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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 purine nucleoside triphosphate binding domain consisting of the conserved Walker A and Walker B motifs. In addition, Cdc6p contains several potential phosphorylation sites in t he N-terminal region. C dc6p is required f or the formation of pre-replicative c omplexes and therefore essen- tial for replication initiation i n 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 c lasses o f protein phosphorylating e nzymes, c yclin- dependent kinases (Cdk) and t he 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 b y 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 o f Cdc6p fluctuate across the yeast 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 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 o f t he 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 D ecember 2001, accepted 19 December 2001) Eur. J. Biochem. 269, 1040–1046 (2002) Ó FEBS 2002 EXPERIMENTAL PROCEDURES Cell culture Human HeLa S 3 cells were grown on plastic dishes in Dulbecco’s modified Eagle’s m edium plus 5% fetal bovine serum. Cells were s ynchronized at the beginning of S phase by a double-thymidine procedure (12 h in 2.2 m M thymi- dine; 9 h w ithout 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 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 M ATP; 20 m M Hepes, pH 7.5). After 15 min on ice and douncing, cells were centrifuged to separate the cytosolic supernatant f rom t he 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 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). 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 B iotech). Immunoprecipitations were performed with extracts from 4–6 · 10 6 cells incubated w ith 2 lg h Cdc6p-specific antibodies for 1 h on i ce. Protein A–Sepharose bead s (50 lL of a 50% suspension; Amersham, Pharmacia Biotech) were then added f or 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 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 distribution of hCdc6p in t he cytoplasm a s well as in t he fractions of soluble (nucleosolic) and structure-bound nuclear proteins (chromatin) from a synchronously prolifer- ating HeLa cells. Chromatin-bound hCdc6p could be mobilized in buffers with 0.25–0.45 M NaCl (Fig. 1B) and was effectively i mmunoprecipitated by h Cdc6p-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 w hether 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 e ither hCdc6p- or hOrc1p-specific antibodies (not shown). This does not exclude the possibility that the two proteins interact when b ound to chromatin. In fact, w e Ó FEBS 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 l arge protein c omplexes that protect DNA against nuclease attack, but dissociate a t high salt concentrations (Fig. 1B). In the e xperiments reported b elow, we prepared H eLa 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). The membranes used for Western b lotting were washed to remove the ECL reagent, dried a nd exposed to X-ray films for autoradiography. T he 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 w as almost evenly distributed between the c ytoplasmic and the chromatin fractions of hCdc6p (Fig. 2C). We conclude that hCdc6p was syn thesized 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 . Sy nthes is of h Cdc 6p. HeLa cells ( 1 · 10 7 ), arrested by a do uble- thymidine block, were released into the cell cycle. At the times indi- cated, 4 · 10 5 HeLa cells were incubated with 15 lg propidium io dide in 0.3 mL phosphate-buffered saline with 0.1% Triton X-100 for 30minoniceandprocessedforFACSanalysis(A).Theremaining cells w ere l abeled with [ 35 S]methionine for 2 h and then fractionated to prepare cytosol (Cy) and nuclear extracts at 0.45 M NaCl. Ext racts were immunoprecipitated. Precipitated pr oteins were analysed by 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 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. A s a n explanation, we considered the possibility that t he 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 prepar ation of nuclear extracts. I n these extracts, the phosphorylated hCdc6p in the control sample was at l east as stable as the phosphatase-treated hCdc6p (Fig. 3C) suggesting that nuclear extracts from S-phase HeLa cellscontain a proteasome-related activity that preferentially attacks the phosphorylated form of hCdc6p. However, more importantly in th e present context, we note that the synth esis of hCdc6p c ontinues through m ost of the cell cycle. Synthesis of hCdc6p during G1 phase is necessary f or the f ormation of pre-replicative complexes, whereas s ynthesis 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 c ompensates for t he S-phase-dependent loss of hCdc6p. We have investigated this point using HeLa cells released from a nocodazole b lock into G 1 and S p hase (Fig. 4A). We found a gradual increase of chromatin-bound hCdc6p 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 form of labeled cytoplasmic hCdc6p r apidly disappeared during the chase (half life < 2 h, Fig. 5B, left). It can assumed that p art of t he labeled c ytoplasmic hCdc6p moved into the nucleus and bound to ch romatin, but another p art may h ave remained in the c ytoplasm to be degraded or Fig. 4. Soluble and chromatin-bound hCdc6p. Cells were arrested by nocodazoleandthenreleasedintoG1andSphaseasmonitoredby FACS analysis (A). Soluble n uclear and chromatin-bou nd proteins (450 m M NaCl) were investigated by denaturing polyacrylamide gel electrophoresis. C oomassie 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. Immunopre cipitates of cytosolic proteins (Cy) and of nuclear extracts (450 m M NaCl) were incubated with buffer only or with buffer p lus lambda phosphatase as indic ated. The proteins were analysed by Western blotting (A) and autor adiography (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 c ell frac- tionation. Immunoprecipitates of cytosolic and nuclear proteins were treated with phosphatase and investigated by Western blotting (C). Ó FEBS 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 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 cells fail to express Cdc6 m RNA and protein, but r eaddition 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 replicatio n. Once proliferation has been initiated, mamm alian 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 pr esence 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 l evels of hCdc6p may below i n 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]. T he 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 b ehaviour of mammalian Cdc6p during S phase has b een 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 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 autoradiographic b ands were evaluate d by densitometry with the pulse values taken a s 100%. Deviation bars give averages o f 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 d ouble- thymidine block and then chased for 4 and 8 h as indicated. Cytosolic (Cy) and chromatin- associated (4 50 m M NaCl) proteins were prepared and investigated by immunoprecipitation. We sho w Western blot (A) an d autoradiogra- phy (B) of the immunoprecipitates. 1044 E. Biermann et al. (Eur. J. Biochem. 269) Ó FEBS 2002 occurs that is closely f ollowed b y the synthesis o f new Cdc6p [28]. Thus, synthesis follows degradation in early G1 phases whereas the production of new hCdc6p s eems 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 r eplication. One function is certainly the assembly of the pre-replicatio n complex, but additional functions seem to be required at or after the i nitiation 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 t o 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 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. 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