Wee1 controls genomic stability during replication by regulating the Mus81-Eme1 endonuclease

Abstract

Correct replication of the genome and protection of its integrity are essential for cell survival. In a high-throughput screen studying H2AX phosphorylation, we identified Wee1 as a regulator of genomic stability. Wee1 down-regulation not only induced H2AX phosphorylation but also triggered a general deoxyribonucleic acid (DNA) damage response (DDR) and caused a block in DNA replication, resulting in accumulation of cells in S phase. Wee1-deficient cells showed a decrease in replication fork speed, demonstrating the involvement of Wee1 in DNA replication. Inhibiting Wee1 in cells treated with short treatment of hydroxyurea enhanced the DDR, which suggests that Wee1 specifically protects the stability of stalled replication forks. Notably, the DDR induced by depletion of Wee1 critically depends on the Mus81-Eme1 endonuclease, and we found that codepletion of Mus81 and Wee1 abrogated the S phase delay. Importantly, Wee1 and Mus81 interact in vivo, suggesting direct regulation. Altogether, these results demonstrate a novel role of Wee1 in controlling Mus81 and DNA replication in human cells.

Figure 1.

Wee1 inhibition triggers a general DDR response. (A) Immunofluorescence analysis of U2OS cells transfected with siRNA oligonucleotides targeting Luciferase (Luc si) or Wee1 (Wee1 si) for 48 h or cells treated with etoposide (20 µM for 1 h) or UV light (40 J/m² for 1 h) as positive controls using the indicated antibodies. For every sample, a field with a similar amount of cells is shown. RPA, replication protein A. (B) Immunoblot analysis using cells treated as described in A. (C) U2OS cells were mock transfected or transfected with a wild-type (wt) or kinase-dead (kd; K328A) version of Flag-Wee1 (top left and bottom) or treated with a Wee1 inhibitor for 4 h (top right). Thereafter, phosphorylation of H2AX was analyzed by immunoblot and immunofluorescence analysis. Bars, 10 µm.

Figure 2.

Wee1 down-regulation affects cell cycle progression during S phase. (A) U2OS transfected with Luciferase siRNA (Luc si) or Wee1 siRNA (Wee1 si) and cell cycle profiles were determined by flow cytometry analysis after staining with propidium iodide. (B) U2OS cells were transfected with Luciferase or Wee1 siRNA oligonucleotides. 48 h later, cells were fixed and analyzed by flow cytometry for γ-H2AX and DNA content by propidium iodide staining. (C and D) U2OS cells were transfected with the indicated siRNAs at the same time as they were synchronized with nocodazole (noc; C) or thymidine (thym; D) and were subsequently released from the arrest. At the indicated times after the release, cells were collected for flow cytometry analysis for propidium iodide staining or for analysis by Western blotting with the indicated antibodies. (E) Flow cytometry analysis for γ-H2AX/propidium iodide (right) or immunoblotting with the indicated antibodies (left) of U2OS cells transfected with Luc or Wee1 siRNA oligonucleotides and a control or Flag-Chk1 plasmid for 48 h.

Figure 3.

Fork movement slowdown by Wee1 depletion. U2OS cells were transfected with control (C) or Wee1 (Wee) siRNA oligonucleotides for the indicated times and thereafter pulsed for IdU and CldU. Fork speed was determined by measuring the length of IdU and CldU tracks on combed DNA molecules. Bar graphs show the percentages of molecules with certain replication speeds from one representative experiment. Above each graph, the average (Av) fork speed with the SEM of three independent experiments is indicated. Western blots show efficiency of Wee1 down-regulation.

Figure 4.

Wee1 protects the stability of stalled replication forks. (A) U2OS cells were treated with different concentrations of HU for 24 h. Thereafter, cells were pulsed for IdU and CldU and analyzed as described in Fig. 3 (top) or stained using the indicated antibodies for immunofluorescence (bottom). Av, average; PCNA, proliferating cell nuclear antigen. (B) U2OS cells were treated with Wee1 inhibitor. After 1 h, HU was added at the indicated concentrations, and cells were harvested and analyzed by immunofluorescence for γ-H2AX or immunoblotting using the indicated antibodies. The graph represents the quantification of the immunofluorescence. Represented is the percentage of γ-H2AX–positive cells. A minimum of 1,000 cells was counted for each sample. Error bars represent the SEM of two independent experiments. Bars, 10 µm.

Figure 5.

Wee1 and Chk1 protect from replication problems in a different manner. (A) Flow cytometry analysis after propidium iodide staining (bottom) and immunoblot analysis (top) of U2OS cells transfected with Luc, Wee1, or Chk1 siRNA oligonucleotides or a combination for 48 h. (B) U2OS cells were transfected with Chk1 with or without Wee1 siRNA oligonucleotides for 48 h and thereafter pulsed for IdU and CldU. (bottom) Fork speed was determined by measuring the length of IdU and CldU tracks on combed DNA molecules. Bar graphs show the percentages of molecules with certain replication speeds from one representative experiment. Above each graph, the mean fork speed with the SEM of three independent experiments is indicated. (top) Levels of the indicated proteins by Western blotting.

Figure 6.

Mus81 knockdown rescues the DNA damage induction and S phase delay in the absence of Wee1. (A) U2OS cells were transfected with Luciferase or Mus81 siRNA (Mus81 si) for 48 h in the presence of thymidine (thym). During the last 24 h, they were transfected with Luciferase or Wee1 siRNA (Wee1 si). After that, cells were released or not released from the thymidine block for 3 h before being lysed and analyzed with the indicated antibodies. (B) U2OS cells were transfected with Luciferase siRNA (Luc si) and Mus81 siRNA for 64 h and/or Wee1 siRNA oligonucleotides during the last 24 h, all in the presence of thymidine. Cells were then released from the block and studied at the indicated times for DNA content by flow cytometry. Western blot analysis with the indicated antibodies of whole-cell extracts at time 0 is also shown. Dotted lines indicate the position of the G1 peak at t = 0.

Figure 7.

Interaction between Wee1 and Mus81. (A) HEK 293T cells were cotransfected with the indicated combinations of plasmids including expression vectors for the wild-type (wt) or kinase-dead (kd; K328A) version of Flag-Wee1, GFP, and GFP-Mus81, and immunoprecipitations (IP) using anti-GFP or anti-FLAG antibodies were performed. Inputs and immunoprecipitations were analyzed with anti-Wee1 and -GFP antibodies. MM, molecular mass; WB, Western blot. (B) Immunoprecipitations of endogenous Mus81 from U2OS cells analyzed by Western blotting using the indicated antibodies. (C) A model for Wee1 regulating DNA replication by controlling Mus81. See Discussion for details.