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Article |
Address correspondence to J. Julian Blow, Wellcome Trust Biocentre, University of Dundee, Dow Street, Dundee DD1 5EH, UK. Tel.: 44-1-382-345-797. Fax: 44-1-382-348-072. email: j.j.blow{at}dundee.ac.uk
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Abstract |
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Key Words: DNA replication; Cdc6; replication licensing; Xenopus; Chk1
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Introduction |
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The fate of Cdc6 during later cell cycle stages varies in different organisms. In yeasts, phosphorylation of Cdc6 (or its homologue Cdc18 in Schizosaccharomyces pombe) by Cdks targets it for ubiquitin-dependent proteolysis (Drury et al., 1997, 2000; Jallepalli et al., 1997; Kominami and Toda, 1997; Baum et al., 1998; Elsasser et al., 1999). In metazoans, chromatin-bound Cdc6 persists throughout S phase and G2 (Coleman et al., 1996; Coverley et al., 2000; Mendez and Stillman, 2000) and is degraded during G1 by the anaphase promoting complex (Petersen et al., 2000). During S phase and G2, however, the majority of the soluble (nonchromatin bound) Cdc6 appears to be exported out of the nucleus in a Cdk-dependent manner (Saha et al., 1998; Petersen et al., 1999; Pelizon et al., 2000). The majority of the data showing Cdc6 nuclear export in S phase were obtained by overexpression of Cdc6. Surprisingly, a recent study focusing entirely on endogenous Cdc6 showed that even nonchromatin-bound Cdc6 may remain nuclear throughout S phase (Alexandrow and Hamlin, 2004). Despite this persistence of Cdc6 on chromatin or in nuclei later in the cell cycle, it has been shown in Xenopus that once DNA has been licensed, efficient DNA replication no longer requires the presence of Cdc6 (Rowles et al., 1999).
Several lines of evidence suggest that Cdc6 may play another role later in the cell cycle to generate appropriate checkpoint signals for regulated cell cycle progression. During mitotic exit in Saccharomyces cerevisiae, Cdc6 cooperates with Sic1 to directly inactivate Cdks (Calzada et al., 2001). In human cells, overexpression of Cdc6 in G2 phase inhibits activation of Cdk1cyclin B and blocks entry into mitosis (Clay-Farrace et al., 2003). This latter effect seems to be mediated by activation of the Chk1 checkpoint kinase, because Cdc6 overexpression induced Chk1 phosphorylation, which is indicative of kinase activation. Moreover, addition of a Chk1 inhibitor overcame the Cdc6-induced inhibition of mitotic entry. Finally, results in S. pombe suggest that the Cdc6 homologue Cdc18 is required for checkpoint activation in response to S phase arrest (Murakami et al., 2002). When cdc18 was inactivated after S phase progression had been blocked with hydroxyurea, activation of the Cds1 (Chk2) checkpoint kinase was abolished. Further, the stalled replication forks became destabilized in the absence of Cdc18. These results suggest that Cdc6/Cdc18 has important roles in regulating cell cycle progression in addition to its well-documented role in origin licensing.
We have performed experiments to examine in detail the function and regulation of Xenopus Cdc6. We show that in Xenopus egg extracts, Cdc6 is displaced from chromatin as a direct consequence of origins first becoming licensed. Rebinding of Cdc6 to chromatin occurs in S phase as a consequence of replication forks progressing away from the origin. Finally, we show that the activation of Chk1 that normally occurs as a consequence of replication fork inhibition is dependent on the continued presence of Cdc6.
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Results |
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It has been shown previously that in Xenopus egg extract, ORC saturates sperm chromatin at approximately one copy per 10 kb (Rowles et al., 1996, 1999). This approximately matches the density of replication origins, which are roughly spaced every 515 kb (Blow et al., 2001). To understand the physical relationship between Cdc6 and ORC, we examined the quantity of Cdc6 bound to chromatin. Because Cdc6 is displaced from chromatin when licensing occurs, we performed the analysis using extracts supplemented with geminin, which prevents licensing by inhibiting Cdt1. Equal aliquots of Xenopus sperm nuclei were incubated in increasing volumes of geminin-treated extract; after 15 min, the chromatin was isolated and immunoblotted for Cdc6. Fig. 2 A shows that, as reported previously for ORC (Rowles et al., 1996, 1999), the quantity of Cdc6 on chromatin increased in proportion to the volume of extract used, until a plateau was reached and the chromatin became saturated with Cdc6. Quantification by comparison with recombinant standards showed that at saturation there was 10 ng Cdc6 bound to each 400 ng of aliquot of DNA (Fig. 2 B), corresponding to
2.4 molecules of Cdc6 per 10 kb. This suggests that before licensing occurs, each molecule of ORC can recruit a dimer of Cdc6 onto chromatin.
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Cdc6 is reloaded shortly after origins initiate
Previous studies in Xenopus egg extracts have shown that Cdc6 rebinds to chromatin during the later stages of an incubation (Coleman et al., 1996; Jares and Blow, 2000). Therefore, we performed a detailed time course to determine the relative timing of Cdc6 reloading compared with other known S phase events (Fig. 4). In these extracts, DNA synthesis starts 3035 min after DNA addition and continues at a maximal rate for a further 2030 min (Fig. 4 A). Fig. 4 B shows immunoblots of various proteins present on chromatin over this period, and Fig. 4 C shows a quantitation of the blots, each normalized to the maximal level. PCNA, an essential replication fork protein, is present at high levels on chromatin 3545 min after DNA addition when DNA synthesis is maximal. Mcm2 is displaced from DNA during S phase with levels falling from 4055 min. Cdt1 and geminin are also displaced from chromatin during S phase. In contrast, Cdc6 is reloaded onto the DNA as it replicates. The loading of Cdc6 mirrors that of PCNA at the start of S phase, though unlike PCNA, Cdc6 levels do not subsequently decline. Fig. 4 D shows that at 120 min, when most of the DNA has replicated, the quantity of Cdc6 on the chromatin is similar to that observed when licensing is blocked by geminin. This behavior is consistent with Cdc6 being reloaded onto origins as they replicate.
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Next, we wanted to confirm that the role of Cdc6 in checkpoint activation is distinct from its role in Mcm2-7 chromatin loading. Although Mcm2-7 loading does not normally occur once S phase has started, it is formally possible that in the presence of aphidicolin Cdc6 is required to reload or stabilize Mcm2-7 complexes at stalled forks. If forks became destabilized in the absence of Cdc6 they may not be able to maintain the checkpoint signal that leads to Chk1 phosphorylation. Thus, Cdc6 might be required for Chk1 activation in response to aphidicolin simply by performing its normal function in Mcm2-7 loading. To address this point, we allowed licensing to occur in Xenopus extracts, and then supplemented aliquots with an excess of a mutant form of geminin, gemininDEL, which is constitutively active (McGarry and Kirschner, 1998; Li and Blow, 2004), to prevent any further Mcm2-7 loading. Fig. 8 A shows that the mutant geminin had no effect on the ability of extracts to maintain the phosphorylation of Chk1 in response to aphidicolin. This indicates that Chk1 phosphorylation is not dependent of ongoing Mcm2-7 loading. To directly investigate the stability of fork proteins in the absence of Cdc6, we added high salt-washed chromatin to nonimmune- and Cdc6-depleted extracts treated with aphidicolin, as in Fig. 7 E. Chromatin was then isolated at different times over a 4-h period and immunoblotted for the presence of the replication fork proteins Mcm2, RPA, Cdc45, and PCNA (Fig. 8 B). There was no evidence for the destabilization of any of these proteins in the absence of Cdc6. Since RPA binds to single-stranded DNA this suggests that there are similar quantities of single-stranded DNA (which in turn is thought to be a major signal for the activation of the ATMATR checkpoint pathway) in the presence or absence of Cdc6. Together, these results suggest that the requirement of Cdc6 for Chk1 phosphorylation represents a function of Cdc6 distinct from its known role in the loading of Mcm2-7 onto chromatin.
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Discussion |
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In normal Xenopus egg extract, an average of 1020 copies of Mcm2-7 are loaded onto chromatin for each replicon (Mahbubani et al., 1997; Edwards et al., 2002). Maximum replication rates can be achieved, however, with an average of only one to two copies of Mcm2-7 per 10-kb replicon. If, as seems likely, the Mcm2-7 hexamer functions as a helicase ahead of each replication fork, then two copies would be needed at each origin to drive its two replication forks. We show here that the affinity of Cdc6 for chromatin declines once the chromatin has obtained the minimal quantity of Mcm2-7 required for efficient DNA replication. However, loading of further Mcm2-7 onto each origin is still dependent on the presence of Cdc6 and ORC. This behavior has some interesting implications for how replication origins are established. Once the first one to two Mcm2-7 hexamers are loaded onto each origin, the origin becomes licensed to support efficient DNA replication in the subsequent S phase (Fig. 9, A and B). The affinity of Cdc6 for the origin then drops, so that Cdc6 will then be targeted toward ORC at other currently unlicensed origins. Therefore, under circumstances where Cdc6 or Mcm2-7 are limiting, this mechanism ensures that most ORC binding sites become licensed and bind at least one to two Mcm2-7 hexamers. This reduces the danger of chromosomes being incompletely replicated as a consequence of too few origins being licensed (Blow, 2001; Blow et al., 2001; Hyrien et al., 2003).
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Reloading of Cdc6 after initiation
Later in the cell cycle, Cdc6 regains its ability to bind chromatin with high affinity (Coleman et al., 1996; Jares and Blow, 2000). We show here that this reassociation with chromatin is dependent on the continued presence of ORC and results in similar levels of Cdc6 on chromatin as is seen on unlicensed DNA, and so probably represents a simple reestablishment of the ORC-Cdc6 interaction that is observed before licensing. The reloading of Cdc6 starts just at the beginning of S phase and is dependent on Cdk activity, suggesting that it is dependent on the initiation of DNA replication. Consistent with this, Cdc6 reloading was also inhibited in extracts treated with low levels of aphidicolin, where an intraS phase checkpoint has been activated which inhibits initiation from late-firing replication origins (Luciani, M.G., personal communication; unpublished data). When the intraS phase checkpoint was abolished by caffeine, only high levels of aphidicolin (>40 µM) could block Cdc6 reloading. This suggests that some DNA synthesis and movement of the replication fork away from origins is required for Cdc6 to rebind with high affinity to ORC (Fig. 9 C).
Previous work has shown that Cdc6 is required to recruit cyclin ECdk2 to chromatin in Xenopus egg extracts (Furstenthal et al., 2001a). Recruitment of cyclin ECdk2 to chromatin is thought to be required for the ubiquitination of proteins such as the Cdk inhibitor Xic1, which are recognized by the SCF (Skp1CullinF-box) ubiquitin ligases (Furstenthal et al., 2001b). Xic1, which binds to cyclin ECdk2, is degraded during S phase after the initiation of replication has taken place (You et al., 2002). Since we show here that Cdc6 reloading is dependent on initiation, this provides a potential explanation for why Xic1 degradation is also dependent on initiation. This system may represent part of a positive feedback loop to ensure complete genome replication, whereby low Cdk levels lead to limited initiation and Cdc6 reloading, causing Xic1 degradation and further Cdk activation (Li and Blow, 2001).
Role of Cdc6 in checkpoint activation
We also show that Cdc6 is required during S phase to activate the Chk1 checkpoint kinase in response to inhibition of replication elongation by the DNA polymerase inhibitor aphidicolin (Fig. 9 C). This is reminiscent of results in S. pombe, which showed that the Cdc6 homologue Cdc18 is required to activate the Cds1 checkpoint kinase in response to the replication inhibitor hydroxyurea (Murakami et al., 2002). It has also been shown in mammalian cells that overexpression of Cdc6 in G2 cells can block progression into mitosis by activation of Chk1 (Clay-Farrace et al., 2003). Therefore, our results add weight to the idea that Cdc6 plays an important regulatory role that is distinct from its contribution to the pre-RC and licensing. The ability of Cdc6 to bind Cdks (Elsasser et al., 1996; Brown et al., 1997; Furstenthal et al., 2001a) raises the possibility that Cdc6 functions in some way to integrate cell cycle checkpoint activity with the activity of Cdks.
The mechanism by which Cdc6 activates checkpoint kinases is currently unclear. We present data suggesting that the requirement of Cdc6 for Chk1 phosphorylation does not depend on its ability to load Mcm2-7 onto chromatin. We also show that activation of Chk1 in response to aphidicolin in Xenopus does not depend on the continued presence of ORC, and so does not require the Cdc6 to be chromatin bound. However, we also show that much endogenous Cdc6 remains associated with nuclei in the absence of ORC, where it is in an ideal position to mediate the phosphorylation of nuclear Chk1 in response to stalled replication forks. This result is in apparent contrast with results reported in a number of different metazoans, including Xenopus, that the majority of the soluble Cdc6 is exported out of the nucleus during S and G2 in a Cdk-dependent manner (Saha et al., 1998; Petersen et al., 1999; Pelizon et al., 2000). However, many of these experiments reported the export of ectopically expressed or recombinant protein, and it is possible that endogenous protein behaves differently (Alexandrow and Hamlin, 2004). Perhaps in the absence of ORC, Cdc6 can either shuttle between nucleus and cytoplasm, or can be retained within the nucleus associated with some nonchromatin structure (Coleman et al., 1996; Fujita et al., 2002) where it can still interact with replication forks and Chk1.
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Materials and methods |
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Recombinant proteins and antibodies
Recombinant proteins were produced as described previously: Xenopus Cdc6 (Gillespie et al., 2001), Xenopus Cdt1 (Tada et al., 2001), and Xenopus gemininDEL (McGarry and Kirschner, 1998; Tada et al., 2001). The antibodies used in this work were as follows: Xenopus Orc1 (Rowles et al., 1996), Xenopus Orc2 (raised against full-length 6X His-tagged Xenopus Orc2), Xenopus Cdc6 (Tada et al., 1999), human Mcm2 (BM28; BD Transduction Labs), Xenopus Mcm3 (Mahbubani et al., 1997), Xenopus Mcm5 (raised against 6X His-tagged amino acids 289721 of Xenopus Mcm5), Xenopus Mcm4, 6, and 7 (Prokhorova and Blow, 2000), Xenopus Cdt1 (Tada et al., 2001), Xenopus geminin (Tada et al., 2001), histones H3 and H4 (rabbit polyclonal; Upstate Biotechnology), PCNA (PC10; Sigma-Aldrich), phospho-Chk1 (Ser345; Cell Signaling Technology), and Chk1 (sc-7898; Santa Cruz Biotechnology, Inc.).
Chromatin and nuclear templates
Demembranated Xenopus sperm nuclei were prepared as described previously (Chong et al., 1997). They were assembled into chromatin by incubation at 520 ng DNA/µl for appropriate times in treated extracts. In the case of immunodepleted extracts, sperm nuclei were incubated at 27 ng DNA/µl, to compensate for the threefold dilution that occurs during depletion. Extract was diluted 1020-fold in nuclear isolation buffer (Chong et al., 1997; NIB: 50 mM KCl, 50 mM Hepes-KOH, pH 7.6, 5 mM MgCl2, 2 mM DTT, 0.5 mM spermidine-3HCl, 0.15 mM spermine-4HCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin), supplemented with 0.1% Triton X-100, 2.5 mM Mg-ATP, and underlayered with the same buffer containing 15% sucrose (wt/vol). The chromatin was pelleted at 2,100 g in a swinging bucket centrifuge for 5 min at 4°C. The diluted extract and the top part of the cushion were carefully removed, and the cushion was carefully washed with 150 µl NIB supplemented with 0.1% Triton X-100, 2.5 mM Mg-ATP. The majority of the overlying cushion was then removed, and the chromatin was recentrifuged at 10,000 g in a fixed-angle rotor. The chromatin pellet was resuspended in loading buffer and subjected to immunoblotting by standard techniques using 412% bis-Tris gradient SDS-PAGE (Invitrogen) and ECL detection (SuperSignal® West Pico Chemiluminescent). For the Cdc6 quantification experiments, the chemiluminescence signal was measured using a cooled CCD camera (model LAS-1000; Fuji).
When chromatin was isolated that had to be reincubated in another extract, the isolation was performed in a similar way except that Triton X-100 was omitted from the buffers and only the first centrifugation was performed, after which the chromatin was resuspended to 100 ng DNA/µl and snap frozen in liquid nitrogen in 5 µl of aliquots for later use. For high salt chromatin isolation the extract was diluted in NIB plus 2.5 mM Mg-ATP and 200 mM KCl, and was underlayered with two cushions: the first containing NIB plus 2.5 mM Mg-ATP, 200 mM KCl, and 5% sucrose (wt/vol); and the second containing NIB plus 2.5 mM Mg-ATP and 10% sucrose (wt/vol).
For isolation of intact nuclei (Kumagai et al., 1998), extract containing nuclei was underlayered with 10 vol of a buffer containing 40% sucrose, 50 mM Hepes-KOH, pH 7.5, 100 mM KCl, and 2.5 mM Mg-MgCl2, and pelleted at 5,000 g in a fixed-angle rotor for 3 min at 4°C. The pellets were resuspended in 1 ml of the same buffer and recentrifuged under the same conditions. For extracts that had been treated with caffeine, 5 mM of caffeine was included in the isolation buffer.
Purification of Xenopus Mcm2-7
Mcm2-7 was purified from interphase Xenopus egg extract by immunoaffinity purification. All purification steps were performed at 4°C. 400 µl recombinant protein A beads (Amersham Biosciences) were mixed with 800 µl of antiserum against Xenopus Mcm3 or Mcm7. 2 ml of interphase egg extract was diluted fourfold in LFB3/10 (20 mM Hepes-KOH, pH 8, 10% (wt/vol) sucrose, 2 mM DTT, 10 mM KCl, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin) before addition to the beads. After incubation for 45 min on a rotary mixer, the beads were washed three times (5 min, 5 min, and 30 min) in 15 ml LFB1/50 (40 mM Hepes-KOH, pH 8, 10% sucrose (wt/vol), 2 mM DTT, 50 mM KCl, 20 mM K2HPO4/KH2PO4, 2 mM MgCl2, 1 mM EGTA, 1 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin) for 5 min, 5 min, and 30 min on the mixer. Elution was performed in three successive one-bead volumes of LFB1/600 (LFB1 containing 600 mM KCl). Mcm3/5 from the anti-Mcm7 antibody beads and Mcm2/4/6/7 from the anti-Mcm3 beads were recovered by centrifugation for 2 min at 700 g in a swinging bucket centrifuge through a 25-µm CellMicroSievesTM mesh (BioDesign Inc. of New York) and dialyzed in LFB3/10. The Mcm2-7 heterohexamer was recovered via stoichiometric addition of Mcm3/5 to Mcm2/4/6/7 after analysis by SDS PAGE, immunoblotting, or gel filtration.
Alkaline agarose gels
For alkaline agarose gel analysis, Xenopus sperm nuclei were incubated at 15 ng/µl for 90 min in 20 µl of aliquots of treated extracts containing -[32P]dATP before addition of 300 µl StopN (20 mM Tris-HCl, pH 8, 200 mM NaCl, 5 mM EDTA, 0.5% SDS) containing 2 µg/ml RNase, and digestion for 10 min at 37°C. Proteinase K was added to 200 µg/ml and incubated for a further 30 min at 37°C. Protein was extracted with 300 µl phenol/chloroform/isoamyl alcohol (25:24:1) using Phase Lock GelTM tubes (Eppendorf). 2.5 vol of ethanol were added and the DNA was precipitated for 20 min in dry ice before centrifugation for 10 min at 16,000 g in a fixed-angle rotor. The pellet was washed in 90% ethanol and resuspended in 20 µl 2x alkaline loading buffer: 50 mM NaOH, 6 mM EDTA, 2.5% Ficoll, 0.025% Bromocresol green. 15 cm 0.9% agarose gels were equilibrated for >1 h at RT in alkaline gel running buffer (50 mM NaCl, 1 mM EDTA). Gels were run at 2 V/cm for 1012 h at 4°C and fixed in 7% TCA (wt/vol) and 1.4% (wt/vol) sodium pyrophosphate for 20 min. Gels were then dried between paper and exposed to X-ray film.
Modeling a random distribution of Mcm2-7
Suppose there are x replication origins and m Mcm2-7 hexamers, such that the ratio r between them is given by m = rx. If the Mcm2-7 hexamers are distributed at random amongst all the origins, the probability P that any randomly selected origin gets no Mcm2-7 hexamers is given by
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For a very large number of origins, consider the case where x
. Since
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Acknowledgments |
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This work was funded by a CR-UK studentship to M. Oehlmann (STU063/001) and CR-UK program grants SP2385/0101 and C303/A3135.
Submitted: 7 November 2003
Accepted: 17 March 2004
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