Human replication protein Cdc6 prevents mitosis through a checkpoint mechanism that implicates Chk1

Abstract

In yeasts, the replication protein Cdc6/Cdc18 is required for the initiation of DNA replication and also for coupling S phase with the following mitosis. In metazoans a role for Cdc6 has only been shown in S phase entry. Here we provide evidence that human Cdc6 (HuCdc6) also regulates the onset of mitosis, as overexpression of HuCdc6 in G(2) phase cells prevents entry into mitosis. This block is abolished when HuCdc6 is expressed together with a constitutively active Cyclin B/CDK1 complex or with Cdc25B or Cdc25C. An inhibitor of Chk1 kinase activity, UCN-01, overcomes the HuCdc6 mediated G(2) arrest indicating that HuCdc6 blocks cells in G(2) phase via a checkpoint pathway involving Chk1. When HuCdc6 is overexpressed in G(2), we detected phosphorylation of Chk1. Thus, HuCdc6 can trigger a checkpoint response, which could ensure that all DNA is replicated before mitotic entry. We also present evidence that the ability of HuCdc6 to block mitosis may be regulated by its phosphorylation.

Fig. 1. In vivo analysis of HuCdc6 overexpression in G₂ cells. (A) pEGFP and (B) pEGFP-HuCdc6 were microinjected into the nucleus of G₂ phase HeLa cells. The behaviour of injected cells was observed by time-lapse fluorescence and DIC microscopy and images were taken every 30 min, or every 3 min after entry into mitosis over a 10 h period. Approximately 100 fluorescent cells expressing GFP or HuCdc6–GFP were classified according to their cell cycle stage: G₂ phase, mitosis and after completion of mitosis (early G₁ phase) during different time points starting from 1 h after microinjection. These numbers were compared with the total number of cells expressing GFP or HuCdc6–GFP (C and D). While 40–45% of cells expressing GFP went through mitosis [arrows in (A) and (B) show mitotic cells], only 3–5% of cells expressing HuCdc6–GFP did so. Representative images, mean values and standard deviations of five independent experiments are shown. (E) Comparison of expression of HuCdc6–GFP to endogenous HuCdc6 levels. A total of 1000 cells were injected with pEGFP-HuCdc6 and after 2 h were directly lysed in SDS buffer, proteins were separated on 10% SDS–PAGE and immunoblotted for HuCdc6. (F) Anti human Cdc6 antibody neutralizes HuCdc6 overexpression and abolishes the G₂ phase arrest. G₂ phase HeLa cells were microinjected with HuCdc6–GFP and a specific anti HuCdc6 antibody. As a control, cells were injected with the anti HuCdc6 antibody and Texas Red dextran. The percentages of cells expressing pEGFP-HuCdc6 in G₂, M and G₁ phases were calculated and compared with the control cells 7 h after injection. Mean values and standard deviation were calculated from at least three independent experiments.

Fig. 2. Constitutively active MPF abolishes the G₂ phase arrest caused by HuCdc6 overexpression. G₂ phase HeLa cells were microinjected with (A) pEGFP-HuCdc6, pCyclin B1 and a constitutively active pCDK1AF or (B) with pEGFP-HuCdc6, pCyclin B1 and pCDK1wt. As controls we co-injected pCyclin B1 and pCDK1AF or pCyclin B1 and pCDK1wt were injected without HuCdc6 as a control (C). Approximately 100 fluorescent cells were counted for each sample. Fluorescence and DIC images were taken 7 h after injection. Arrows show mitotic cells. (C) The numbers of cells in G₂ phase, mitosis and in early G₁ phase were scored and calculated as a percentage of the total of injected cells. At least three independent experiments were performed and representative images are shown.

Fig. 3. Overexpression of Cdc25 abolishes the G₂ phase arrest caused by HuCdc6–GFP. G₂ phase cells were microinjected with pCdc25B and pEGFP-HuCdc6 (A) or with pEGFP-Cdc25C and pHuCdc6 (B). Arrows in (A) and (B) show mitotic cells. Cells were followed by time-lapse fluorescence and DIC microscopy as described in Figure 1, starting from 2 h after microinjection during a 7 h time course. Sixty per cent of cells entered mitosis prematurely and arrested in mitosis whether they expressed HuCdc6–GFP and Cdc25B (C) or Cdc25B alone (E). (D) Forty five per cent of cells expressing Cdc25C and HuCdc6–GFP entered and progressed through mitosis, but often with a 4 h delay compared with the 2 h delay of the control cells expressing Cdc25C only (F).

Fig. 4. An inhibitor of Chk1 kinase activity, UCN-01, abolishes HuCdc6-mediated G₂ arrest. (A) G₂ phase HeLa cells were microinjected with pEGFP-HuCdc6 or pEGFP as a control (not shown). UCN-01 (300 nM) was added to the medium immediately after the injections. Fluorescence and DIC images were taken during a 10 h period. G₂ phase and mitotic cells (marked with an arrow) were counted from a total of ∼100 fluorescent cells over a 10 h period. (B) Percentage of cells progressing through mitosis expressing HuCdc6 or GFP. Twenty-one percent of cells expressing HuCdc6–GFP entered and progressed through mitosis comparable with the 24% of GFP expressing cells. Two experiments were performed and representative images are shown. (C) HuCdc6-mediated phosphorylation of Chk1. One thousand cells were injected with pEGFPHuCdc6 or pEGFP, uninjected but untreated or treated with HU (control cells). Proteins were separated on SDS–PAGE and immunoblotted for Chk1. Lane 1 shows a mobility shift due to phosphorylation after HU treatment, whereas control cells do not (lanes 2, 4 and 6). HuCdc6 shows a similar mobility shift as HU-treated cells (lane 1), whereas GFP alone does not (lane 3). (D) The HuCdc6-mediated G₂ phase arrest is maintained in the presence of caffeine and/or wortmannin. Cells were microinjected with pEGFP-HuCdc6 in the presence or absence of 5 mM caffeine and/or 25 µM wortmannin. As a control, cells were γ-irradiated (100 kVp/min) for 15 min and caffeine and/or wortmannin were added. Cells were followed for 10 h counting cells entering and progressing through mitosis as one (%M + G₁). Uninjected cells and irradiated cells entered into mitosis prematurely in the presence of caffeine and/or wortmannin. The cells expressing HuCdc6 remained arrested in G₂ phase in the presence of caffeine and/or wortmannin.

Fig. 5. Behaviour of HuCdc6 mutants in G₂ cells. (A) Schematic drawing of HuCdc6 depicting features such as the CDK phosphorylation sites serines (S) 54, 74 and 106, destruction box, KEN box, the cyclin-binding-motif, the ATPase/ORC homology domain and leucine-zipper, all previously identified. (B) The pEGFP-HuCdc6 mutants S54A, S74A, S106A and Δcy-motif were injected into G₂ HeLa cells and their progression into mitosis compared with GFP or wtHuCdc6–GFP-expressing cells. Cells expressing the mutants S75A and Δcy-motif cannot arrest cells in G₂, whereas cells expressing S54A and S106A do arrest cells in G₂ in the same fashion to wtHuCdc6. (C) Illustration of the localization of the mutants. Two independent experiments were performed and representative images are shown.

Fig. 6. Model for potential checkpoint function of HuCdc6. Ongoing DNA replication is monitored either by proteins at the replication fork, including ATR, or by soluble factors, including HuCdc6. ATR and HuCdc6 might work in parallel pathways, both leading to the activation of Chk1 and consequent inactivation of Cdc25. The major role of the ATR pathway would be in response to stalled replication forks. Chk1 may also increase the activity of Wee1 that further prevents the activation of MPF.