ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair

ERCC1-XPF endonuclease is required for nucleotide excision repair(NER) of helix-distorting DNA lesions. However, mutations inERCC1 or XPF in humans or mice cause a more severe phenotypethan absence of NER, prompting a search for novel repair activitiesof 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 repairpathways: microhomology-mediated end joining (a Ku86-independentmechanism) and single-strand annealing. To determine if ERCC1-XPFparticipates in DSB repair in mammals, mutant cells and micewere screened for sensitivity to gamma irradiation. ERCC1-XPF-deficientfibroblasts were hypersensitive to gamma irradiation, and ${gamma}$ H2AXfoci, a marker of DSBs, persisted in irradiated mutant cells,consistent with a defect in DSB repair. Mutant mice were alsohypersensitive to irradiation, establishing an essential rolefor ERCC1-XPF in protecting against DSBs in vivo. Mice defectivein both ERCC1-XPF and Ku86 were not viable. However, Ercc1^–/–Ku86^–/– fibroblasts were hypersensitive to gammairradiation compared to single mutants and accumulated significantlygreater chromosomal aberrations. Finally, in vitro repair ofDSBs with 3' overhangs led to large deletions in the absenceof ERCC1-XPF. These data support the conclusion that, as inyeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanismthat is Ku86 independent.

INTRODUCTION

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INTRODUCTION

ERCC1-XPF is a highly conserved endonuclease identified forits essential role in nucleotide excision repair (NER) of helix-distortingDNA lesions, in particular, UV-induced damage (4, 74). Defectsin NER cause xeroderma pigmentosum (XP), a rare disorder characterizedby photosensitivity, a dramatically increased risk of skin cancer,and neurodegeneration in severe cases. In contrast, the onlyreported patient with a mutation in ERCC1 had severe congenitalanomalies (cerebro-oculo-facial-skeletal syndrome) (33). Patientswith subtle mutations in XPF have a mild form of XP (46), consistentwith only a partial defect in NER. However, a mutation in XPFthat severely compromises protein levels causes dramaticallyaccelerated aging (53). This observation implies additionalfunctions for mammalian ERCC1-XPF distinct from NER. Consistentwith that, ERCC1- and XPF-deficient mice have a much more severephenotype than mice defective in NER. Xpa^–/– micewith undetectable NER are indistinguishable from wild-type (WT)mice until challenged with carcinogens (12). In contrast, Ercc1^–/–and Xpf^–/– mice have a constellation of progeroidsymptoms affecting the musculoskeletal, dermatologic, hepatobiliary,renal, and hematopoietic systems (48, 53, 79, 84) and die ofliver failure before sexual maturation (72).

XPF contains the catalytic domain of the nuclease (18), whereasERCC1 is required for DNA binding and stabilization of XPF (51,80). The endonuclease is structure specific, incising double-strandedDNA 5' to a junction with single-stranded DNA. Thus, ERCC1-XPFcan remove 3' single-stranded flaps from DNA ends (11) and cleavesthe 5' side of a bubble in NER to excise the lesion (74). Incisionby ERCC1-XPF creates a 3' OH group that is used to prime DNAsynthesis to replace excised bases (74). Neither ERCC1 nor XPFhas structural domains that suggest that the protein functionsother than as a nuclease (73). Thus, novel functions of ERCC1-XPFthat protect against rapid aging are likely contributions toother DNA repair mechanisms.

Indeed, ERCC1-XPF is required for the repair of DNA interstrandcross-links (ICLs) via a mechanism distinct from NER (47, 54)and ICLs are implicated in contributing to the dramatic prematureaging phenotype caused by ERCC1-XPF deficiency (53). However,orthologs of ERCC1-XPF, AtErcc1-AtRad1 in Arabidopsis thaliana(29), DmERCC1-MEI-9 in Drosophila melanogaster (3), and Rad10-Rad1in Saccharomyces cerevisiae (22, 32) are also implicated indouble-strand break (DSB) repair. DNA DSBs are extremely cytotoxiclesions because both strands of the double helix are affected.DSBs are caused by both environmental and endogenous processes,including ionizing radiation (IR), radiomimetic drugs, programmedcleavage by endonucleases during meiotic recombination and V(D)Jrecombination, and replication of DNA containing single-strandbreaks, ICLs, or topoisomerase I-induced lesions (6, 30). Failureto repair DSBs can lead to the accumulation of chromosomal aberrationsor cell death (6, 30, 37). Thus, humans with genetic defectsin DSB recognition or repair, or model organisms mimicking thesehuman syndromes, are prone to cancer and segmental prematureaging (21, 30).

There are two major mechanisms of DSB repair in eukaryotes:homologous recombination (HR)-mediated repair and nonhomologousend joining (NHEJ) (6). HR is an error-free mechanism in whichsequence information lost at a broken end is recovered fromthe sister chromatid. Therefore, HR is primarily restrictedto S and G₂ phases of the cell cycle, when a sister chromatidis available. NHEJ, on the other hand, rejoins two broken endsvia ligation. Thus, it is not restricted to proliferating cellsand this repair mechanism is frequently used in mammals (36).Since NHEJ does not require sequence homology, inappropriateends can potentially be joined, leading to chromosomal translocations(6, 30). In addition, bases may be lost at broken ends, resultingin deletions. However, NHEJ appears to be primarily error free(27, 81).

There are several well-defined error-prone mechanisms of DSBrepair in yeast (28). Both single-strand annealing (SSA) andmicrohomology-mediated end-joining (MMEJ) pathways align twobroken DNA ends by pairing homologous sequences at or near theDSB. In both mechanisms, if the homology is not immediatelyat the broken end, Rad10-Rad1 endonuclease, the ortholog ofERCC1-XPF, is required to remove the 3' flap of nonhomologoussequence from the end, permitting DNA synthesis and ligationand thereby creating a deletion. Despite their similarity, SSAand MMEJ appear to have distinct genetic requirements. SSA requiresRad52 and Rad10-Rad1, while for short patches of homology, Rad59,Msh2, and Msh3 are also implicated (28). In contrast, MMEJ isnot dependent on Rad52 or Ku86 but requires Rad10-Rad1, Mre11-Rad50-Xrs2(28), the flap endonuclease Sae2 (40), and mismatch repair proteinsMsh2 and Pms1 (10). DSB repair events that utilize short patchesof sequence homology were recognized in mammalian cells forquite some time (65). Evidence exists for SSA in mammalian cells(34, 42), particularly when HR or NHEJ is defective (76). NHEJmutant mammalian cells can still support end joining of DSBs(35) by utilizing short sequences of microhomology at the brokenends (38), demonstrating that MMEJ also occurs in mammaliancells. Recently, this alternative end-joining mechanism of microhomology-mediatedDSB repair was shown to support class switch recombination inNHEJ-deficient B cells (87). The genetic requirements for SSAand MMEJ and the biological significance of these pathways inmammals remain unknown.

In this study, we investigated if the 3' flap endonuclease ERCC1-XPFis involved in DSB repair in mammalian cells. ERCC1-XPF-deficientChinese hamster ovary cell lines were reported to be moderatelyhypersensitive to IR, particularly under hypoxic conditions(50, 86). In contrast, human XP-F fibroblasts and murine Ercc1^–/–embryonic stem (ES) cells are not hypersensitive to IR (50,52). To look more systematically for a role of the mammaliannuclease in DSB repair, we screened Ercc1^–/– mouseembryonic fibroblasts (MEFs), XPF-deficient human fibroblasts,and ERCC1-deficient mice for sensitivity to IR. We also useda genetic approach to determine if ERCC1-XPF is epistatic withNHEJ proteins with respect to sensitivity to IR.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Generation of cell lines and mice. Ercc1^–/– mouse ES cells were generated and culturedas previously described (52). Ercc1^–/–, DNA-Pk_cs^–/–,Ku86^–/–, and Csb^–/– primary MEFs weredeveloped from embryonic day 12 to 15 embryos derived from crossinginbred C57BL/6 mice heterozygous for each null allele, as previouslydescribed (13, 41, 43, 84). Ercc1^–/– Ku86^–/–and Ercc1^–/– DNA-Pk_cs^–/– double-knockoutprimary MEFs were created from embryos derived from crossingdouble-heterozygous mice in an inbred C57BL/6 background. GenomicDNA was isolated from a tissue sample of each embryo with theNucleoSpin DNA extraction system (Macherey-Nagel, Inc.). Genotypingof the Ercc1 allele was done by PCR coamplification of the 3'end of exon 7 from the WT allele and the neomycin resistancemarker cloned into exon 7 of the targeted allele with primersspecific for exon 7, neo^r, and intron 7 (5'-AGCCGACCTCCTTATGGAAA,5'-TCGCCTTCTTGACGAGTTCT, and 5'-ACAGATGCTGAGGGCAGACT, respectively).WT (0.25-kb) and mutant (0.4-kb) products were separated byelectrophoresis on a 2% agarose gel. Genotyping of the Ku86alleles by PCR was done as previously described (82). Genotypingof DNA-Pk_cs alleles was done by PCR with forward (5'-GCATCGCCTTCTATCGCCTT)and reverse (5'-GCTGAGACATCCTGGACTGAA) primers for the nullallele (0.4 kb) and the forward primer 5'-GGAATTGACTTTGGACATGCGwith the same reverse primer as for the WT allele.

MEFs were cultured in a 1:1 mixture of Dulbecco's modified Eagle'smedium and Ham's F10 with 10% fetal calf serum and antibioticsand incubated at 3% oxygen (59). Each experimental replica wasdone with a new MEF line, created from a unique embryo, in itssecond or third passage. Cell lines derived from WT littermateembryos were used as controls in all experiments. A spontaneouslytransformed Ercc1^–/– MEF line was stably transfectedwith a plasmid expressing human ERCC1 cDNA fused in frame withenhanced yellow fluorescent protein (YFP) (52). Human fibroblastsimmortalized with hTert derived from a normal individual (C5RO)and a patient with severe progeria caused by a mutation in XPF(XFE) (53) were cultured in Ham's F10 with 10% fetal calf serumand antibiotics and incubated at 3% oxygen.

Double-heterozygous (Ercc1^+/ Ku86^+/– and Ercc1^+/–Ku86^+/–) mice in a mixed genetic background (50:50 C57BL/6and FVB/n) were bred to recover Ercc1^–/ Ku86^–/–mice. DNA was extracted from an ear plug of 2-week-old pupsand genotyped by PCR as described above. The mutant allele ofErcc1 ( ${Delta}$ ) was amplified by adding a fourth primer (5'-CTAGGTGGCAGCAGGTCATC)to amplify the neo cassette in the ${Delta}$ allele (0.5 kb).

Clonogenic survival assays. Early-passage primary MEFs were seeded in 6-cm dishes in triplicateat 10³ to 10⁴ per plate, depending on the dose of genotoxin.After 16 h, cells were irradiated with a ¹³⁷Cs source. Sevento 10 days later, cultures were fixed and stained with 50% methanol,7% acetic acid, and 0.1% Coomassie blue. Colonies (defined as $≥$ 10 cells) were counted with a Nikon SMZ 2B stereomicroscopewith a 10x eyepiece. The data were plotted as the number ofcolonies that grew on the treated plates relative to untreatedplates ± the standard error of the mean for at leastthree independent experiments each with a unique cell line.

Immunofluorescence. Cells were trypsinized and seeded at 25% confluence on glasscoverslips. Sixteen hours later, the cells were irradiated with2 Gy of gamma rays. The cells were incubated in fresh mediumat 37°C for the indicated amount of time and then fixedwith 2% paraformaldehyde in phosphate-buffered saline, pH 7.4,for 15 min. Cells were permeabilized with 0.1% Triton X-100in phosphate-buffered saline, and the phosphorylated form ofH2AX ( ${gamma}$ H2AX) was detected with polyclonal anti- ${gamma}$ H2AX (1:1,000;Upstate Biotechnology) and Alexa 488-conjugated goat anti-rabbitimmunoglobulin (1:500; Molecular Probes) in phosphate-bufferedsaline with 0.15% glycine and 0.5% bovine serum albumin. ${gamma}$ H2AXfoci were counted with an Olympus BX51 fluorescent microscopewith a 60x to 100x objective.

Animal survival. Six-week-old WT and mutant animals (six per genotype) were exposedto 6 Gy of whole-body IR from a ¹³⁷Cs source, and their healthwas monitored daily. Animals were euthanized when deemed terminalin accordance with the University of Pittsburgh IACUC standards.Survival data were recorded as a Kaplan-Meier curve.

Histological analysis and immunohistochemistry. Tissues (liver, femur, and bowel) were collected from terminalgamma-irradiated Ercc1^–/ mice or 2 days postirradiation.Age-matched untreated and treated WT and untreated Ercc1^–/mice were sacrificed simultaneously as controls. Tissues werefixed with 10% formalin and embedded in paraffin. Five-micrometersections were cut and stained with hematoxylin and eosin. Proliferationof cells within the crypts of the small intestine was visualizedby immunostaining with a rat anti-mouse Ki67 monoclonal antibody(clone TEC-3; Dako North America, Inc., Carpinteria, CA) asrecommended by the manufacturer.

Cytogenetics. MEFs were transformed by stable transfection with a plasmidexpressing the simian virus 40 large T antigen and exposed to2 Gy (WT and Ercc1^–/–) or 0.4 Gy (Ku86^–/–and Ercc1^–/– Ku86^–/–) of IR. Bromodeoxyuridine(10 µM) and colcemid (0.1 µg/10 ml) were added tothe medium 48 and 36 h, respectively, prior to harvesting ofthe cells. The cells were harvested by trypsinization, and metaphasespreads were prepared as previously described (15). The numbersof fragments, breaks, fusions, radials, and marker chromosomesper diploid metaphase spread were determined.

Calculation of population doubling number. WT and mutant MEFs were plated at a density of 0.5 x 10⁶ per6-cm dish. Cells were trypsinized at confluence, counted, andreplated at the same density until double-knockout cells stoppedgrowing. The total number of cells at each passage was calculatedas follows: (no. of cells at previous passage/no. of cells plated)x no. of cells at current passage. The total cell number wasplotted as the log cell number.

In vitro DNA repair assay. WT and Ercc1^–/– MEFs were grown on 6-cm dishes andtransfected by using either a Nucleofector MEF II kit (AmaxaBiosystems, Gaithersburg, MD) or Lipofectamine 2000 reagent(Invitrogen, Carlsbad, CA) and following the manufacturer'sinstructions. pEYFP-N1 (Clontech Laboratories Inc., MountainView, CA) was linearized by digesting between the promoter andcoding sequence of YFP with SmaI (blunt ends), HindIII (5' complementary),or KpnI-SacI (3' noncomplementary ends). The linear productswere gel purified and transfected into WT or mutant MEFs. After48 h, cells were sorted and the fraction of cells expressingYFP was determined by a DakoCytomation MoFLo high-speed cellsorter (Dako North America, Carpinteria, CA). The efficiencyof repair was calculated as the percentage of YFP-positive cellsafter transfection with a linear plasmid, relative to transfectionwith the circular plasmid, from at least three independent experiments.In parallel, transfected plasmid DNA was recovered from MEFsby alkaline lysis (31). After phenol-chloroform extraction,DNA was ethanol precipitated and treated with lambda exonucleaseto remove any linear plasmid DNA (16, 44). The DNA was thenamplified in Escherichia coli, and individual plasmids wereisolated. The plasmids were screened by digestion with SmaI(blunt ends), HindIII (5' complementary ends), or EcoRI (3'noncomplementary ends) to distinguish between error-free (sensitiveto digestion) and error-prone (resistant to digestion) DSB repair.The plasmids were next screened for deletions by digestion withApaLI and ClaI, which yields two products of 2.9 and 1.7 kb,the latter of which will be diminished in size if a deletionoccurred at the site of the DSB. For plasmids containing smalldeletions, a 475-bp region flanking the site of the DSB wasPCR amplified with the forward primer 5'-TACATCAATGGGCGTGGATAand the reverse primer 5'-GAACTTCAGGGTCAGCTTGC. The amplifiedfragments were run on a 3% agarose gel to estimate the sizeof small deletions; the lower limit of detection was approximately25 bp. Finally, the junction where DSB repair occurred was sequencedfor 20 to 30 plasmids from each group (blunt or 5' or 3' overhangs)to determine the frequency with which microhomology was utilizedand bases were inserted.

RESULTS

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RESULTS

Sensitivity of ERCC1-XPF-deficient cells to IR. As a first screen to determine if ERCC1-XPF participates inDSB repair, cells harboring mutations in either protein subunitwere tested for sensitivity to IR by clonogenic survival assay.Telomerase-immortalized XPF-deficient human fibroblasts weresignificantly more sensitive to IR than were WT fibroblastsor HeLa cells (Fig. 1A), even though these cells are not completelydevoid of XPF (53). Similarly, Ercc1^–/– primaryMEFs, in which XPF protein is also undetectable (53), were 2.5-foldmore sensitive to IR relative to congenic WT MEFs (Fig. 1B).The hypersensitivity was rescued by stable transfection of theErcc1^–/– cells with human ERCC1 cDNA. In contrast,Ercc1^–/– mouse ES cells were not sensitive to IRrelative to a congenic WT cell line (Fig. 1C), as previouslyreported (52). These data reveal heterogeneity in sensitivityto genotoxic stress between different types of cells with defectsin ERCC1-XPF and indicate that this endonuclease is requiredto protect differentiated mammalian cells from IR.

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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.

Because IR induces numerous types of DNA lesions in additionto DSBs (e.g., single-strand breaks and oxidative base damage[23]), Ercc1^–/– cells were also screened for sensitivityto the oxidizing agent paraquat (see Fig. S1 in the supplementalmaterial). Ercc1^–/– ES cells were hypersensitiveto paraquat and H₂O₂ (53), whereas Ercc1^–/– primaryMEFs were not. The cell type-specific pattern of sensitivityis contrary to that of IR, indicating that the hypersensitivityof ERCC1-XPF-deficient MEFs to IR is not due to a failure torepair oxidative base damage.

Quantitation of IR-induced DSBs in ERCC1-XPF-deficient cells. To further probe the cause of the IR sensitivity of ERCC1-XPF-deficientcells, we measured phosphorylated histone variant H2AX, a markerof DSBs (63), in WT and ERCC1-XPF-deficient cells at multipletime points following IR exposure. ${gamma}$ H2AX foci provide a quantitativemeasurement of DSB induction and repair at low doses of irradiation(66). Cells were exposed to 2 Gy of IR, and ${gamma}$ H2AX foci were detectedby immunofluorescence (see Fig. S2 in the supplemental material).Foci were counted at 0, 4, 12, 24, and 48 h following irradiation.Cells were categorized as having no foci, one or two foci, ormore than two foci (Fig. 2). A 2-Gy dose induced multiple ${gamma}$ H2AXfoci in 100% of WT and XPF mutant fibroblasts. By 12 h postirradiation,75% of the WT cells no longer had ${gamma}$ H2AX foci (Fig. 2A) whereas>60% of the XPF-deficient cells still had multiple foci,suggesting continued accumulation or persistence of DSBs (Fig.2B). Similar results were obtained with Ercc1^–/–primary MEFs (Fig. 2C and D). In both human and murine mutantfibroblasts, the fraction of cells with ${gamma}$ H2AX foci decreasedat 24 and 48 h postirradiation, consistent with repair of DSBs,albeit substantially delayed relative to WT controls. Therewas no difference in the number of ${gamma}$ H2AX foci in WT and Ercc1^–/–ES cells at any time point following irradiation (Fig. 2E and F),correlating with the lack of sensitivity of these cells to IR.These data support a role for the ERCC1-XPF nuclease in facilitatingDSB repair in mammalian fibroblasts.

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FIG. 2. Quantitation of ${gamma}$ H2AX 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.

Sensitivity of ERCC1-deficient mice to IR. To determine if ERCC1-XPF contributes to protection againstIR in vivo, 6-week-old Ercc1^–/ mice, which are hypomorphicfor ERCC1-XPF (14, 84), and WT littermates (n = 6) were exposedto a single dose of 6 Gy of total-body irradiation. At thisage, Ercc1^–/ mice are healthy and have no obvious abnormalphenotype other than growth retardation (unpublished data).A 6-Gy dose of radiation did not acutely affect the survivalof WT mice (Fig. 3A and reference 41) but caused 100% lethalityof Ercc1^–/ mice by 11 days postirradiation. Although difficultto compare because of genetic background differences, the sensitivityof Ercc1^–/ mice is similar to that of DNA-Pk_cs^–/–mice (41) but less than that of Ku86^–/– mice (56),both of which lack a protein required for NHEJ. Therefore, ERCC1-XPFmakes a substantial contribution to protecting mammals fromradiation-induced DNA damage in vivo.

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FIG. 3. Sensitivity of ERCC1-deficient mice to IR. (A) Six-week-old Ercc1^–/ 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^–/ 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^–/ mice. (D) BM of untreated mice. (E) BM of mice exposed to IR demonstrating fatty replacement in Ercc1^–/ mice. (F) Livers of untreated mice. (G) Livers of mice exposed to IR demonstrating centrilobular necrosis in Ercc1^–/ mice. (H) Hepatocyte nuclei demonstrating ${gamma}$ H2AX foci in Ercc1^–/ mice 11 days after IR.

To determine the cause of death of the Ercc1^–/ mice, gastrointestinaltract, bone marrow (BM), and liver tissue sections from WT andErcc1^–/ mice 11 days postirradiation were examined. Thesetissues were indistinguishable between untreated Ercc1^–/and WT littermates (Fig. 3B, D, and F). However, following IR,Ercc1^–/ mice had dramatically fewer intestinal villi thanWT mice (Fig. 3C). Immunostaining for the proliferation markerKi67 (69) revealed considerably fewer positive cells in Ercc1^–/crypts compared to WT mice at 11 days postirradiation, but notat 2 days post-IR or in unexposed animals (see Fig. S3 in thesupplemental material). Postirradiation, the femoral BM of WTmice displayed hypercellularity of all hematopoietic lineagesconsistent with reactive hyperplasia (Fig. 3D and E). In contrast,the BM of Ercc1^–/ mice was markedly hypocellular withresidual cells displaying dysplastic changes including internuclearbridging, multinuclearity, and megablastoid changes, most prominentlyaffecting erythropoiesis. Granulopoiesis displayed a shift toimmature cell types. In the livers of Ercc1^–/ but notWT mice, this dose of IR induced centrilobular necrosis (Fig.3F and G). Immunostaining of liver sections for ${gamma}$ H2AX revealednuclear foci in hepatocytes of Ercc1^–/ mice but not WTlittermates (Fig. 3H), indicative of persistent DSBs. Collectively,these data indicate that proliferative tissues of Ercc1^–/mice are vulnerable to IR-induced genotoxic stress and havediminished regenerative capacity following damage.

ERCC1 deficiency causes embryonic lethality in a Ku86^–/– background. Very recent evidence indicates that ERCC1-XPF facilitates SSAbetween direct repeats in mammalian cells (2) as do the orthologsRad10-Rad1 in S. cerevisiae (28). In yeast, Rad10-Rad1 alsoparticipates in MMEJ, a Ku-independent mechanism of DSB repair(45). This pathway was recently confirmed in mammals (87). Totest if this function of ERCC1-XPF is conserved in mammals,we attempted to breed mice that are doubly deficient in ERCC1and Ku86. Surprisingly, no live Ercc1^–/ Ku86^–/–mice were recovered (see Table S1 in the supplemental material;0 of 220 offspring, 14 expected; P < 0.001). Genotyping ofpups that died within 24 h of birth also failed to identifydouble-mutant mice. This was unexpected since both Ku86^–/–and Ercc1^–/ mice are born with Mendelian frequency andlive into adulthood, although they have reduced life spans (88and 30 weeks, respectively [82, 84; unpublished results]). Inmammals, combined loss of Ku86 and ERCC1 thus results in lethality,similar to the effect of deleting Ku and Rad1 in budding yeast(45). This supports the conclusion that ERCC1-XPF contributesto a DSB repair pathway that is Ku independent, distinct fromNHEJ and other Ku-dependent mechanisms.

Ercc1^–/– Ku86^–/– MEFs senesce prematurely. To generate Ercc1^–/– Ku86^–/– MEFs, double-heterozygousErcc1^+/– Ku86^+/– mice were crossed. Ercc1^–/–Ku86^–/– embryos (embryonic days 13 to 16) were recoveredat Mendelian frequency (see Table S2 in the supplemental material),indicating that deficiency of both ERCC1 and Ku86 causes lethalitylate in embryonic development. MEFs derived from Ercc1^–/–Ku86^–/– embryos grew extremely poorly in culturecompared to single-mutant cell lines derived from littermates(Fig. 4A; see Fig. S4 in the supplemental material). Even whenthe MEFs were cultured at 3% O₂, which alleviates prematuresenescence due to oxidative stress (59), Ercc1^–/–Ku86^–/– MEFs did not proliferate beyond passage5. This indicates that simultaneous deletion of Ercc1 and Ku86causes premature senescence of primary cells and that cellularproliferation in an oxidative environment requires either Ku-dependentor ERCC1-dependent DNA repair.

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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.

Ercc1^–/– Ku86^–/– MEFs are hypersensitive to IR. To determine if Ercc1 is epistatic with Ku86 or DNA-Pk_cs withrespect to DSB repair, Ercc1^–/– Ku86^–/–and Ercc1^–/– DNA-Pk_cs^–/– cells werescreened for sensitivity to IR. Since primary Ercc1^–/–Ku86^–/– MEFs rapidly senesce, one primary cell lineof each genotype was transformed by stable transfection witha plasmid expressing simian virus 40 large T antigen (83). TransformedErcc1^–/–, Ku86^–/–, and DNA-Pk_cs^–/–single-mutant MEFs were hypersensitive to IR (Fig. 4B), as shownin Fig. 1B and as previously reported (8, 41, 43). Unexpectedly,Ercc1^–/– DNA-Pk_cs^–/– MEFs appear tobe equally as sensitive to IR as DNA-Pk_cs^–/– cells.However, Ercc1^–/– Ku86^–/– MEFs weresignificantly more sensitive to IR than either Ercc1^–/–or Ku86^–/– cells, consistent with the additive effectof both mutations in vivo. These results provide further evidencethat ERCC1-XPF participates in a DSB repair mechanism that providesa critical back-up for Ku-dependent DSB repair, as do the yeastorthologs Rad10-Rad1.

Genomic instability in Ercc1^–/– MEFs after IR. To further investigate whether the hypersensitivity of ERCC1-XPF-deficientcells to IR is caused by DSBs, cells were examined for chromosomalabnormalities after exposure to low-dose IR. Transformed WTand Ercc1^–/– MEFs were treated with 2 Gy, whileKu86^–/– and Ercc1^–/– Ku86^–/–MEFs were treated with 0.4 Gy due to their extreme hypersensitivityto IR (Fig. 4A and reference 43). Sister chromatid exchanges(SCEs), which occur via HR of DSBs (75), were significantlyincreased in Ercc1^–/– MEFs relative to WT cells(Table 1). These data demonstrate that homologous recombinationis not affected by loss of ERCC1-XPF nuclease. In contrast,the frequency of SCEs was not increased in irradiated Ercc1^–/–Ku86^–/– cells relative to Ku86^–/– cells.This may reflect inefficient processing of radiation-inducedbreaks lacking 3' OH groups, which are necessary for HR, whenboth Ku and ERCC1-XPF are present. Chromosomal aberrations weresignificantly increased (about fivefold) in Ercc1^–/–MEFs treated with IR compared to WT cells (Table 1 and Fig.5A and B). These abnormalities include gaps, fragments, breaks,fusions, and radials, all characteristic of unrepaired or misrepairedDSBs. Gaps, breaks, fusions, and radials were also significantlyincreased in Ercc1^–/– Ku86^–/– MEFs relativeto Ku86^–/– cells (Table 1 and Fig. 5C and D), providingfurther evidence that ERCC1-XPF participates in a DSB repairmechanism that functions as a back-up for Ku86-dependent repair.

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TABLE 1. Frequency of chromosomal aberrations in transformed MEFs exposed to IR

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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).

ERCC1-XPF facilitates removal of noncomplementary 3' overhangs. To determine if ERCC1-XPF participates in an end-joining mechanismof DSB repair similar to Rad10-Rad1, we used an in vitro DNAend-joining assay (67). A plasmid expressing YFP (pYFP-N1) waslinearized by digestion with restriction enzymes that produceblunt, complementary 5' overhangs or noncomplementary 3' overhangs.These linear plasmids were transfected into WT, Ercc1^–/–,Ku86^–/–, and Ercc1^–/– Ku86^–/–cells, and repair events were scored as the percentage of YFP-expressingcells (see Fig. S5 in the supplemental material). Creation ofa DSB between the promoter and YFP cDNA reduced the recoveryof YFP⁺ cells by threefold. There was no difference in the frequencyof repair of DSBs with either blunt or single-strand overhangingends. Furthermore, there was no difference in the frequencyof repair events in WT, Ercc1^–/–, or Ku86^–/–cells transfected with any of the repair substrates. The onlysignificant difference observed was decreased recovery of YFP⁺Ercc1^–/– Ku86^–/– cells after transfectionwith a linear plasmid containing single-strand overhangs. Thisindicates that, in the absence of both ERCC1-XPF and Ku86, DSBsthat cannot be directly ligated are inefficiently repaired.

Repaired pYFP-N1 plasmid DNA was recovered from WT and Ercc1^–/–YFP⁺ cells 48 h after transfection. The plasmid DNA was digestedwith the same enzyme that was used for linearization prior totransfection to score for error-free repair events. Approximatelyhalf of the repair events were error free for DNA with bluntor complementary single-strand overhanging ends in WT and Ercc1^–/–cells (Table 2), while all of the repair events were error pronefor the DNA with noncomplementary single-strand overhangs, asexpected. Error-prone events were divided into three categoriesbased on the size of the deletion, as determined by agarosegel electrophoresis: <100 bp, 100 to 500 bp, and >500bp. There was no difference in the size of the deletions resultingfrom repair of blunt ends or 5' complementary overhangs betweenWT and Ercc1^–/– cells. However, there was a significantincrease in very large deletions in substrates with 3' noncomplementaryoverhangs repaired in Ercc1^–/– cells relative toWT cells. This indicates that, in the absence of ERCC1-XPF,repair of DSBs with 3' ends that cannot be directly ligatedis impaired, leading to increased resection and larger deletions.This is consistent with the substrate specificity of ERCC1-XPFas a 3' flap endonuclease (11, 74).

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TABLE 2. Size of linear plasmid with blunt or overhanging DNA ends after repair in WT or Ercc1^–/– cells

A subset of the plasmids with deletions were sequenced to determinethe frequency with which DSB repair occurred via a mechanismthat utilizes sequence homology between the broken ends (seeTables S3 to S5 in the supplemental material). Short stretchesof microhomology (one to five nucleotides) were identified atthe broken ends in 70 to 90% of the error-prone DSB repair events,regardless of whether the ends were blunt or had overhangs (Table3). There was not a significant difference in the utilizationof microhomology to repair DSBs in WT and Ercc1^–/–cells. However, significantly fewer of the repair events inErcc1^–/– cells resulted in the insertion of basesrelative to the WT. This observation is consistent with ERCC1-XPFbeing required to remove 3' nonhomologous flaps to create anend that can be used to prime DNA synthesis (74).

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TABLE 3. Sequence analysis of blunt, complementary, and noncomplementary DNA ends after repair in WT or Ercc1^–/– cells

DISCUSSION

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DISCUSSION

Mammalian ERCC1-XPF participates in DSB repair. ERCC1-XPF is a structure-specific endonuclease that incisesdouble-stranded DNA at a junction with single-stranded DNA (3'),nicking bubble structures and 3' single-strand overhangs (11).This activity is essential for excising damaged DNA in NER (74)and for creating a 3' end that can be used to prime DNA synthesisto replace the excised bases. In addition to the bubble substratesin NER, ERCC1-XPF incises stem-loop structures (11), cross-linkedY structures (39), and R loops in vitro (78) and removes 3'single-strand overhangs of nonhomologous sequence that otherwisewould prevent HR (1, 52, 68). It has also been proposed thatERCC1-XPF incises D loops (52), G-rich telomeric overhangs (88),and intermediates of DNA ICL repair (54). Such a flap endonucleaseactivity could facilitate DSB repair, specifically, of endswith 3' single-strand noncomplementary overhangs or withouta 3' hydroxyl group necessary for DNA synthesis and/or ligation.Indeed, orthologs of ERCC1-XPF in S. cerevisiae (Rad10-Rad1)(22, 32, 58), Arabidopsis thaliana (AtErcc1-AtRad1p) (17, 29),and Drosophila melanogaster (DmERCC1-MEI-9) (24, 61) do facilitateDSB repair and confer resistance to IR. Herein, we provide invitro, genetic and in vivo evidence that this function is conservedin murine and human cells.

Role of ERCC1-XPF in DSB repair. ERCC1-XPF-deficient cells are exquisitely sensitive to cross-linkingagents and accumulate DSBs in response to cross-link damage(54). We postulated that in ICL repair, ERCC1-XPF is requiredto incise the cross-link lesion after a DSB is created by stalledreplication, before the DSB can be repaired. Here we provideevidence for a distinct role for ERCC1-XPF in DSB repair. ERCC1-XPF-deficientmurine and human cells are hypersensitive to IR (Fig. 1), similarto cells defective in HR-mediated or NHEJ DSB repair (77). Murinefibroblasts, but not ES cells, are IR sensitive (Fig. 1). Thispattern of cell type-specific sensitivity is similar to thatof DNA-Pk_cs mutant lines defective in NHEJ (25) but the oppositeof that of Rad54^–/– cells defective in HR (19).This suggests that ERCC1-XPF may participate in an end-joiningmechanism of DSB repair that is more frequently utilized indifferentiated cells than in ES cells. In further support ofthis, SCEs are significantly elevated in irradiated Ercc1^–/–MEFs (Table 1), indicating that HR-mediated DSB repair is possiblein the absence of ERCC1-XPF. This increase in SCEs might reflectpreferential repair of DSBs by HR when end-joining mechanismsare impaired (85). ERCC1-XPF, however, is clearly not part ofthe core NHEJ machinery, as there is no evidence of severe combinedimmunodeficiency, a pathognomonic feature of NHEJ deficiency(87), in Ercc1^–/– or Xpf^–/– mice (70,79) or humans with XPF deficiency (53). Furthermore, Ercc1^–/–MEFs are less sensitive to IR than are DNA-Pk_cs^–/–or Ku86^–/– MEFs, in which NHEJ is absent (Fig. 4B).

IR induces a variety of lesions in addition to DSBs (23). Thus,to confirm that the IR sensitivity of ERCC1-XPF-deficient cellsis due, at least in part, to a defect in DSB repair, we quantitated ${gamma}$ H2AX foci, a marker of DSBs (63), in cells following IR. ${gamma}$ H2AXfoci persist in irradiated ERCC1-XPF-deficient fibroblasts (Fig.2) and in hepatocytes of irradiated ERCC1-deficient mice (Fig.3H) relative to congenic WT controls, consistent with impairedDSB repair in the absence of ERCC1-XPF both in vitro and invivo. Furthermore, irradiated Ercc1^–/– MEFs hadincreased gaps, breaks, fragments, and radial structures comparedto WT cells (Table 1). These chromosome abnormalities are aconsequence of DSBs (49) and thus demonstrate a defect in DSBrepair in Ercc1^–/– cells. Interestingly, there isno difference in the number of ${gamma}$ H2AX foci in Ercc1^–/–and WT ES cells at any time point following exposure to IR,correlating with their lack of sensitivity to IR. There is increasingevidence that homologous recombination is essential for genomestability in ES cells (6, 26, 62), further suggesting that HRoccurs in ERCC1-deficient cells.

Biological significance of ERCC1-XPF in DSB repair. A single dose of 6 Gy of IR is universally lethal to mice hypomorphicfor ERCC1 (Fig. 3A), whereas 2 Gy is sublethal (data not shown).This level of sensitivity is similar to that of mice in whichDNA-PK_cs, an essential component of NHEJ, is deleted (41), eventhough ERCC1-XPF expression is reduced only 85% in the hypomorphicstrain (84). This establishes an important role for ERCC1-XPFin the radioprotection of mammals. IR causes loss of intestinalvilli and BM hypocellularity in ERCC1-deficient mice, as anticipated(Fig. 3). In addition, ERCC1-deficient mice display centrilobularnecrosis of the liver, which is caused by occlusion of smallvenules with cellular debris, following IR (20). Liver pathologyis life limiting for Ercc1^–/– mice (72), explainingthe particular hypersensitivity of Ercc1^–/ mouse liverto IR. Mice defective in DSB repair have impaired hematopoieticstem cell function and regenerative capacity after stress (55,64). We observed significantly reduced proliferative reservein the BM of Ercc1^–/– mice (60) and in the intestinefollowing IR (see Fig. S3 in the supplemental material). Thus,the hypersensitivity of Ercc1^–/ mice to IR likely is aconsequence not only of increased cell death/senescence in responseto unrepaired DNA damage but also reduced capacity to regeneratedamaged tissues.

Although ERCC1 hypomorphic mice are equally as sensitive toIR as DNA-Pk_cs^–/– mice are, they are less sensitivethan Ku86^–/– mice (9, 56). DNA-Pk_cs^–/–mice do not have a spontaneous phenotype other than severe combinedimmunodeficiency (25, 41), whereas Ku86^–/– micedisplay growth retardation and progeroid symptoms in additionto immunodeficiency (82). Ercc1^–/– mice have a moresevere phenotype than either DNA-Pk_cs^–/– or Ku86^–/–mice, including growth retardation, liver dysfunction, renalinsufficiency, epidermal atrophy, impaired hematopoiesis, sarcopenia,kyphosis, and neurodegeneration (48, 53, 60, 84), making itunlikely that the phenotype of Ercc1^–/– mice canbe ascribed in its entirety to a defect in DSB repair. Moreplausibly, it is the combined defects in multiple DNA repairpathways including global-genome and transcription-coupled NER,ICL repair, and DSB repair, documented here, that contributeto the severe phenotype of Ercc1^–/– mice, XFE progeria(53), and a recently identified ERCC1 patient (33).

ERCC1-XPF participates in error-prone, Ku-independent end joining of DSBs. ERCC1-XPF endonuclease facilitates DSB repair and protects againstIR in vivo. Yet the two major mechanisms of DSB repair in mammaliancells, HR and NHEJ, are intact in ERCC1-XPF-deficient cells.Thus, the following question remains: how does this nucleasecontribute to DSB repair? In yeast, the orthologs of ERCC1-XPF,Rad10-Rad1, are required for two error-prone DSB repair mechanisms:SSA (22, 32, 57) and MMEJ (45). Rad10-Rad1 are required to removenonhomologous sequence from the 3' ends of the break to permitDNA synthesis and/or ligation. MMEJ in yeast is independentof the HR protein Rad52 and the NHEJ protein Ku86 and criticalfor protection against IR (45). In rad52; yku70 strains, DSBrepair is error prone, leading to large deletions and utilizingshort homologies between ends (5). Recently, an alternativemechanism of end joining, distinct from NHEJ, was identifiedin mammals (87). This mechanism supports class switch recombinationin the absence of NHEJ, is error prone, and utilizes microhomologyto join broken ends, analogous to MMEJ in yeast. Linearizedplasmids and I-Sce-I-induced genomic DSBs are repaired in cellsdeficient in NHEJ, most notably Ku86, leading to deletions andinsertions and utilizing microhomology (27, 35, 71). However,the biological significance of this pathway in mammals, whenNHEJ is present, remains uncertain.

Our data provide several lines of evidence that support theconclusion that ERCC1-XPF contributes to this alternative mechanismof end joining in mammalian cells. First, Ercc1^–/–Ku86^–/– MEFs are hypersensitive to IR relative tosingle-mutant cells (Fig. 4B), similar to yeast (45). In addition,Ercc1^–/– Ku86^–/– cells accumulate significantlymore chromosomal aberrations in response to low-dose IR comparedto cells defective in NHEJ only (Ku86^–/– cells;Table 1). Furthermore, Ercc1^–/– Ku86^–/–primary MEFs proliferate poorly, surviving only to passage 5,even when cultured at 3% oxygen (Fig. 4A). These data are consistentwith the conclusion that ERCC1-XPF participates in a mechanismof DSB repair that is Ku86 independent and important for protectionagainst IR and oxidative stress, analogous to the role of Rad10-Rad1in yeast.

The second line of evidence that supports a role for ERCC1-XPFin MMEJ comes from the in vitro end-joining assay. Joining ofblunt 5' or 3' overhangs is not significantly reduced in Ercc1^–/–or Ku86^–/– cells compared to WT cells (see Fig.S5 in the supplemental material). However, joining of ends withoverhangs is significantly impaired in Ercc1^–/–Ku86^–/– cells, suggesting loss of two distinct mechanismsof end joining in these double-mutant cells. Ercc1^–/–cells are defective in joining ends that cannot be directlyligated, i.e., ends with 3' noncomplementary overhangs but notblunt ends or ends with 5' complementary overhangs. In the absenceof ERCC1-XPF, joining of nonhomologous 3' overhangs leads tovery large deletions (Table 2), similar to yeast mutants (45).Furthermore, in Ercc1^–/– cells, insertion of nucleotidesat the joints is significantly reduced (Table 3), consistentwith the conserved role of this nuclease in creating a 3' endthat can prime DNA synthesis (58). In addition, there is a trendtoward decreased use of microhomology in end joining in Ercc1^–/–cells relative to WT cells (Table 3). Deletion of Rad1 diminishesMMEJ to a greater extent in yeast but does not abrogate MMEJ,indicating the presence of redundant 3' flap endonucleases inyeast (45) and therefore likely also in mammals. Recently, itwas demonstrated that telomere fusion likely occurs via a Ku-independentend-joining mechanism (7). Thus, the participation of ERCC1-XPFin this alternative end-joining mechanism is also supportedby the fact that ERCC1-XPF trims unprotected telomeric overhangs,allowing telomeric fusion via end joining (88).

In total, these data demonstrate a novel function of ERCC1-XPFnuclease in a Ku86-independent mechanism of DSB repair. Further,our data, in combination with a recent report establishing arole for ERCC1-XPF in SSA (2), indicate that Rad10-Rad1 activitiesin DSB repair are conserved in mammals. The data also suggestthat alternative end joining of DSBs is important for protectionagainst DSBs even when NHEJ and HR repair can occur.

ACKNOWLEDGMENTS

This work was supported by the University of Pittsburgh CancerInstitute, NCI and NIA CA103730 and CA111525, the PennsylvaniaDepartment of Health, and The Ellison Medical Foundation (AG-NS-0303).J.H.J.H. is supported by the Nederlandse Organisatie voor WetenschappelijkOnderzoek (NOW), Cancer Genomics Center, the Association forInternational Cancer Research, and the European Commission (IP512113), RISC-RAD contract F16R-CT-2003-508842.

We thank Richard D. Wood for careful reading of the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: University of Pittsburgh Cancer Institute, Hillman Cancer Center, Research Pavilion 2.6, 5117 Centre Avenue, Pittsburgh, PA 15213-1863. Phone: (412) 623-7763. Fax: (412) 623-7761. E-mail: niedernhoferl{at}upmc.edu

Published ahead of print on 9 June 2008.

Supplemental material for this article may be found at http://mcb.asm.org/.

REFERENCES

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