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Synthesis and turn-over of the replicative Cdc6 proteinduring the HeLa cell cycle Esther Biermann, Martina Baack, Sandra Kreitz and Rolf Knippers Department of Biology, Universita¨t Kons

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 [35S]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

of Cdc6p in the HeLa cell cycle

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)

E X P E R I M E N T A L P R O C E D U R E S

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 mM

thymi-dine; 9 h without thymithymi-dine; 14 h in 2.2 mMthymidine) or at

mitosis with a nocodazole block (12 h in 2.2 mMthymidine;

9 h release; 3 h at 40 ngÆmL)1nocodazole) 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 [35S]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 lMMG-132 (Calbiochem) for 6 h

Cell fractionation

Cells were washed with phosphate-buffered saline (NaCl/Pi)

and suspended in buffer A (20 mM NaCl; 5 mM MgCl2;

1 mMATP; 20 mMHepes, 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 (0.3Msucrose;

0.5 mM MgCl2; 1 mM ATP; 20 mM Hepes, pH 7.5) plus

NaCl in concentrations of 0.1–0.45Mas indicated below

All extraction buffers contained phosphatase inhibitors:

1 mM NaF, 1 mM 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 mM CaCl2

and 100 mM 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 mMEDTA 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) were used in a

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· 106 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 0.45MNaCl 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 0.45MNaCl in buffer B as above and then with phosphatase buffer (100 mM NaCl; 0.1 mM MnCl2; 0.1 mM EGTA; 50 mM

Tris/HCl, pH 7.5) Treatment with lambda protein phos-phatase (400 U; New England BioLabs) was in 0.05 mL buffer for 30 min on ice and 30 min at 30°C under shaking The immunocomplexes were then washed in 0.45MNaCl buffer B and processed for electrophoresis as described above

R E S U L T S 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 0.25–0.45MNaCl (Fig 1B) and was effectively immunoprecipitated by hCdc6p-specific antibodies (Fig 1C)

We note that 0.25–0.45M 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

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

0.45M 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

[35S]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 [35S]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

30 min on ice and processed for FACS analysis (A) The remaining 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.

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 [35S]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 35S-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 nocodazole and then released into G1 and S phase as monitored by 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

[35S]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).

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

D I S C U S S I O N

We show here that [35S]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 [35S]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 ÔoldÕ hCdc6p 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 [35S]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.

occurs that is closely followed by the synthesis of new Cdc6p

[28] Thus, synthesis follows degradation in early G1 phases

whereas the production of new hCdc6p seems to occur

simultaneously with the displacement of old hCdc6p from

chromatin during S phase

Why does hCdc6p go through a cycle of synthesis,

chromatin-binding and release within one S phase? This

must be somehow connected with functions that hCdc6p

performs in genome replication One function is certainly

the assembly of the pre-replication complex, but additional

functions seem to be required at or after the initiation of

replication This has been concluded because the

micro-injection of mutant unphosphorylatable hCdc6p interferes

with DNA replication [32] and ectopic expression of mutant

hCdc6p leads to a delay in S phase entry [27] Therefore,

phosphorylation may be necessary for a S-phase related

function of hCdc6p, e.g the activation of late origins

Because phosphorylation also causes the 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 is also involved, and the Cdt1

protein is effectively sequestered during S phase by the

regulator protein geminin [40] It will therefore certainly be

of interest to determine the biochemical function that

hCdc6p performs during S phase

A C K N O W L E D G E M E N T S

We thank Christine Peinelt for composing Fig 6 This work was

supported by Deutsche Forschungs-Gemeinschaft.

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