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

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.

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.