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 a t similar rates during the G1 phase
and the S phase of the cell cycle. Newly synthesized Cdc6p
associates with chromatin. Pulse–chase e xperiments show
that chromatin-bound newly synthesized Cdc6p has a half
life of 2 –4 h. The r esults indicate that, once bound to
chromatin, pulse-labeled n ew C dc6p 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

cell cycle. They rapidly decrease w ith the entry of yeast cells
into S phase and increase again during t he 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 i n 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 a mounts
of hCdc6p are present in early G 1 phase cells when hCdc6p
is rapidly degraded by ubiquitin-dependent proteolysis
[28,29]. Although more recent data suggest that t he 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 l oading factor for M cm
proteins in human cells, but performs additional functions
during replication. This was concluded because ectopic
expression or microinjection of mutant hCdc6p lacking the
phosphorylation s ites interferes with DNA replication
[27,32]. A continuous requirement of hCdc6p for mamma-
lian genome replication may explain w hy hCdc6p is present
until the end of a c ell 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 t hat
enough hCdc6p must always be synthesized to replace the
fraction of hCdc6p that dissociates from chromatin during
S phase. T o investigate this possibility we have m etabolically

9hrelease;3hat40ngÆmL
)1
nocodazole). The block was
released by washing c ells three times with medium.
For metabolic labeling, cells on 94-mm plates were
washed with methionine-free med ium (Gibco, Life T ech-
nologies) and labeled with 20 0 lCi [
35
S]methionine (ICN)
for 2 h in 5 mL methionine-free medium with d ialysed
bovine serum. For a chase, t he radioactive medium was
removed, and cells were washed several times with normal
medium and then grown un der standard conditions.
For proteasome inhibition, HeLa cells were synchronized
by a double t hymidine-block and released into f resh
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

vanadate and an EDTA-free protease
inhibitor cocktail in concentrations suggested by the man-
ufacturer (Roche Molecular B iochemicals).
For n uclease 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 1 4 °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 S 2 a nd 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. E xtracted DNA was
analysed by PAGE and ethidium bromide st aining.
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) a nd expressed in
bacteria. The purified polypeptide was used as an antigen to
raise a ntibodies in rabbits. Monospecific antibodies were
prepared from the crude antisera by affinity chromatogra-
phy with the antigen immobilized on the SulfoLink gel
(Pierce).

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
bufferfor30minoniceand30minat30°C under shaking.
The immunocomplexes were then washed in 0.45
M
NaCl
buffer B and processed f or electrophoresis as described
above.
RESULTS
HCdc6p on chromatin
We have prepared monospecific antibodies against recom-
binant hCdc6 protein. T o demonstrate their specificity and
efficiency, we presen t immunoblotting (Western) experi-
ments showing t hat the antibodies specifically recognize the
antigen in crude extra cts of bacteria expressing his-t agged
hCdc6p (Fig. 1A, lane 1). Western blots o f whole protein
extracts from HeLa cells frequently resulted in two bands
(Fig. 1A, lane 2), but, as control e xperiments 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 i mmunoblotting the

cell extracts a s in Fig. 1B and separated a cytos olic fraction
from the nuclear fraction w hich w as t hen t r eated w ith
0.45
M
NaCl to mobilize chromatin-bound h Cdc6p. The
presence of hCdc6p in these preparations was determined by
immunoprecipitation.
Rates of hCdc6p synthesis
To investigate w hether the synthesis of hCdc6p was
restricted to specific phases of t he c ell cyc le, He La cells
were arrested by a double-thymidine procedure at the G1
phase/S phase transition, and then released i nto the cycle
after removing e xcess 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 e nd of mitosis and during the early G1 phase
(12–14 h) of the next cycle (Fig. 2A).
Cytoplasmic and chromatin e xtracts were immunopre-
cipitated with s pecific antibodies an d transferred to n itro-
cellulose membranes. Immunostaining showed that cells in
all cell cycle phas es possess substantial amounts o f 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 p revious work
which had shown that hCdc6p dissociates from chromatin
and migrates to the cytoplasm during S phase [25,31] (see
introduction) (Fig. 2B, left).

Western blotting (B) and autoradiography (C).
Fig. 1. Characterization of antibodies and cell fractionation. (A) Identification of hCdc6p by immunoblo tting. Lane 1, H is-tagged recombin ant
hCdc6p; l ane 2, nuclear extracts pre pared at 0.45
M
NaCl staine d with h Cdc6p-spec ific antibodies; lan e 3, as in lane 2 except that only t he secondary
antibody w as used. (B) Cell fractionation (see Experimen tal procedures). Cy, cytosol; Nu, soluble nuclear proteins (nucleo sol); last three l anes,
extracts prep ared with 100, 250 and 450 m
M
NaCl from NP40-treated nuclei. The experiment w as performed with 2 · 10
6
HeLa cells. Five hundred
nanograms of protein per lane were investigated by immunoblotting. (C) Immunoprecipitation. A n uclear extract (2 · 10
6
cells) prepared a t
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) Ó FEBS 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 t he
faster moving species (Fig. 3B). In c ontrast, 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 c hromatin-bound hCdc6p appears t o b e

during G1 phase followed b y a d ecrease in early S phase
[24]. With the continuation of S phas e, however, the amount
of hCdc6p on chromatin remained constant (Fig. 4C)
implying that newly synth esized 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 r eleased from a double-thymidine block
and l abeled wit h [
35
S]methionine for 2 h. The radioactive
medium was t hen removed and r eplaced by s tandard 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 c hase period (not shown).
We present an experiment where the label period was 4–6
h after release f rom the thymidine block followed by chase
periods of 4 and 8 h (Fig. 5). Total h Cdc6p, as determined
by Western blotting, was similar in the pulse and in the
chase s amples (Fi g. 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

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 o f t he autoradiographic signals in labeled
chromatin-associated hCdc6p. With t he 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 t ransferred to chromatin where i t
shares the fate of o ld hCdc6p, namely dissociation, trans-
port to the cytoplasm and degradation. Continued protein
synthesis guarantees that the amount of chromatin-associ-
ated hCdc6p remains h igh.
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 c ycle. 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

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 m ost likely r egulated by phosphorylation [ 27]
involving the cyclin A-dependent protein kinase CDK2
which h as 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 o f pulse-labeled Cdc6p on chromatin were similar
in G1 phase and S-phase cells. This r esult s uggests that the
fraction of Ôold Õ hCdc6p that dissociates from chromatin
and is transferred to the cytoplasm during S phase is at least
partially replaced by newly s ynthesized 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 m easured for hCdc6p at the mitosis/G1 phase
transition when a sudden massive destruction of Cdc6p
Fig. 6. Half-life of c hromatin-bound hCdc6p in S p hase. HeLa cells
were labeled with [
35
S]methionine for 2 h im mediately 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 a utoradiography. T he

phosphorylation may be necessary for a S-phase related
function of hCdc6p, e.g. the activation of late origins.
Because phosphorylation also causes t he relocalization of
hCdc6p from the nucleus to the cytoplasm [22,24,27],
continued synthesis would be necessary to provide enough
hCdc6p for S phase progression. The S-phase function of
hCdc6p must be distinct from Mcm loading because Mcm
proteins dissociate from their chromatin sites during S
phase. Indeed, while hCdc6p may be necessary for Mcm
loading during G1 phase, it is certainly not sufficient
because the Cdt1 protein i s also i nvolved, and t he Cdt1
protein is e ffectively sequestered during S phase by th e
regulator protein geminin [40]. It will therefore certain ly b e
of interest to determine the biochemical function that
hCdc6p performs during S phase.
ACKNOWLEDGEMENTS
We thank C hristine Peinelt for composing Fig. 6. This work was
supported by Deutsche Forschungs-Gemeinschaft.
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