Synthesis and turn-over of the replicative Cdc6 protein
during the HeLa cell cycle
Esther Biermann, Martina Baack, Sandra Kreitz and Rolf Knippers
Department of Biology, Universita
¨t Konstanz, Germany
The human replication protein Cdc6p is translocated from
its chromatin sites to the cytoplasm during the replication
phase (S phase) of the cell cycle. However, the amounts of
Cdc6p on chromatin remain high during S phase implying
either that displaced Cdc6p can rebind to chromatin, or that
Cdc6p is synthesized de novo. We have performed metabolic
labeling experiments and determined that [
35
S]methionine is
incorporated into Cdc6p at similar rates during the G1 phase
and the S phase of the cell cycle. Newly synthesized Cdc6p
associates with chromatin. Pulse–chase experiments show
that chromatin-bound newly synthesized Cdc6p has a half
life of 2–4 h. The results indicate that, once bound to
chromatin, pulse-labeled new Cdc6p behaves just as old
Cdc6p: it dissociates and eventually disappears from the
nucleus. The data suggest a surprisingly dynamic behaviour
ofCdc6pintheHeLacellcycle.
Keywords: cell cycle; DNA replication; hCdc6; phospho-
rylation; turn-over.
The eukaryotic replication initiation protein Cdc6 (Cdc6p)
is a member of the large AAA
+
family of ATPases [1]. Like
other members of this family, Cdc6p possesses a bipartite
purine nucleoside triphosphate binding domain consisting
of the conserved Walker A and Walker B motifs. In
addition, Cdc6p contains several potential phosphorylation
sites in the N-terminal region. Cdc6p is required for the
formation of pre-replicative complexes and therefore essen-
tial for replication initiation in eukaryotic cells.
Pre-replicative complexes are assembled in a stepwise
manner during the G1 phase of the eukaryotic cell cycle.
Cdc6p associates with the chromatin-bound six-subunit
origin recognition complex (ORC) and promotes, together
with the Cdt1 protein [2,3], the subsequent loading of the
Mcm protein complex. The fully assembled pre-replicative
complex is induced to activate replication origins by at least
two classes of protein phosphorylating enzymes, cyclin-
dependent kinases (Cdk) and the Dbf4-Cdc7 kinase [4–6].
In yeasts, Cdc6p is expressed during the G1 phase [7,8],
associates with stationary ORC [9,10] and loads Mcm
initiation proteins in reactions requiring an intact nucleotide
binding domain [11–13]. Once replication begins, yeast
Cdc6p is phosphorylated and then rapidly destroyed by
ubiquitin-mediated protein degradation. The regulated
destruction of Cdc6p effectively prevents the binding of
Mcm proteins, and therefore prevents the re-replication of
DNA sections that had already replicated during the same S
phase [14–22]. In fact, overexpression of the wild-type
Cdc6p homolog (cdc18) in the yeast Schizosaccharomyces
pombe [17], and certain mutant alleles of the Saccharomyces
cerevisiae gene CDC6 induce the repeated activation of
replication origins within one cell cycle [23]. Normally,
however, the amounts of Cdc6p fluctuate across the yeast
cell cycle. They rapidly decrease with the entry of yeast cells
into S phase and increase again during the following G1
phase with the synthesis of new Cdc6p.
In contrast, the rapid S-phase-related elimination of
Cdc6p that is characteristic for the yeast cell cycle does not
occur in mammalian cells, and levels of human Cdc6p
(hCdc6p) in cycling human cells remain fairly stable during
S phase, G2 phase and mitosis [24–27], but lower amounts
of hCdc6p are present in early G1 phase cells when hCdc6p
is rapidly degraded by ubiquitin-dependent proteolysis
[28,29]. Although more recent data suggest that the reported
rapid degradation could be an extraction artefact [30].
Nuclear hCdc6p is phosphorylated during S phase
[27,31,32] and transported to the cytoplasm [31]. However,
at the same time, a considerable portion of hCdc6p is found
to be bound to chromatin [29], and it has been argued that
hCdc6p does not only serve as a loading factor for Mcm
proteins in human cells, but performs additional functions
during replication. This was concluded because ectopic
expression or microinjection of mutant hCdc6p lacking the
phosphorylation sites interferes with DNA replication
[27,32]. A continuous requirement of hCdc6p for mamma-
lian genome replication may explain why hCdc6p is present
until the end of a cell cycle.
The fact that the amounts of hCdc6p on chromatin
remain fairly constant during S phase while considerable
fractions are translocated to the cytoplasm implies that
enough hCdc6p must always be synthesized to replace the
fraction of hCdc6p that dissociates from chromatin during
S phase. To investigate this possibility we have metabolically
labeled hCdc6p and followed its fate in cycling HeLa cells
by pulse–chase experiments. We found that hCdc6p is
synthesized at similar rates during various stages of the cell
cycle, and determined a half life of newly synthesized
hCdc6p of 2–4 h in S phase. The data suggest a surprisingly
dynamic behaviour of hCdc6p in the HeLa cell cycle.
Correspondence to E. Biermann, Department of Biology, Universita
¨t
Konstanz, D-78457, Konstanz, Germany. Fax: + 49 7531 88 4036,
Tel.: + 49 7531 88 2127,
E-mail: Esther.Biermann@uni-konstanz.de
Abbreviations: ORC, origin recognition complex.
(Received 22 October 2001, revised 17 December 2001, accepted
19 December 2001)
Eur. J. Biochem. 269, 1040–1046 (2002) ÓFEBS 2002
EXPERIMENTAL PROCEDURES
Cell culture
Human HeLa S3 cells were grown on plastic dishes in
Dulbecco’s modified Eagle’s medium plus 5% fetal bovine
serum. Cells were synchronized at the beginning of S phase
by a double-thymidine procedure (12 h in 2.2 m
M
thymi-
dine; 9 h without thymidine; 14 h in 2.2 m
M
thymidine) or at
mitosis with a nocodazole block (12 h in 2.2 m
M
thymidine;
9hrelease;3hat40ngÆmL
)1
nocodazole). The block was
released by washing cells three times with medium.
For metabolic labeling, cells on 94-mm plates were
washed with methionine-free medium (Gibco, Life Tech-
nologies) and labeled with 200 lCi [
35
S]methionine (ICN)
for 2 h in 5 mL methionine-free medium with dialysed
bovine serum. For a chase, the radioactive medium was
removed, and cells were washed several times with normal
medium and then grown under standard conditions.
For proteasome inhibition, HeLa cells were synchronized
by a double thymidine-block and released into fresh
medium with 5 l
M
MG-132 (Calbiochem) for 6 h.
Cell fractionation
Cells were washed with phosphate-buffered saline (NaCl/P
i
)
and suspended in buffer A (20 m
M
NaCl; 5 m
M
MgCl
2
;
1m
M
ATP; 20 m
M
Hepes, pH 7.5). After 15 min on ice and
douncing, cells were centrifuged to separate the cytosolic
supernatant from the nuclear pellet. Nuclei were resus-
pended in buffer A with 0.5% NP40 and kept on ice for
15 min to lyse the nuclear envelope. Centrifugation yielded
supernatant nucleosolic proteins and an insoluble nuclear
pellet including chromatin. To dissociate bound proteins,
the nuclear pellet was washed with buffer B (0.3
M
sucrose;
0.5 m
M
MgCl
2
;1m
M
ATP; 20 m
M
Hepes, pH 7.5) plus
NaCl in concentrations of 0.1–0.45
M
as indicated below.
All extraction buffers contained phosphatase inhibitors:
1m
M
NaF, 1 m
M
vanadate and an EDTA-free protease
inhibitor cocktail in concentrations suggested by the man-
ufacturer (Roche Molecular Biochemicals).
For nuclease treatment, nuclei, prepared as above, were
resuspended in buffer B supplemented with 2 m
M
CaCl
2
and 100 m
M
NaCl and incubated for 10 min with 30 U
micrococcal nuclease at 14 °C. Digested chromatin was
recovered in the supernatant (S1) of low speed centrifuga-
tion. The pellet was resuspended in 5 m
M
EDTA and again
centrifuged to obtain supernatant S2 and a pellet [33,34].
The supernatants and pellets were investigated by Western
blotting using hCdc6p-specific antibodies (see below) and
used for the extraction of DNA. Extracted DNA was
analysed by PAGE and ethidium bromide staining.
Preparation and use of antibodies
A cDNA sequence encoding a 30-kDa-fragment (amino-
acid residues 278–561) of hCdc6p was cloned in the
expression vector pRSET (Invitrogen) and expressed in
bacteria. The purified polypeptide was used as an antigen to
raise antibodies in rabbits. Monospecific antibodies were
prepared from the crude antisera by affinity chromatogra-
phy with the antigen immobilized on the SulfoLink gel
(Pierce).
For immunoblotting (Western blotting), proteins were
first separated on a 7.2% denaturing polyacrylamide gel and
then transferred onto a Protran nitrocellulose transfer
membrane (Schleicher and Schuell). For staining, hCdc6p-
monospecific antibodies (0.22 lgÆlL
)1
)wereusedina
1 : 200 dilution and visualized by goat anti-rabbit Ig
(Jackson Immuno Research) with the enhanced chemi-
luminescence system (ECL) as suggested by the manu-
facturer (Amersham Pharmacia Biotech).
Immunoprecipitations were performed with extracts
from 4–6 ·10
6
cells incubated with 2 lg hCdc6p-specific
antibodies for 1 h on ice. Protein A–Sepharose beads (50 lL
of a 50% suspension; Amersham, Pharmacia Biotech) were
then added for 1 h. The immunocomplexes were precipi-
tated and washed several times with 0.45
M
NaCl in buffer B
on ice. Proteins were eluted in Laemmli electrophoresis
buffer and investigated by denaturing PAGE as above.
Phosphatase treatment
Immunocomplexes were washed first with 0.45
M
NaCl in
buffer B as above and then with phosphatase buffer
(100 m
M
NaCl; 0.1 m
M
MnCl
2
;0.1m
M
EGTA; 50 m
M
Tris/HCl, pH 7.5). Treatment with lambda protein phos-
phatase (400 U; New England BioLabs) was in 0.05 mL
bufferfor30 minoniceand30 minat30 °C under shaking.
The immunocomplexes were then washed in 0.45
M
NaCl
buffer B and processed for electrophoresis as described
above.
RESULTS
HCdc6p on chromatin
We have prepared monospecific antibodies against recom-
binant hCdc6 protein. To demonstrate their specificity and
efficiency, we present immunoblotting (Western) experi-
ments showing that the antibodies specifically recognize the
antigen in crude extracts of bacteria expressing his-tagged
hCdc6p (Fig. 1A, lane 1). Western blots of whole protein
extracts from HeLa cells frequently resulted in two bands
(Fig. 1A, lane 2), but, as control experiments showed, only
the upper band corresponded to hCdc6p whereas the lower
of the two bands was unspecific (because the secondary
mouse anti-rabbit Ig react with an unknown cellular protein
(Fig. 1A, lane 3). We analysed by immunoblotting the
distribution of hCdc6p in the cytoplasm as well as in the
fractions of soluble (nucleosolic) and structure-bound
nuclear proteins (chromatin) from asynchronously prolifer-
ating HeLa cells. Chromatin-bound hCdc6p could be
mobilized in buffers with 0.25–0.45
M
NaCl (Fig. 1B) and
was effectively immunoprecipitated by hCdc6p-specific
antibodies (Fig. 1C).
We note that 0.25–0.45
M
NaCl is also required to
dissociate human Orc proteins from chromatin [34], and the
question arises whether human Orc1p and hCdc6p in the
salt-extracts are bound to each other as both proteins are
known to physically interact under in vitro conditions
[25,32]. However, we were unable to detect coimmunopre-
cipitations of hCdc6p and hOrc1p (or other Orc proteins)
using either hCdc6p- or hOrc1p-specific antibodies (not
shown). This does not exclude the possibility that the two
proteins interact when bound to chromatin. In fact, we
ÓFEBS 2002 The fate of hCDC6p during the HeLa cycle (Eur. J. Biochem. 269) 1041
determined that hCdc6p resides in a nuclease-resistant
compartment of chromatin (data not shown) [26] just like
hOrc1p and hOrc2p as previously shown [34]. It is therefore
possible that hCdc6p together with other replication
initiation proteins occur in large protein complexes that
protect DNA against nuclease attack, but dissociate at high
salt concentrations (Fig. 1B).
In the experiments reported below, we prepared HeLa
cell extracts as in Fig. 1B and separated a cytosolic fraction
from the nuclear fraction which was then treated with
0.45
M
NaCl to mobilize chromatin-bound hCdc6p. The
presence of hCdc6p in these preparations was determined by
immunoprecipitation.
Rates of hCdc6p synthesis
To investigate whether the synthesis of hCdc6p was
restricted to specific phases of the cell cycle, HeLa cells
were arrested by a double-thymidine procedure at the G1
phase/S phase transition, and then released into the cycle
after removing excess thymidine. Cells were labeled with
[
35
S]methionine for 2 h at the beginning (0–2 h after
thymidine-block) and in the middle of S phase (4–6 h), as
well as at the end of mitosis and during the early G1 phase
(12–14 h) of the next cycle (Fig. 2A).
Cytoplasmic and chromatin extracts were immunopre-
cipitated with specific antibodies and transferred to nitro-
cellulose membranes. Immunostaining showed that cells in
all cell cycle phases possess substantial amounts of chroma-
tin-bound hCdc6p although the amount of chromatin-
bound hCdc6p appeared to be lower in early G1-phase
(Fig. 2B, right) [28,29]. Cytoplasmic hCdc6p was detected
mainly in S-phase cells in agreement with previous work
which had shown that hCdc6p dissociates from chromatin
and migrates to the cytoplasm during S phase [25,31] (see
introduction) (Fig. 2B, left).
The membranes used for Western blotting were washed
to remove the ECL reagent, dried and exposed to X-ray
films for autoradiography. The results show that similar
amounts of [
35
S]methionine were incorporated into hCdc6p
during the cell cycle phases tested. Moreover, in all cases the
incorporated radioactivity was almost evenly distributed
between the cytoplasmic and the chromatin fractions of
hCdc6p (Fig. 2C).
We conclude that hCdc6p was synthesized during the
four cell-cycle stages examined, and that about one half of
the newly synthesized hCdc6p associated with chromatin
during the 2-h label period.
Fig. 2. Synthesis of hCdc6p. HeLa cells (1 ·10
7
), arrested by a double-
thymidine block, were released into the cell cycle. At the times indi-
cated, 4 ·10
5
HeLa cells were incubated with 15 lg propidium iodide
in 0.3 mL phosphate-buffered saline with 0.1% Triton X-100 for
30minoniceandprocessedforFACSanalysis(A).Theremaining
cells were labeled with [
35
S]methionine for 2 h and then fractionated to
prepare cytosol (Cy) and nuclear extracts at 0.45
M
NaCl. Extracts
were immunoprecipitated. Precipitated proteins were analysed by
Western blotting (B) and autoradiography (C).
Fig. 1. Characterization of antibodies and cell fractionation. (A) Identification of hCdc6p by immunoblotting. Lane 1, His-tagged recombinant
hCdc6p; lane 2, nuclear extracts prepared at 0.45
M
NaCl stained with hCdc6p-specific antibodies; lane 3, as in lane 2 except that only the secondary
antibody was used. (B) Cell fractionation (see Experimental procedures). Cy, cytosol; Nu, soluble nuclear proteins (nucleosol); last three lanes,
extracts prepared with 100, 250 and 450 m
M
NaCl from NP40-treated nuclei. The experiment was performed with 2 ·10
6
HeLa cells. Five hundred
nanograms of protein per lane were investigated by immunoblotting. (C) Immunoprecipitation. A nuclear extract (2 ·10
6
cells) prepared at
450 m
M
NaCl (input) was treated with 2 lg antibodies for immunoprecipitation. Equal aliquots of the supernatants and the immunoprecipitates
were immunoblotted and stained with hCdc6p-specific antibodies.
1042 E. Biermann et al. (Eur. J. Biochem. 269)ÓFEBS 2002
In most experiments, soluble labeled hCdc6p in S phase
appeared in two electrophoretic bands (Fig. 2C, left). The
labeled hCdc6p species in the upper band was phosphory-
lated because phosphatase-treatment converted it into the
faster moving species (Fig. 3B). In contrast, the labeled
hCdc6p in early G1-phase always appeared in one faster
moving electrophoretic band (Fig. 2C, left panel) and was
therefore un- or underphosphorylated. The changes in the
electrophoretic mobilities of phosphatase-treated prepara-
tions indicate that not only labeled cytoplasmic hCdc6p, but
also labeled chromatin-bound hCdc6p appears to be
phosphorylated during S phase (Fig. 3B) [26,28].
As in Fig. 3A,B we have repeatedly observed in other
similar experiments that more hCdc6p can be immunopre-
cipitated from phosphatase-treated nuclear extracts than
from control extracts of S-phase HeLa cells. As an
explanation, we considered the possibility that the phos-
phorylated form of hCdc6p was prone to degradation
during the in vitro incubation. This could be due to
proteasome-mediated degradation. To investigate this pos-
sibility, we treated HeLa cells with the proteasome-inhibitor
MG-132 prior to the preparation of nuclear extracts. In
these extracts, the phosphorylated hCdc6p in the control
sample was at least as stable as the phosphatase-treated
hCdc6p (Fig. 3C) suggesting that nuclear extracts from
S-phase HeLa cells contain a proteasome-related activity that
preferentially attacks the phosphorylated form of hCdc6p.
However, more importantly in the present context, we
note that the synthesis of hCdc6p continues through most of
the cell cycle. Synthesis of hCdc6p during G1 phase is
necessary for the formation of pre-replicative complexes,
whereas synthesis during S phase may be needed to replace
that fraction of hCdc6p that is transferred to the cytoplasm
and eventually degraded. It can therefore be predicted that
the amounts of hCdc6p on chromatin increase during G1
phase, but remain unchanged during S phase because
de novo synthesis compensates for the S-phase-dependent
loss of hCdc6p.
We have investigated this point using HeLa cells released
from a nocodazole block into G1 and S phase (Fig. 4A). We
found a gradual increase of chromatin-bound hCdc6p
during G1 phase followed by a decrease in early S phase
[24]. With the continuation of S phase, however, the amount
of hCdc6p on chromatin remained constant (Fig. 4C)
implying that newly synthesized hCdc6p (Fig. 2) first
associates with chromatin, and then turns over like old
hCdc6p. We have addressed this point performing pulse–
chase experiments.
Fate of newly synthesized hCdc6p
HeLa cells were released from a double-thymidine block
and labeled with [
35
S]methionine for 2 h. The radioactive
medium was then removed and replaced by standard culture
medium. Cells were collected immediately after the 2-h-
pulse and after cultivation for several hours in medium with
excess methionine. Note that a 2-h-pulse-period under
methionine-free conditions causes a delay in cell cycle
progression with the consequence that cells are still in
S phase after a 8-h chase period (not shown).
We present an experiment where the label period was 4–6
h after release from the thymidine block followed by chase
periods of 4 and 8 h (Fig. 5). Total hCdc6p, as determined
by Western blotting, was similar in the pulse and in the
chase samples (Fig. 5A) whereas
35
S-labeled hCdc6p
decreased during the chase period (Fig. 5B).
Just as shown in Fig. 2, about one half of the pulse-label
appeared in cytoplasmic hCdc6p, and the other half in
chromatin-bound hCdc6p. The phosphorylated upper-band
form of labeled cytoplasmic hCdc6p rapidly disappeared
during the chase (half life < 2 h, Fig. 5B, left). It can
assumed that part of the labeled cytoplasmic hCdc6p moved
into the nucleus and bound to chromatin, but another part
may have remained in the cytoplasm to be degraded or
Fig. 4. Soluble and chromatin-bound hCdc6p. Cells were arrested by
nocodazoleandthenreleasedintoG1andSphaseasmonitoredby
FACS analysis (A). Soluble nuclear and chromatin-bound proteins
(450 m
M
NaCl) were investigated by denaturing polyacrylamide gel
electrophoresis. Coomassie staining (B) served as a loading control and
Western blotting (C) to analyse for hCdc6p.
Fig. 3. Phosphorylated hCdc6p in S phase. HeLa cells (1 ·10
7
)were
released from a double-thymidine block, and labeled for 2 h with
[
35
S]methionine. Immunoprecipitates of cytosolic proteins (Cy) and of
nuclear extracts (450 m
M
NaCl) were incubated with buffer only or
with buffer plus lambda phosphatase as indicated. The proteins were
analysed by Western blotting (A) and autoradiography (B). Cells were
released from a double-thymidine block as above, but cultivated for 6 h
in the presence of the proteasome-inhibitor MG-132 before cell frac-
tionation. Immunoprecipitates of cytosolic and nuclear proteins were
treated with phosphatase and investigated by Western blotting (C).
ÓFEBS 2002 The fate of hCDC6p during the HeLa cycle (Eur. J. Biochem. 269) 1043
converted into the more phosphorylated form (half life:
4 h) (Fig. 5B, left).
In either case, the amount of labeled chromatin-bound
hCdc6p decreased during the chase with an estimated half
life of 2–4 h (Fig. 5B, right). This value appears to be similar
for cytosolic and salt-extracted hCdc6p.
We have performed several pulse–chase experiments and
quantitated the results by densitometry to determine the
relative strengths of the autoradiographic signals in labeled
chromatin-associated hCdc6p. With the pulse value taken
as 100% we determined that half of the labeled chromatin-
bound hCdc6p disappears during chase periods of 2–4 h
(Fig. 6).
A likely explanation is that a fraction of labeled
cytoplasmic hCdc6p is transferred to chromatin where it
shares the fate of old hCdc6p, namely dissociation, trans-
port to the cytoplasm and degradation. Continued protein
synthesis guarantees that the amount of chromatin-associ-
ated hCdc6p remains high.
DISCUSSION
We show here that [
35
S]methionine is incorporated into
hCdc6p of HeLa cells at various times after release from a
thymidine-block, and conclude that hCdc6p is newly
synthesized at similar rates during most stages of the HeLa
cell cycle. This information adds to the growing knowledge
of hCdc6p expression in mammalian cells.
The expression of hCdc6p in mammalian cells is strictly
associated with cell proliferation. Quiescent mammalian
cells fail to express Cdc6 mRNA and protein, but readdition
of serum to serum-starved cells and dilution of contact-
inhibited primary cells induce the expression of Cdc6p
[24,35,36]. This reaction is controlled by E2F transcription
factors [35] which are responsible for the expression of a
large number genes involved in DNA replication. Once
proliferation has been initiated, mammalian cells express
Cdc6 mRNA at all stages of the cell cycle with a several fold
increase in mRNA abundance at the onset of S phase
[25,28].
Consistent with the continuous presence of mRNA, levels
of Cdc6p remain high in poliferating human cells at most
stages of the cell cycle [24,25,27,35,37] although more recent
experiments suggest that the levels of hCdc6p may below in
early G1 cells due to the mitotic destruction of most hCdc6p
[28–30]. In spite of this, chromatin from nocodazole-
arrested HeLa cells still carries some hCdc6p (Fig. 4),
which is apparently required for an early loading of Mcm
proteins [29]. The amount of hCdc6p on chromatin
increases after removal of nocodazole as cells traverse the
G1 phase [28,29] (Fig. 4). This was demonstrated here by
the incorporation of [
35
S]methionine.
More surprisingly, hCdc6p continues to be synthesized at
similar rates during S phase (Fig. 2). The behaviour of
mammalian Cdc6p during S phase has been investigated
over the past few years. Williams et al. [24] have noted that
chromatin-bound hCdc6p decreases in S phase, and Saha
et al. [25] found hCdc6p in the nucleus during pre-replica-
tion phase, and in the cytoplasm after origins had started to
fire in S phase. The subcellular distribution of Cdc6p during
the cell cycle is most likely regulated by phosphorylation [27]
involving the cyclin A-dependent protein kinase CDK2
which has been shown to bind to and specifically phospho-
rylate mammalian Cdc6p [31,38]. However, in spite of the
S-phase-related nuclear–cytoplasmic transfer, substantial
amounts of mammalian Cdc6p remain on chromatin [29,39]
(Fig. 4). One reason for this is that hCdc6p is synthesized at
high rates during S phase (Fig. 2). In fact, we found that the
amounts of pulse-labeled Cdc6p on chromatin were similar
in G1 phase and S-phase cells. This result suggests that the
fraction of ÔoldÕhCdc6p that dissociates from chromatin
and is transferred to the cytoplasm during S phase is at least
partially replaced by newly synthesized hCdc6p.
Once bound to chromatin, pulse-labeled new hCdc6p
behaves just as old hCdc6p, i.e. it dissociates and eventually
disappears from the nucleus with a half life of < 4 h
(Fig. 6). This is substantially longer than the half life of
30 min measured for hCdc6p at the mitosis/G1 phase
transition when a sudden massive destruction of Cdc6p
Fig. 6. Half-life of chromatin-bound hCdc6p in S phase. HeLa cells
were labeled with [
35
S]methionine for 2 h immediately after release
from a double-thymidine block and chased for the times indicated
(squares). Proteins were extracted with 450 m
M
NaCl from chromatin
and analysed by immunoprecipitation and autoradiography. The
autoradiographic bands were evaluated by densitometry with the pulse
values taken as 100%. Deviation bars give averages of three inde-
pendent experiments. The results of the experiment in Fig. 5 are
included (circles).
Fig. 5. The fate of labeled hCDC6p. HeLa cells were first labeled with
[
35
S]methionine at 6 h after release from a double-thymidine block and
then chased for 4 and 8 h as indicated. Cytosolic (Cy) and chromatin-
associated (450 m
M
NaCl) proteins were prepared and investigated by
immunoprecipitation. We show Western blot (A) and autoradiogra-
phy (B) of the immunoprecipitates.
1044 E. Biermann et al. (Eur. J. Biochem. 269)ÓFEBS 2002