in budding yeast(1) . Mutations in MCM affect the
function of each ARS in an ARS-specific manner. This unique phenotype
indicates that MCM gene products are involved in the initiation of DNA
replication at ARSs rather than in the elongation step(2) .
Among them, MCM2, MCM3, and MCM5 (identical to CDC46), together with
the fission yeast cdc21, share striking homology and compose a protein
family implicated in the initiation of DNA replication (3, 4, 5, 6) and conserved even in
higher eukaryotes (7, 8) .
CDC46/MCM5, MCM2, and
MCM3 proteins show unique behavior during the cell cycle; they are
localized in the nucleus during G phase but disappear from
this site at the beginning of the S phase, to reappear at the end of
the M phase(9, 10) . This behavior is reminiscent of
that of the putative replication licensing factor, which was first
proposed from results with an in vitro replication system with Xenopus egg extracts(11) . In this latter model, the
licensing factor can enter the nucleus during nuclear membrane
breakdown in mitosis. It is excluded, however, by an intact nuclear
membrane and allows DNA to replicate only once in the S phase by
evoking the initiation step and afterward being immediately
inactivated.
Recently, a number of mammalian homologues of the MCM family have been identified, including MCM2, MCM3, CDC46/MCM5, and cdc21(7, 12, 13, 14, 15, 16, 17, 18, 19) . Microinjection of antibodies against murine MCM3 (mMCM3) or human MCM2 (hMCM2) protein inhibits cellular DNA synthesis, suggesting that both proteins are related to replication in mammalian cells(13, 14) . mMCM3 protein is present in the nucleus as a soluble form as well as in tight association with nuclear structures, becoming dissociated during S phase(14, 19) . Similar observations were also obtained for hMCM2(20) . Like yeast MCM proteins, mammalian MCM proteins have been also implicated in the regulatory machinery allowing DNA to replicate only once in the S phase(7, 12, 13, 14, 15, 16, 17, 18, 19, 20) .
We have isolated a cDNA, named hCDC47 (human CDC47), encoding a
putative human homologue of budding yeast CDC47 replication protein. ()A part of hCDC47 cDNA was previously identified as
P1.1Mcm3, a member of the mammalian MCM homologues(12) . CDC47
belongs to the yeast MCM family, and its cell cycle-dependent
subcellular localization pattern closely resembles that of other yeast
MCM proteins(21, 22) . In the present study, we
performed an initial characterization of hCDC47.
For
synchronization, exponentially growing WI38 cells were starved of serum
for 48 h prior to addition of fresh medium with 10% FCS. HeLa cells
were synchronized at the G/S boundary by means of a double
thymidine/hydroxyurea block. HeLa cells at log phase were incubated
with 2.5 mM thymidine for 22 h, released for 10 h, and then
treated with 1.5 mM hydroxyurea for a further 16 h. The cells
were washed and incubated in fresh medium with 5% FCS to reenter the
cell cycle.
For affinity purification of anti-hCDC47 antibodies, the glutathione S-transferase-fused hCDC47 protein produced in E. coli was coupled to CNBr-activated Sepharose 4B (Pharmacia) according to the manufacturer's instruction. The antiserum was applied to the glutathione S-transferase-hCDC47 Sepharose column, washed, and eluted.
Rabbit polyclonal antibodies against the
oligopeptide VVCIDEFDKMSDMDRTA derived from hMCM3 protein were
generated by the procedure described by Hu et al.(12) except that bovine serum albumin-peptide
conjugate-coupled Sepharose 4B was used for purification. Antibodies
obtained after two immunizations preferentially reacted against
authentic 105-kDa hMCM3 protein but did not cross-react with other hMCM
members by immunoblotting.
Figure 1:
Expression of hCDC47 mRNA in the cell
cycle after release from serum starvation. Quiescent human fibroblast
WI38 cells, starved in serum-free medium for 48 h, were stimulated by
addition of fresh medium with 10% FCS and harvested at the indicated
times. A, cell cycle pattern analyzed by flow cytometry. B, Northern blots of total RNA (30 µg) probed with a P-labeled hCDC47 cDNA fragment or a rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment
as a control. Positions of 28 S and 18 S rRNA are indicated on the left.
Figure 2:
Characterization of anti-hCDC47 rabbit
antiserum. A, immunoprecipitation from a S-labeled extract of WI38 cells with a preimmune rabbit
serum (lane 1) or anti-hCDC47 antiserum (lane 2). B, immunoblot analysis of whole cell lysates, equivalent to
approximately 1
10
cells, with the anti-hCDC47
antiserum. Lane 1, SiHa; lane 2, C33A; lane
3, WI38. C, partial proteolytic map of hCDC47. In
vitro translated [
S]Met-labeled hCDC47 (lane 1) or the immunoprecipitate obtained from a
[
S]Met-labeled HeLa cell extract with
anti-hCDC47 antibodies (lane 2) was subjected to SDS-PAGE. The
83-kDa doublet bands were excised and subjected to partial digestion
with V8 protease (lanes 3 and 4, 25 ng; lanes 5 and 6, 250 ng). Lanes 3 and 5 were
loaded with in vitro translated hCDC47, and lanes 4 and 6 were loaded with immunoprecipitated hCDC47 from
HeLa cells. Molecular weight markers are shown on the left.
The arrow head indicates hCDC47.
Using the antiserum, we examined whether the total protein levels of hCDC47 changed during the cell cycle along with the mRNA levels in synchronized WI38 cells. hCDC47 protein was clearly identified even in the quiescent WI38 cells starved of serum for 48 h, and, when the cells entered S phase, only a slight increase in the amount of the protein was observed (Fig. 3).
Figure 3:
Levels of hCDC47 protein in the cell cycle
after release from serum starvation. WI38 cells were treated as
described in Fig. 1. Whole cell lysates equivalent to
approximately 1 10
cells were analyzed by
immunoblotting with the anti-hCDC47
antiserum.
Figure 4: Localization of hCDC47 in human cells. Asynchronous WI38 cells were fixed with formaldehyde, permeabilized with 0.2% Triton X-100, and reacted with either the anti-hCDC47 antiserum or the preimmune serum followed by fluorescein isothiocyanate-conjugated goat anti-rabbit IgG antibodies. After the immunostaining, the samples were further treated with RNase and counterstained with PI to identify the nuclei. The samples were then analyzed by confocal microscopy. FITC, fluorescein thiocyanate.
Figure 6:
A, preparation of G/S, mid S,
and late S/G
-enriched HeLa cell populations. HeLa cells
were synchronized at the G
/S boundary by a
thymidine/hydroxyurea double block, released and harvested at 0, 4, and
8 h after release. DNA contents of these cells determined by flow
cytometry are shown. B, localization of hCDC47 protein in HeLa
cells in G
/S and late S/G
phases.
G
/S or late S/G
-enriched HeLa cells were
immunostained with the anti-hCDC47 polyclonal antibodies as described
in Fig. 4and analyzed by conventional
microscopy.
Nuclear mMCM3 protein consists of two forms that can be differentiated by non-ionic detergent extraction(14) . We were, therefore, interested in whether this was also the case for hCDC47. Asynchronously growing WI38 cells were first extracted with hypotonic buffer containing 0.5% Nonidet P-40, and then the pellets were successively extracted with the same buffer containing NaCl at the indicated concentrations. The amount of hCDC47 protein in each sample was determined by immunoblotting (Fig. 5). The supernatant fraction of the Nonidet P-40 extraction contained three-fourths of the total hCDC47 protein amount, and most of the remaining protein in the pellet fraction was extractable with 0.5 M NaCl, demonstrating that hCDC47 protein also has two forms like mMCM3, a form extractable by Nonidet P-40 and the nucleus-bound form.
Figure 5: Differential extraction of hCDC47 protein. WI38 cells were extracted with hypotonic Nonidet P-40 buffer to obtain the Nonidet P-40-extractable fraction (NP-40/sup). The remaining pellet fraction (NP-40/pellet) was extracted with Nonidet P-40 buffer adjusted to the indicated NaCl concentration. The amounts of hCDC47 protein in each sample were determined by immunoblot analysis with the anti-hCDC47 antiserum.
Nonidet P-40-extracted supernatant and
remaining pellet fractions were prepared from these three populations
and subjected to SDS-PAGE followed by Coomassie Blue staining. The
extraction efficiency was confirmed to be virtually equal (data not
shown). Examination of the levels of hCDC47 protein in these samples by
immunoblotting with the anti-hCDC47 antiserum demonstrated no great
variation through the S phase in the supernatant fraction, which
contained three-fourths of the total hCDC47 protein in the
G/S population. In contrast, the levels of hCDC47 protein
in the pellet fraction decreased remarkably along with progression
through the S phase (Fig. 7). In the late
S/G
-enriched population, the amount of the nucleus-bound
hCDC47 was one-fifth that in the G
/S population. With
parallel immunoblotting, the levels of hMCM3 protein in the pellet
fraction were found to be coincidentally decreased with that of hCDC47
during the S phase (Fig. 7).
Figure 7:
Dissociation of the nucleus-bound hCDC47
protein during the S phase in HeLa cells. G/S, mid S, and
late S/G
-enriched HeLa cell populations, prepared as in Fig. 6A, were extracted with hypotonic Nonidet P-40
buffer to obtain the Nonidet P-40-extractable fraction (NP-40/sup) and
the remaining pellet fraction (NP-40/pellet). Each fraction was
immunoblotted with anti-hCDC47 antibodies, anti-hMCM3 antibodies, or
anti-PCNA monoclonal antibody. The loaded amount of the Nonidet
P-40-extractable fraction (equivalent to approximately 1
10
cells/lane) was set at one-third that of the Nonidet
P-40/pellet fraction (approximately 3
10
cells/lane).
PCNA, the auxiliary protein of
DNA polymerase required for DNA
replication(26, 27) , also has two forms that can be
differentiated by non-ionic detergent extraction. The detergent
extraction-resistant form is probably associated with DNA replication
sites and is thought to play a fundamental role in DNA
synthesis(28) . To establish whether the change in nuclear
association of hCDC47 and hMCM3 during S phase is specific for these
MCM proteins, we examined the levels of nucleus-bound PCNA with
parallel immunoblotting (Fig. 7). PCNA also showed cell
cycle-regulated association with the nucleus, but the kinetics of
change proved to be different from those of hMCM proteins. The amount
of the nucleus-bound PCNA was most abundant in the mid S-enriched
population, and in the late S/G
-enriched population it
decreased to only two-thirds that in the G
/S population.
Therefore, the changing nuclear association pattern of hMCM proteins
seems to be specific for them.
hCDC47 is a putative human homologue of yeast CDC47 (21, 22) and a member of the MCM protein family(7, 12, 13, 14, 15, 16, 17, 18, 19, 20) . In this study, we investigated the biological properties of hCDC47. Immunochemical analysis of cultured human cells showed that hCDC47 protein is present in the nucleus in two different forms: one extractable by Nonidet P-40 and the other resistant to Nonidet P-40 extraction and thus tightly associated with the nucleus. In synchronized HeLa cells, the levels of the nucleus-bound form were gradually diminished during S phase progression, although the total amount of nuclear hCDC47 did not vary greatly, suggesting that the nucleus-bound hCDC47 is dissociated from nuclear structures during DNA replication. Although the question of whether the findings for hCDC47 observed in HeLa cells, which are transformed, can be generalized for normal cell cycle regulation remains, similar change in nuclear association of mammalian MCM3 to that found for hCDC47 has been observed not only in HeLa cells(18, 19) but also in untransformed murine Swiss 3T3 cells(14) . Overall, these findings for hCDC47 closely resemble those reported for other mammalian MCMs(14, 18, 19, 20) . Very recently, a Xenopus MCM (XMCM) complex including XMCM2, -3, and -5 was reported as a component of the licensing factor for replication in the Xenopus egg extract (19, 29, 30) . In this system, only chromatin-bound XMCMs are active for the initiation of replication, becoming detached as replication proceeds. By analogy with the Xenopus system, the nucleus-bound form of mammalian MCMs including hCDC47 may be required for DNA replication. Recently, it has also been demonstrated that MCM proteins form complexes with each other in mammalian cells(16, 17, 18) , which may be similarly regulated and function in the regulation of DNA replication. The precise role of the MCM proteins, however, still remains mostly unknown. In the case of yeast, different MCM mutants are arrested at different points of S phase, which indicates specific and indispensable functions of each MCM for DNA replication(3, 4, 6, 9, 21) . It is very informative that the phenotype of mammalian cells in which each MCM is repressed be elucidated.
In higher eukaryotes, MCM
proteins may play a regulatory role through their
association-dissociation with the nuclear structure. Kimura et al.(14) has proposed that association of mMCM3 with the
nuclear structure is regulated by its phosphorylation(14) . On
the other hand, it has been reported that mCDC46 coprecipitating with
mMCM3 is not phosphorylated(14, 16, 18) .
Furthermore, while hcdc21 coprecipitating with hCDC47 is
phosphorylated(17) , hCDC47 was not evidently phosphorylated
when the cells were labeled in vivo during S phase. The mechanisms regulating the nuclear association of MCM proteins
thus remain to be clarified.
In the present study, hCDC47 mRNA was
not expressed in quiescent cells but was expressed at the late G to S phase, as reported for other mammalian MCM genes (7, 15, 18) and also for other DNA
synthesis-related genes. The 5`-region of the mMCM3 gene has two
binding sites for E2F(14) , which is considered to control the
expression of some genes involved in DNA synthesis(31) . We
also detected two E2F sites in the 5`-region of the hCDC47
gene
, and thus the expression of mammalian MCMs might be
simultaneously regulated by factor(s) such as E2F. In contrast to the
mRNA expression, the fluctuation in hCDC47 protein levels appeared
small during the cell cycle, which is in agreement with the findings
for expression of other mammalian MCMs during the cell
cycle(15, 18) . One simple and possible explanation
for the mRNA/protein difference may be that hCDC47 is a particularly
abundant and stable protein like hMCM3(15, 16) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D55716[GenBank].