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. 2013 Jul 15;73(14):4362-71.
doi: 10.1158/0008-5472.CAN-12-3154. Epub 2013 Apr 10.

Structure-specific Endonucleases Xpf and mus81 Play Overlapping but Essential Roles in DNA Repair by Homologous Recombination

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Free PMC article

Structure-specific Endonucleases Xpf and mus81 Play Overlapping but Essential Roles in DNA Repair by Homologous Recombination

Koji Kikuchi et al. Cancer Res. .
Free PMC article

Abstract

DNA double-strand breaks (DSB) occur frequently during replication in sister chromatids and are dramatically increased when cells are exposed to chemotherapeutic agents including camptothecin. Such DSBs are efficiently repaired specifically by homologous recombination (HR) with the intact sister chromatid. HR, therefore, plays pivotal roles in cellular proliferation and cellular tolerance to camptothecin. Mammalian cells carry several structure-specific endonucleases, such as Xpf-Ercc1 and Mus81-Eme1, in which Xpf and Mus81 are the essential subunits for enzymatic activity. Here, we show the functional overlap between Xpf and Mus81 by conditionally inactivating Xpf in the chicken DT40 cell line, which has no Mus81 ortholog. Although mammalian cells deficient in either Xpf or Mus81 are viable, Xpf inactivation in DT40 cells was lethal, resulting in a marked increase in the number of spontaneous chromosome breaks. Similarly, inactivation of both Xpf and Mus81 in human HeLa cells and murine embryonic stem cells caused numerous spontaneous chromosome breaks. Furthermore, the phenotype of Xpf-deficient DT40 cells was reversed by ectopic expression of human Mus81-Eme1 or human Xpf-Ercc1 heterodimers. These observations indicate the functional overlap of Xpf-Ercc1 and Mus81-Eme1 in the maintenance of genomic DNA. Both Mus81-Eme1 and Xpf-Ercc1 contribute to the completion of HR, as evidenced by the data that the expression of Mus81-Eme1 or Xpf-Ercc1 diminished the number of camptothecin-induced chromosome breaks in Xpf-deficient DT40 cells, and to preventing early steps in HR by deleting XRCC3 suppressed the nonviability of Xpf-deficient DT40 cells. In summary, Xpf and Mus81 have a substantially overlapping function in completion of HR.

Figures

Figure 1
Figure 1. Xpf is essential for the maintenance of genome stability
(A) Targeted disruption of the chicken XPF gene. The chicken XPF locus, the two targeting constructs and the resulting targeted locus are shown. The black boxes represent the exons of the XPF gene. The triangles flanking the blastidine-resistance (bsrR) and histidinol-resistance (hisDR) genes represent the loxP sequences, the recognition site of the Cre recombinase. (B) Growth curve after addition of tamoxifen (TAM) to XPF−/−GdXPF-loxP cells at time zero for the excision of the GdXPF-loxP transgene. (C) Flow-cytometric analysis of cell-cycle distribution after BrdU pulse-labeling in XPF−/−GdXPF-loxP cells. (D) Spontaneous chromosomal aberrations in XPF−/−GdXPF-loxP cells. Top panels, a representative chromatid-type break (shown by arrowhead) and a chromosome-type break (shown by arrow). Breaks are magnified in the lower panels. Bottom panels, measurement of spontaneous chromosomal aberrations. A chromatid-type break indicates a discontinuity in one of the two sister chromatids, and a chromosome-type break indicates discontinuities at the same site of both sisters. Exchange indicates chromosomal translocation. The vertical axis shows the number of aberrations per cell.
Figure 2
Figure 2. A defect in homologous-recombination-dependent DSB repair in Xpf-depleted cells
(A) Camptothecin (CPT)-induced chromosomal aberrations in Xpf-depleted cells. XPF−/−GdXPF-loxP cells were continuously exposed to TAM for 33 h, during which the cells were treated with 100 nM CPT for the last 9 h, and with colcemid for the last 3 h before harvest of mitotic cells. Left panels, measurement of chromosomal aberrations after treatment with or without CPT. Right panels, CPT-induced chromosomal aberrations were calculated by subtracting the number of spontaneously occurring chromosomal aberrations from the number of chromosomal aberrations observed in the CPT-treated sample of the same genotype. 100 mitotic cells were examined for each analysis. The vertical axis shows the number of aberrations per cell in A and B. (B) Ionizing radiation (IR)-induced chromosomal aberrations in Xpf-depleted cells. Cells were exposed to TAM for 24 h, exposed to γ-rays, then treated with colcemid for 3 h. Left panels, measurement of chromosomal aberrations after exposure to γ-rays. Right panels, IR-induced chromosomal aberrations were calculated by subtracting the number of spontaneously occurring chromosomal aberrations from the number of chromosomal aberrations observed in the γ-rays-exposed sample of the same genotype. Results of IR-induced chromosomal aberrations in RAD54−/− cells were described in a previous report (26). (C) The formation of γ-induced Rad51 foci in Xpf-expressing and Xpf-depleted cells. Left panels, a fraction of the γ-irradiation-induced Rad51 subnuclear foci persists for extended periods. Cells were exposed to TAM for 2 days, irradiated with 4 Gy γ-ray, fixed at 3 and 6 h post-IR, and subjected to immuno-cytochemistry using anti-Rad51 antibody. Right panels, quantification of Rad51 foci number at 6 h post-IR. 100 cells were examined for each analysis. (D) Xpf depletion reduces sister-chromatid exchange (SCE) events induced by CPT. The distribution of SCE events per cell is shown for the indicated cell samples to the left panels. Blue bars represent no CPT treatment, and red and green bars represent data for 5 nM and 100 nM CPT treatment, respectively. Mean values and photo of a representative SCE (shown by arrowhead) are shown to the right panels.
Figure 3
Figure 3. Deletion of the XRCC3 reverses the mutant phenotype of XPF−/− cells
(A) Growth curve after adding TAM to XPF−/−GdXPF-loxP/XRCC3−/− cells at time zero to inactivate the GdXPF-loxP transgene. (B) Spontaneous chromosomal aberrations in XPF−/−GdXPF-loxP/XRCC3−/− cells were measured as described in Figure 1D. Results of spontaneous chromosomal aberrations for XRCC3−/− cells were described in a previous report (23). (C) Growth curve after adding TAM to the indicated cells at time zero. +RusA represents XPF−/−GdXPF-loxP cells expressing RusA. (D) Spontaneous chromosomal aberrations in the indicated cells were measured as described in Figure 1D. (E) IR-induced chromosomal aberrations in XPF−/−/RusA cells at 1 day after addition of TAM. Chromosomal aberrations induced by γ-rays were measured and calculated as described in Figure 2B.
Figure 4
Figure 4. Ectopic expression of HsXpf-Ercc1 or HsMus81-Eme1 suppresses the lethality in XPF−/− cells
(A) Growth curve after addition of TAM to the indicated cells at time zero. + HsXPF alone and +HsXPF-ERCC1 represent XPF−/−GdXPF-loxP cells expressing HsXpf alone and XPF−/−GdXPF-loxP cells expressing HsXpf-Ercc1, respectively. (B) Spontaneous chromosomal aberrations in the indicated cells were measured as described in Figure 1D. (C) Growth curve after addition of TAM to the indicated cells at time zero. +HsMUS81-EME1 represents XPF−/−GdXPF-loxP cells expressing HsMus81-Eme1. (D) Spontaneous chromosomal aberrations in the indicated cells were measured as described in Figure 1D.
Figure 5
Figure 5. Ectopic expression of H HsXpf-Ercc1 or HsMus81-Eme1 reverses the mutant phenotype of XPF−/− cells
(A) IR-induced chromosomal aberrations in XPF−/−/HsXPF-ERCC1 cells at 1 day after addition of TAM. Chromosomal aberrations induced by γ-rays were measured and calculated as described in Figure 2B. (B) IR-induced chromosomal aberrations in XPF−/−/HsMUS81-EME1 cells at 1 day after addition of TAM. Chromosomal aberrations induced by γ-rays were measured and calculated as described in Figure 2B. (C) CPT-induced SCE events at 2 days after addition of TAM. The histograms to the left display the distribution of SCEs per cell following treatment with 5 nM CPT. Blue and red bars represent XPF−/− and XPF−/−/HsMUS81-EME1 cells, respectively. Mean values are shown to the right panels.
Figure 6
Figure 6. Xpf and Mus81 compensate for each other in the completion of HR in human and mouse cell lines
(A) After treatment with the indicated siRNAs in human HeLa cells, aberrant chromosomes in metaphase cells (n=200) were analyzed. The vertical axis shows the number of aberrations per cell in A and B.(B) After treatment with the indicated siRNAs in mouse wild-type or MUS81−/− cells, aberrant chromosomes in metaphase cells (n=100) were analyzed. A representative chromatid-type break and a chromosome-type break are magnified in the middle panels and are shown by arrow.

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