XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair

Abstract

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

FIG. 1.

FANCD2 is monoubiquitinated in XPF-ERCC1- and MUS81-EME1-deficient cells after cross-link damage. (A) Wild-type and Ercc1 mutant CHO cells were exposed to 15 μM nitrogen mustard (HN2) for 1 h, and then WCEs were collected 18 or 24 h later for the immunodetection of FANCD2. FANCD2-L is the monoubiquitinated protein, and FANCD2-S is the nonubiquitinated form. U indicates cells that were untreated. (B) Wild-type HeLa cells were transfected with control siRNA, with one of two siRNAs against hSNM1A as further controls for specificity, or with siRNA against Ercc1. After confirming the specific knockdown of ERCC1 by immunoblotting, cells were treated with 1 μM MMC for 1 h, and WCEs were collected at multiple time points after exposure for the immunodetection of FANCD2. (C) Wild-type and Ercc1^−/− MEFs were exposed to 0.3 μM MMC for 12 h, and then WCEs were collected at multiple time points after exposure for the immunodetection of FANCD2. (D) Wild-type, Mus81^−/−, and Ercc1^−/− mouse ES cells were exposed to 3 μM MMC for 1 h, and then WCEs were collected 24 h later for the immunodetection of FANCD2.

FIG. 2.

Monoubiquitination of FANCD2 persists in XPF-ERCC1-deficient cells. (A) AA8 (wild type), UV135 (Xpg mutant), UV96 (Ercc1 mutant), and UV47 (Xpf mutant) CHO cells were exposed to 1 μM MMC for 12 h or 5 μM nitrogen mustard (HN2) for 1 h, and then WCEs were collected at multiple time points following exposure for the immunodetection of FANCD2. U indicates cells that were untreated. (B) Wild-type, XP-A (XP25RO), and XP-F (XP51RO and XP42RO) immortalized human fibroblasts were exposed to 0.3 μM MMC for 12 h, and then WCEs were collected at multiple time points after exposure for the immunodetection of FANCD2. (C) CHO cells were treated as described for panel A, fixed at the same time points, and stained with propidium iodide for cell cycle analysis by flow cytometry. (D) The efficiency of the unhooking of interstrand cross-links, as determined by modified comet assay following 1 h of treatment with 5 μM HN2 in Xpg, Xpf, and Ercc1 mutant and wild-type parent cell lines.

FIG. 3.

FANCD2 focus formation is impaired in XPF-ERCC1-deficient cells. Wild-type (C5RO) and XP-F (XP51RO) human fibroblasts were seeded on glass coverslips. Sixteen hours later, the cells were exposed to 3 μM MMC for 1 h and then fixed at multiple time points. FANCD2 was detected by immunofluorescence. Cells with FANCD2 foci were counted. Representative images are shown, and the averages from three experiments are plotted. A paired Student's t test was used to calculate significance. DAPI, 4′,6′-diamidino-2-phenylindole.

FIG. 4.

Chromatin localization of FANCD2 is impaired in XPF-ERCC1-deficient cells exposed to cross-linking agents but not ionizing radiation. (A) HeLa cells depleted for ERCC1 or mock depleted with a control siRNA duplex were treated with 1 μM HN2 for 1 h. At 18 h after exposure, WCEs were prepared or cells were fractionated to isolate the soluble nuclear and chromatin-bound protein fractions. Samples were immunoblotted for FANCD2. The immunodetection of ORC2 (origin recognition complex 2) was used as a loading control for chromatin-bound protein and GRB2 for cytoplasmic protein. U indicates cells that were untreated. (B) Wild-type (C5RO) and XP-F (XP51RO) human fibroblasts were exposed to 3 μM MMC for 1 h and then fractionated 12 h later. The fractions were immunoblotted for FANCD2. Nucleophosmin and histone H3 were used as loading controls for the soluble nuclear and chromatin fractions, respectively. (C) Wild-type and Ercc1^−/− MEFs were exposed to 3 μM MMC for 1 h and fractionated at multiple time points after exposure. Fractions were immunoblotted for FANCD2 and the loading controls. (D) Wild-type (IB10) and Ercc1^−/− (clone 49) mouse ES cells were exposed to 3 μM MMC for 1 h and fractionated at 24 h after exposure. Fractions were immunoblotted for FANCD2 with TATA binding protein (TBP) as the loading control. (E) Wild-type and Xpf mutant CHO cells were exposed to 5 μM HN2 for 1 h or 10 Gy of ionizing radiation (IR). Cells were processed as described for panel A at 18 and 24 h after radiation, and samples were immunoblotted for FANCD2. Tubulin was used as a loading control for WCE, and ORC2 was used as a loading control for the chromatin fraction.

FIG. 5.

Model of how XPF-ERCC1 and MUS81-EME1 nucleases function in the same S-phase-specific DNA interstrand cross-link repair mechanism as the FA proteins. See the text for more details.