ERCC1-XPF endonuclease facilitates DNA double-strand break repair

Abstract

ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-) Ku86(-/-) fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3' overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.

FIG. 1.

Clonogenic survival assays after exposure of cells to increasing doses of IR. Error bars represent the standard error of the mean for three or more independent experiments. (A) WT immortalized human fibroblasts (C5RO), HeLa cells, and immortalized fibroblasts derived from an XPF-deficient patient (XFE). (B) Two independent, early-passage, primary WT and Ercc1^−/− MEF lines and transformed Ercc1^−/− MEFs stably corrected with human ERCC1 cDNA. (C) WT and Ercc1^−/− murine ES cells.

FIG. 2.

Quantitation of γH2AX foci in WT and ERCC1-XPF-deficient cells exposed to IR. Histograms indicate the fractions of cells with no foci (blue), one or two foci (green), or more than two foci (red) at 0, 4, 12, 24, and 48 h postirradiation. Panels: A, immortalized WT human fibroblasts (C5RO); B, immortalized XPF-deficient human fibroblasts (XFE); C, WT primary MEFs; D, Ercc1^−/− primary MEFs; E, WT ES cells; F, Ercc1^−/− ES cells.

FIG. 3.

Sensitivity of ERCC1-deficient mice to IR. (A) Six-week-old Ercc1^−/Δ mice and WT littermates (n = 6 per group) were exposed to 6 Gy of IR, and survival was recorded in days after exposure. (B to G) Tissue sections from 7- to 8-week-old WT and Ercc1^−/Δ mice ± exposure to 6 Gy of IR. (B) Small intestines of untreated mice. (C) Small intestines of mice exposed to IR demonstrating loss of villi in Ercc1^−/Δ mice. (D) BM of untreated mice. (E) BM of mice exposed to IR demonstrating fatty replacement in Ercc1^−/Δ mice. (F) Livers of untreated mice. (G) Livers of mice exposed to IR demonstrating centrilobular necrosis in Ercc1^−/Δ mice. (H) Hepatocyte nuclei demonstrating γH2AX foci in Ercc1^−/Δ mice 11 days after IR.

FIG. 4.

Growth and IR sensitivity of cells in which the ERCC1 and NHEJ proteins have been deleted. (A) Cell number with each passage of WT, Ercc1^−/−, Ku86^−/−, and Ercc1^−/− Ku86^−/− primary MEFs cultured at 3% oxygen. (B) Clonogenic survival of transformed WT, Ercc1^−/−, DNA-Pk_cs^−/−, Ku86^−/−, Ercc1^−/− Ku86^−/−, and Ercc1^−/− DNA-Pk_cs^−/− MEFS after exposure to IR.

FIG. 5.

Chromosomal aberrations in transformed MEFs after IR. Subconfluent cultures of WT and Ercc1^−/− cells were exposed to 2 Gy of IR, subconfluent cultures of Ku86^−/− and Ercc1^−/− Ku86^−/− cells were exposed to 0.4 Gy, and the cells were analyzed 48 h later. (A) Representative metaphase spread from WT MEFs exposed to IR. (B) Metaphase spread from an Ercc1^−/− cell demonstrating IR-induced gaps (G), fragments (Fr), and radials (R). (C) Ku86^−/− cell demonstrating IR-induced fragments. (D) Ercc1^−/− Ku86^−/− cell demonstrating IR-induced fragments, gaps, fusions (Fu), radials, and marker chromosomes (M).