Haploinsufficiency of the Mus81-Eme1 endonuclease activates the intra-S-phase and G2/M checkpoints and promotes rereplication in human cells

Abstract

The Mus81-Eme1 complex is a structure-specific endonuclease that preferentially cleaves nicked Holliday junctions, 3'-flap structures and aberrant replication fork structures. Mus81-/- mice have been shown to exhibit spontaneous chromosomal aberrations and, in one of two models, a predisposition to cancers. The molecular mechanisms underlying its role in chromosome integrity, however, are largely unknown. To clarify the role of Mus81 in human cells, we deleted the gene in the human colon cancer cell line HCT116 by gene targeting. Here we demonstrate that Mus81 confers resistance to DNA crosslinking agents and slight resistance to other DNA-damaging agents. Mus81 deficiency spontaneously promotes chromosome damage such as breaks and activates the intra-S-phase checkpoint through the ATM-Chk1/Chk2 pathways. Furthermore, Mus81 deficiency activates the G2/M checkpoint through the ATM-Chk2 pathway and promotes DNA rereplication. Increased rereplication is reversed by the ectopic expression of Cdk1. Haploinsufficiency of Mus81 or Eme1 also causes similar phenotypes. These findings suggest that a complex network of the checkpoint pathways that respond to DNA double-strand breaks may participate in some of the phenotypes associated with Mus81 or Eme1 deficiency.

Figure 1

Generation of HCT116 cell lines deficient in Mus81 or Eme1 by gene targeting. (A) Schematic representation of the Mus81 locus, the targeting vectors, and the targeted alleles. Relevant restriction sites and the position of the probes used for Southern blot analysis are shown. (B) Southern blot analysis confirming targeted integration at the Mus81 locus. DNAs were digested with SacI or BamHI and hybridized with the probes depicted in (A). (C) Northern blot analysis confirming the expression levels of Mus81. Poly(A)⁺ RNAs were isolated and hybridized with the full-length Mus81 cDNA. Northern blotting for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was performed to confirm equal loading. (D) Western blot analysis confirming the protein expression levels of Mus81. Western blotting for actin was also carried out to confirm equal loading. (E) Schematic representation of the Eme1 locus, the targeting vector and the targeted allele. Relevant restriction sites and the position of the probes used for Southern blot analysis are shown. (F) Southern blot analysis confirming targeted integration at the Eme1 locus. DNAs were digested with SphI and hybridized with the probes depicted in (E). (G) Northern blot analysis confirming the expression levels of Eme1. Poly(A)⁺ RNAs were isolated and hybridized with the full-length Eme1 cDNA.

Figure 2

Sensitivity to DNA-damaging agents. (A–F) Sensitivities to MMC, MMS, cisplatin, UV radiation, ionizing radiation and hydroxyurea. Values represent the means ± the standard error of the mean for three independent experiments. Mus81^+/− (#653), Mus81^−/− (#150) and Eme1^+/− (#376) cells were used. (G) D37 values of sensitivity to DNA-damaging agents. Graph Pad Prism4 software was used to calculate the values.

Figure 3

Chromosomal aberrations in Mus81^−/− (#150) cells. The arrow in the left panel indicates a chromosome break. The arrow in the middle panel indicates a chromatid gap. Rereplication is shown in the right panel. In the left and middle panels, only a part of metaphase chromosome image is shown.

Figure 4

Effects of Mus81 or Eme1 deficiency on cell cycle progression. Mus81^+/− (#653), Mus81^−/− (#150) and Eme1^+/− (#376) were examined. (A) Growth curves. The results show the means ± the standard error of the mean for three independent experiments. (B) Cell cycle distribution. The cells were synchronized in G₁/S by double-thymidine block and released. Samples were taken at the indicated time points and subjected to FACS analysis.

Figure 5

Activation of the intra-S-phase checkpoint. (A) Cyclin E and cyclin A kinase activities with histone H1 as the substrate. Wild-type and Mus81^−/− (#150) cells were synchronized in G₁/S by double-thymidine block and released. (B) Western blot analysis of synchronized wild-type and Mus81^−/− (#150) cell extracts using anti-phospho-Chk1 (Ser-317). The experiments in (A) and (B) were performed three times, and representative results are shown. (C) Immunofluorescence of Mus81^−/− cells synchronized in S phase using anti-phospho-Chk1 (Ser-317). The frequencies of positive staining for phospho-Chk1 are shown in the right panel. (D) Western blot analysis of unsynchronized Mus81^−/− (#150) cells transfected with siRNAs. The experiment was performed three times. (E) Immunofluorescence of Mus81^−/− cells synchronized in S phase using anti-phospho-Chk2 (Thr-68). The frequencies of the positive staining of phospho-Chk2 are shown in the right panel. In (C) and (E), cells were fixed 2 h after release, and a total of 500 cells were examined for each cell line. The results represent the means ± standard deviation of three independent experiments. IB, immunoblot; IP, immunoprecipitation; p-Chk, phospho-Chk; WT, wild-type.

Figure 6

Activation of the G₂/M checkpoint. (A) Cyclin B kinase activity with histone H1 as the substrate. Wild-type and Mus81^−/− (#150) cells were synchronized in G₁/S by double-thymidine block and released. (B) Chk2 kinase activity using GST-Cdc25C (200–256) as the substrate and whole-cell extracts from wild-type and Mus81^−/− (#150) cells synchronized in G₁/S and released. The experiments in (A) and (B) were performed five times. (C) Chk2 kinase activity on GST-Cdc25C (200–256) of synchronized cells harvested 6 h after release. For caffeine treatment, cells were incubated in the presence of 0.5 mM caffeine for 1 h prior to cell lysis. (D) The effects of ATM and ATR on Chk2 activity. Shown is the Chk2 kinase activity using GST-Cdc25C (200–256) as a substrate in extracts of Mus81^−/− (#150) cells harvested 48 h after transfection with siRNA. (E) Chk1 kinase activity using GST-Cdc25C (200–256) as a substrate in extracts from wild-type and Mus81^−/− (#150) cells synchronized in G₁/S and released. Cells treated with UV radiation are used as positive controls. The treated cells were harvested 5 h after UV radiation (40 J/m²). The experiments in (C), (D) and (E) were performed twice, and representative results are shown. (F) Western blot analysis of extracts from unsynchronized cells using an anti-p21 antibody. (G) Cyclin B kinase activity with histone H1 as a substrate in extracts from unsynchronized cells. In (F) and (G), Mus81^−/− (#150) cells were used.