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. 2006 Jan 27;7(4):425–430. doi: 10.1038/sj.embor.7400624

The functional role of Cdc6 in S–G2/M in mammalian cells

Eric Lau 1,2, Changjun Zhu 1, Robert T Abraham 1, Wei Jiang 1,a
PMCID: PMC1456921  PMID: 16439999

Abstract

The Cdc6 protein is required for licensing of replication origins before the onset of DNA replication in eukaryotic cells. Here, we examined whether Cdc6 has other roles in mammalian cell-cycle progression from S to G2/M phase. Using RNA interference, we showed that depletion of Cdc6 in synchronous G1 cells blocks G1 to S transition, confirming the essential role of Cdc6 in the initiation of DNA replication. In contrast, depletion of Cdc6 in synchronous S-phase cells slowed DNA replication and led to mitotic lethality. The Cdc6-depleted S-phase cells showed fewer newly fired origins; however, established replication forks remained active, even during chromatin condensation. Despite such DNA replication abnormalities, loss of Cdc6 failed to activate Chk1 kinase. These results show that Cdc6 is not only required for G1 origin licensing, but is also crucial for proper S-phase DNA replication that is essential for DNA segregation during mitosis.

Introduction

In all eukaryotes, DNA replication initiates at several origins, and each segment of DNA replicates once and once only during each cell cycle. Before DNA replication, pre-replicative complexes (pre-RCs) are assembled at replication origins in G1 phase of the cell cycle. Origin licensing is sequential with origin-recognition complex (ORC) binding to replication origins, which recruits the loading factors, Cdc6 and Cdt1, thereby promoting the loading of the DNA replicative helicase minichromosome maintenance protein (MCM) complex (Bell & Dutta, 2002; Blow & Dutta, 2005). The licensed origins are then triggered to initiate DNA replication by the concerted actions of two S-phase-promoting kinases, Cdks and Ddk.

Previous studies suggested that regulation of the abundance and/or subcellular localization of Cdc6 is essential both for licensing origins and for preventing of DNA re-replication in the same cell cycle. In mammalian cells, Cdc6 resides in the nucleus in G1, and is essential for pre-RC formation and replication initiation (Williams et al, 1997; Cook et al, 2002). During S and G2/M phases, Cdc6 translocates to the cytoplasm in a Cdk phosphorylation-dependent manner and is subsequently degraded by the anaphase-promoting complex (Jiang et al, 1999; Petersen et al, 1999, 2000). However, recent reports show that unlike the exogenous Cdc6 used in previous studies, a subpopulation of endogenous Cdc6 remains chromatin bound in the nucleus after the cells enter S phase (Fujita et al, 1999; Mendez & Stillman, 2000; Okuno et al, 2001; Alexandrow & Hamlin, 2004). These results indicate that Cdc6 may be important for events in post-G1 progression, such as checkpoint surveillance (Clay-Farrace et al, 2003; Oehlmann et al, 2004). In this study, we explored the S–G2/M functions of Cdc6 during the cell cycle in human cells.

Results and Discussion

Effects of Cdc6 depletion on cell-cycle progression

To explore the functions of Cdc6 during the cell cycle in human cells, we depleted Cdc6 expression using endonuclease-prepared small interfering RNA (esiRNA; Yang et al, 2002) in HeLa cells. Transfection of Cdc6-targeted (Cdc6) esiRNA but not mock (water) or luciferase (Luc) esiRNA significantly reduced endogenous protein levels of Cdc6 (Fig 1A). The depletion of Cdc6 was specific in that no reduction in Orc2, Cdt1, MCM7 or α-tubulin was detected in the esiRNA-treated cells. Fluorescence-activated cell sorting (FACS) analysis indicated that Cdc6-depleted cells accumulated in G1/S–S phases, together with a marked induction of sub-G1 (apoptotic) cells (Fig 1B). Microscopic analysis confirmed that about 20% of the cells transfected with Cdc6 esiRNA showed apoptotic morphology after 72 h in culture (Fig 1C). Similar results were obtained from HCT116 cells (Fig 1C). Thus, these results indicate that depletion of Cdc6 perturbs cell-cycle progression, and also causes cell death in human cells.

Figure 1.

Figure 1

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Depletion of Cdc6 in human cells results in G1/S block and cell death. (A) HeLa cells transfected for 72 h with mock (water) or 120 nM luciferase (Luc) or Cdc6 endonuclease-prepared small interfering RNA (esiRNA) were subjected to immunoblot analysis with anti-Cdc6, anti-Orc2, anti-Cdt1, anti-MCM7 or anti-α-tubulin. (B) HeLa cells transfected as in (A) were fixed and analysed by fluorescence-activated cell sorting. (C) HeLa and HCT116 cells transfected as in (A) were fixed and stained with 4,6-diamidino-2-phenylindole. The percentages of catastrophic cells were scored (>500 cells) using a fluorescence microscope.

To characterize the S-phase-specific consequences of Cdc6 depletion, we ablated Cdc6 expression by Cdc6 esiRNA in synchronous HeLa cells. Previous studies demonstrated that human Cdc6 is an unstable protein with a half-life of ∼1–2 h (Biermann et al, 2002; Mailand & Diffley, 2005). HeLa cells were synchronized in G2/M phase by a sequential thymidine–nocodazole block and then released into early G1 phase by the addition of fresh medium for 3 h (supplementary Fig S1A online). The early G1 cells were transfected with mock or Luc or Cdc6 esiRNA, and after 9 h, cells were collected and Cdc6 expression and cell-cycle profiles were examined by immunoblotting and FACS analyses. Cdc6 was not detected in early G1 cells because Cdc6 was degraded after mitotic exit, as described previously (Petersen et al, 2000; Mailand & Diffley, 2005). In cells transfected with mock or Luc esiRNA, Cdc6 protein was expressed, consistent with the presence of S and G2/M cells in these samples (Fig 2, left panel). In contrast, the Cdc6 esiRNA-transfected cells showed reduced Cdc6 expression and a concomitant cell-cycle arrest at G1/S phase (Fig 2, left panel). Subcellular fractionation experiments showed that chromatin-bound Cdc6 and MCM2 were also significantly reduced in Cdc6-depleted cells when compared with control cells (supplementary Fig S2 online). FACS and microscopic (not shown) analyses indicated that these Cdc6-depleted cells remained non-apoptotic. Thus, depletion of Cdc6 in synchronous G1 cells blocks G1/S transition, confirming the essential role of Cdc6 in the initiation of DNA replication.

Figure 2.

Figure 2

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Depletion of Cdc6 in early G1 phase blocks G1 progression, and depletion of Cdc6 in early S phase results in mitotic cell death. (Left) HeLa cells were synchronized at G2/M by a thymidine–nocodazole block and released into early G1 phase by the addition of fresh medium for 3 h. The early-G1-phase cells were transfected with mock or luciferase (Luc) or Cdc6 endonuclease-prepared small interfering RNA (esiRNA), and grown for another 9 h before fluorescence-activated cell sorting (FACS) analysis or immunoblotting using anti-Cdc6 antibodies. (Right) HeLa cells were synchronized at G1/S by a double-thymidine block and released into early S phase by the addition of fresh medium for 1 h. The early-S-phase cells were transfected with mock or Luc or Cdc6 esiRNA, and grown for another 19 h before FACS analysis or immunoblotting using anti-Cdc6 antibodies.

Next, we synchronized HeLa cells at the G1/S transition with a double-thymidine block and then released cells into early S phase (supplementary Fig S1B online). These cells were transfected with mock or Luc or Cdc6 esiRNA, and were collected after 19 h. The early-S-phase cells expressed Cdc6 and the mock-or Luc esiRNA-transfected cells contained virtually identical amounts of this protein after 19 h in culture (Fig 2, right panel, lanes 1–3; supplementary Fig S2 online). The latter cell populations had also progressed through S and G2/M phases, and entered G1 phase of the next cell cycle. In contrast, transfection of Cdc6 esiRNA during early S phase effectively inhibited Cdc6 expression and grossly perturbed cell-cycle progression (Fig 2, right panel; supplementary Fig S2 online). A substantial proportion (∼20%) of these cells also contained <2N DNA content, indicating increased cell death than in controls (∼2%).

We next monitored S–G2/M progression in synchronous S-phase cells treated with Luc or Cdc6 esiRNA using time-lapse microscopy. Time-lapse videos showed that early-S-phase cells treated with Luc esiRNA entered mitosis and cytokinesis after 11–12 h (supplementary Video S1 online). Unexpectedly, Cdc6 esiRNA-treated cells also entered mitosis around 11–12 h later (supplementary Video S2 online), indicating that depletion of Cdc6 during S phase did not cause any significant delay of S–G2/M progression in these cells. Immunoblotting analysis showed that there were no significant differences in cyclin B1 expression levels in S–G2/M progression in Luc or Cdc6 esiRNA-treated cells (supplementary Fig S3 online). However, time-lapse microscopy showed that Cdc6-depleted cells failed to complete mitosis and instead underwent cell death (supplementary Video S2 online).

S-phase depletion of Cdc6 perturbs new origin firing

We sought to determine how depletion of Cdc6 in S phase caused mitotic lethality. To this end, we pulse-labelled esiRNA-transfected early-S-phase cells with 5-bromo-2′-deoxyuridine (BrdU) for 10 min at hourly intervals until the cells entered mitosis (Fig 3A). As expected, >95% of Luc or Cdc6 esiRNA-treated cells incorporated BrdU into DNA at 5 h after release from the double-thymidine block. Whereas virtually all Luc esiRNA-treated cells had completed S phase after 9–12 h, a considerable fraction (∼10–25%) of the Cdc6-depleted cell population still incorporated BrdU at 9–11 h after S-phase entry. After 12 h, nearly all of the Cdc6-depleted cell population failed to incorporate BrdU, indicating that cells had completed S phase and/or undergone cell death.

Figure 3.

Figure 3

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Depletion of Cdc6 in S phase results in prolonged DNA synthesis but inhibited new origin firing. HeLa cells grown on coverslips were synchronized at early S phase and then transfected with luciferase or Cdc6 endonuclease-prepared small interfering RNA as in Fig 2. (A) Luciferase (Luc) or Cdc6 endonuclease-prepared small interfering RNA (esiRNA)-transfected cells were pulse-labelled with 10 mM 5-bromo-2′-deoxyuridine (BrdU; 20 min), fixed at hourly intervals and immunostained with an anti-BrdU antibody. The percentages of BrdU-positive cells were scored (>300 cells) at the indicated time points using a fluorescence microscope. (B) At 5 and 10 h after transfection, cells were labelled sequentially with 10 μM 5-chloro-2′-deoxyuridine (CldU) followed by 15 μM 5-iodo-2′-deoxyuridine (IdU; 10 min pulse each). DNA fibres were immunostained with anti-CldU and anti-IdU antibodies. CldU/IdU-positive DNA fibres (>150) were visualized and quantified using a fluorescence microscope. Expression levels of Cdc6 at the respective time points were determined by immunoblot analysis.

To investigate the mechanism whereby Cdc6 depletion slows continuing DNA replication, we used a DNA fibre-labelling assay that allows the quantitative assessment of origin firing and fork progression in active replicons (Li et al, 2003; Merrick et al, 2004). Early-S-phase HeLa cells (1 h after release from double-thymidine block) were transfected with Luc or Cdc6 esiRNA. At 5 or 10 h after transfection, cells were labelled sequentially with two modified nucleotide precursors, 5-chloro-2′-deoxyuridine (CldU) and 5-iodo-2′-deoxyuridine (IdU). The labelled chromatin was immunostained with anti-CldU and anti-IdU monoclonal antibodies. Chromatin fibres that were actively synthesizing DNA during the CldU and IdU pulses appeared as double labelled tracks on the DNA (active replicons), which could be interpreted as (i) newly fired origins, (ii) progressing forks and (iii) colliding forks/terminations (Fig 3B).

At 5 h after release from a double-thymidine block, 37% of total active replicons represented newly fired origins and 63% of total active replicons represented established, active forks in the Luc esiRNA-treated cell population (Fig 3B). In contrast, only 23% of labelled replicons represented newly fired origins in the Cdc6-depleted cells, whereas established forks increased to 77% (Fig 3B). Consistent with this notion, active replicons detected in Cdc6 esiRNA-treated cells at 10 h after release from a double-thymidine block were almost all ongoing forks. In contrast, no active replicons were detected in Luc esiRNA-treated cells at the same time point (Fig 3B). These data indicated that S-phase depletion of Cdc6 interfered with new origin firing during S phase.

S-phase Cdc6 depletion does not activate Chk1

The effect of Cdc6 depletion on new origin firing and continuing DNA replication prompted us to investigate the status of the S–G2/M checkpoint mechanisms in these cells. Early-S-phase HeLa cells (1 h after release from double-thymidine block) were transfected with esiRNA, and after 5 h, selected samples were treated with aphidicolin to block DNA synthesis (Luciani et al, 2004). The cells were cultured for further 3 h, and activation of Chk1 was monitored by immunoblotting with anti-pS317-Chk1 antibodies (Zhao & Piwnica-Worms, 2001). As shown in Fig 4, cells transfected with Cdc6 esiRNA did not show constitutive Chk1 phosphorylation, suggesting that loss of Cdc6 alone did not activate an ATR–Chk1-dependent DNA damage response. However, treatment of control or Cdc6 esiRNA-transfected S-phase cells with aphidicolin triggered a marked increase of Ser 317 phosphorylation, accompanied by the appearance of several Chk1 bands. These latter results indicated that reduction of Cdc6 expression did not disrupt signalling from stalled replication forks to the ATR–Chk1 pathway.

Figure 4.

Figure 4

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Depletion of Cdc6 in S phase neither activates nor functionally abolishes Chk1 surveillance. HeLa cells in early S phase were transfected with luciferase (Luc) or Cdc6 endonuclease-prepared small interfering RNA (esiRNA). After 5 h, cells were treated for further 3 h with or without 5 μg/ml aphidicolin. Cellular proteins were immunoblotted with anti-pS317-Chk1, anti-Chk1, anti-Cdc6 or anti-PLCγ1 (control) antibodies.

S-phase Cdc6 depletion results in specific mitotic defects

To further define the mitotic abnormalities observed in Cdc6-depleted cells, we examined spindle formation and chromosome segregation in these cells. Luc or Cdc6 esiRNA-transfected S–G2/M cells were fixed at hourly intervals and immunostained for phosphorylation of histone H3 at Ser 10, a mitotic marker. Phosphorylation of histone H3 peaked at about 11 h post S-phase entry in both Luc and Cdc6 esiRNA-transfected cells (Fig 5A), suggesting that depletion of Cdc6 did not affect the timing of mitosis in HeLa cells. Next, we pulse-labelled Luc or Cdc6 esiRNA-transfected cells with BrdU (1 h pulses) and subsequently co-stained these cells with anti-BrdU and anti-phospho-H3 antibodies at hourly intervals up to mitosis. As shown in Fig 5B, cells transfected with Luc esiRNA showed almost no dual BrdU/phospho-H3 staining at 9–12 h post S-phase entry because these cells had completed DNA replication. In contrast, ∼40–50% of phospho-H3-positive Cdc6 esiRNA-transfected cells showed BrdU staining at these time points, indicating that Cdc6-depleted cells entered mitosis while they were still actively replicating DNA. Thus, these results indicate that loss of Cdc6 in S phase prolongs replication, but does not offset the timing of mitotic entry.

Figure 5.

Figure 5

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Depletion of Cdc6 in S phase does not affect G2/M progression and chromosome condensation, but results in abnormal spindle formation and aberrant chromosomal congression. (A) HeLa cells grown on coverslips were synchronized and transfected with luciferase (Luc) or Cdc6 endonuclease-prepared small interfering RNA (esiRNA) at early S phase. Cells were fixed at hourly intervals and immunostained with an anti-phospho-histone-H3 antibody. The percentages of phospho-H3-positive cells were determined by microscopic evaluation of >300 cells/sample. (B) HeLa cells grown on coverslips were synchronized and transfected with esiRNA at early S phase. Cells were pulse-labelled with 10 μM 5-bromo-2′-deoxyuridine (BrdU; 1 h), fixed at hourly intervals and immunostained with anti-BrdU and anti-phospho-H3 antibodies. The percentages of BrdU/phospho-H3-positive cells at the indicated time points were scored (>250 cells) using a fluorescence microscope. (C) Cells were pulse-labelled with BrdU at 11–12 h post transfection, and were fixed and immunostained with the indicated reagents. DAPI, 4,6-diamidino-2-phenylindole.

We then examined mitotic spindle formation and chromosome segregation events in cells transfected with Luc or Cdc6 esiRNA. Similar to untransfected cells, Luc esiRNA-treated cells showed normal bipolar spindles with bi-oriented chromosomes aligned perfectly at the metaphase plate during mitosis (Fig 5C; supplementary Fig S4 online). The cells completed chromosome segregation and cytokinesis normally (data not shown; supplementary Video S1 online). In contrast, most of the cells transfected with Cdc6 esiRNA showed abnormal spindle formation, where the bipolar spindles were often disorganized and disarrayed (Fig 5C; supplementary Fig S4 online). Misaligned chromosomes were observed between the metaphase plate and spindle poles (Fig 5C; supplementary Fig S4 online). Chromosome segregation was also clearly affected, and ultimately, the cells underwent mitotic cell death (data not shown; supplementary Video S2 online). Thus, depletion of Cdc6 perturbed mitotic spindle formation and chromosome congression and segregation, resulting in mitotic cell death.

Taken together, our results indicate that depletion of Cdc6 by Cdc6-specific esiRNA causes two distinct abnormalities in HeLa cells, depending on the timing of Cdc6 loss. Depletion of Cdc6 in synchronous G1 cells blocked cells at G1/S transition, consistent with the well-known functions of Cdc6 in origin licensing and replication initiation (Coleman et al, 1996; Cook et al, 2002). In contrast, Cdc6 depletion in synchronous early-S-phase cells inhibited new origin firing, prolonged DNA synthesis and promoted mitotic cell death, indicating that Cdc6 is not only required for early origin firing at G1/S phase, but is vital for regulating mid- and late origin firing during S phase. Earlier studies showed that ectopically expressed Cdc6 translocates from the nucleus to the cytoplasm in a Cdk phosphorylation-dependent manner during the G1/S-phase transition (Jiang et al, 1999; Petersen et al, 1999). It is thought that this translocation mechanism inhibits Cdc6 binding to chromatin in S to G2/M, thereby preventing DNA re-replication in the same cell cycle (Jiang et al, 1999; Bell & Dutta, 2002). However, recent studies indicated that endogenous Cdc6 has more complex regulation than ectopically expressed Cdc6 during the cell cycle. Endogenous Cdc6 binds to chromatin not only in G1, but also in S–G2/M in mammalian and Drosophila cells, suggesting that Cdc6 is positively involved in regulating cell-cycle progression beyond G1 (Fujita et al, 1999; Mendez & Stillman, 2000; Okuno et al, 2001; Alexandrow & Hamlin, 2004; Crevel et al, 2005). The observation of continued fork progression in S-phase Cdc6-depleted cells suggests that Cdc6 depletion does not affect replication elongation. Moreover, the replication abnormalities induced by Cdc6 depletion are not perceived by the genome surveillance machinery, as indicated by the absence of Chk1 activation. We speculate that this may be because Cdc6 depletion does not result in accumulation of long stretches of single-stranded DNA sufficient to activate the ATR–Chk1 checkpoint. However, the ATR–Chk1 pathway remained functional in Cdc6-depleted cells, consistent with previous results obtained with cultured Drosophila cells (Crevel et al, 2005). In contrast, Cdc6 seems to be crucial for activation of S-phase checkpoints in a Xenopus cell-free DNA replication system (Oehlmann et al, 2004). The retention of ATR–Chk1 signalling in our studies may reflect incomplete knockdown of Cdc6 in HeLa cells (Fig 4). Ultimately, S-phase Cdc6-depleted cells with replicating DNA entered mitosis, resulting in aberrant mitotic spindle formation and abnormal chromosome congression and segregation, leading to mitotic cell death. A similar ‘reductional anaphase' phenotype was previously observed in the budding yeast when Cdc6p was suppressed in S phase (Piatti et al, 1995). Recently, perturbation of other DNA replication proteins, such as Orc2, Cdt1 and Cdc7, was also reported to cause abnormal mitosis and cell death (Feng et al, 2003; Montagnoli et al, 2004; Prasanth et al, 2004; Crevel et al, 2005). Thus, our results together with these findings indicate that DNA replication proteins are involved in regulating the spatio-temporal coordination of precise replication of DNA during S phase and accurate segregation of the duplicated DNA during mitosis.

Methods

esiRNA synthesis and antibodies. esiRNAs targeting the coding regions of Luc (538–983 bp) and Cdc6 (842–1,252 bp) were synthesized as previously described (Yang et al, 2002; Zhu & Jiang, 2005). Polyclonal anti-human Cdc6 antibodies were previously described (Jiang et al, 1999). Anti-Orc2 was purchased from Oncogene Research (Calbiochem, San Diego, CA, USA). Anti-Cdt1, -MCM2, -MCM7, and anti-human CREST antisera (ADA) were purchased from Abcam (Cambridge, MA, USA). Anti-Chk1 and anti-pS317-Chk1 were purchased from Cell Signaling Technology Inc. (Danvers, MA, USA). Anti-PLCγ was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Anti-tubulin was purchased from Upstate (Dundee, UK). Anti-BrdU was purchased from Sigma-Aldrich (St Louis, MO, USA). Anti-CIdU and anti-IdU were purchased from Molecular Probes (Carlsbad, CA, USA) and CellTech (UCB, Brussels, Belgium), respectively. All secondary antibodies were purchased from Southern Biotech (Birmingham, AL, USA) and Jackson Immuno-Research Laboratories (Westgrove, PA, USA).

CldU/IdU double labelling and preparation of DNA fibres. CldU/IdU double labelling and preparation of DNA fibres were performed as described previously (Li et al, 2003; Merrick et al, 2004). Briefly, S–G2 cells were sequentially pulsed with 10 μM CldU and then 15 μM IdU, for 10 min each. After washing, cells were incubated for 15 min and collected by trypsinization. Cells were mixed 1:8 with unlabelled cells to a final concentration of 625 cells/μl. A total of 1,500 cells were lysed on a glass slide with 7.5 μl spreading buffer (0.5% SDS in 200 mM Tris–HCl, pH 7.5, 50 mM EDTA). Slides were incubated for 9.5 min and then tilted by 15°. DNA spreads were air dried for 30 min, fixed in methanol/acetic acid (1:3) and refrigerated overnight. Slides were then stained with anti-CldU and anti-IdU antibodies as described (Li et al, 2003; Merrick et al, 2004). Imaging was carried out on a Leica DMIRE2 fluorescent microscope using Simple PCI software.

Supplementary information is available at EMBO reports online (http://www.nature.com/embor/journal/vaop/ncurrent/extref/7400624-s1.pdf).

Supplementary Material

Supplementary Information

7400624-s1.pdf (1.7MB, pdf)

Acknowledgments

We are grateful to Dr T. Tsuji and Ms N. Sai for laboratory support. This work was supported by grants from the National Institute of Health to E.L. (predoctoral training grant 2T32 CA77109-06A2), to R.T.A. and W.J. (grant CA97950) and to W.J. (grant GM67859), and from the National Science Foundation to W.J. (grant NSF-0233997).

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Supplementary Materials

Supplementary Information

7400624-s1.pdf (1.7MB, pdf)

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