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Previous Article | Next Article 
Molecular and Cellular Biology, September 2005, p. 7569-7579, Vol. 25, No. 17
0270-7306/05/$08.00+0 doi:10.1128/MCB.25.17.7569-7579.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Hui Gao,2,
Dieder Moechars,3
Michel Janicot,1
Jorge Vialard,1 and
Clare H. McGowan2,4*
Department of Molecular Biology,2 Department of Cell Biology, The Scripps Research Institute, La Jolla, California 92037,4 Oncology Discovery Research and Early Development Europe,1 Functional Genomics Johnson & Johnson Pharmaceutical Research and Development, Turnhoutseweg 30, B-2340 Beerse, Belgium3
Received 16 May 2005/ Returned for modification 3 June 2005/ Accepted 15 June 2005
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ABSTRACT |
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INTRODUCTION |
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The subunits of the mammalian Mus81-Eme1 endonuclease complexes were identified by sequence similarity to Mus81-Eme1 (Mms4) from Saccharomyces cerevisiae and Schizosaccharomyces pombe (1, 7, 12, 14, 44). Haploid yeast Mus81 mutants are sensitive to agents that result in replication fork collapse but not to those that generate DSBs (9, 20, 28, 36, 57). Thus, Mus81-Eme1 is specifically involved in the process of replication restart in yeast. In addition, fission yeast Mus81-Eme1 is essential for meiotic recombination (8, 24, 46, 53). In budding yeast, Mus81 and its partner, Mms4, have a lesser role in meiotic recombination (17, 18); however, as in fission yeast, Mus81-Mms4 mutants have a severe recombination-dependent meiotic defect (28, 30). In vitro, Mus81-Eme1 cleaves a number of synthetic DNA substrates, including 3' flaps, replication forks, nicked Holliday junctions, and Holliday junctions (12, 15, 24, 30, 46). These structures are designed to mimic potential in vivo substrates; however, how closely in vitro specificity reflects in vivo function is not known. To more directly determine the function of Mus81-Eme1 in mammalian cells, we have disrupted the mouse Mus81 gene and analyzed the effect Mus81 deficiency has in vitro and in vivo. We find that Mus81-deficient mice and cells have significantly increased sensitivities to MMC-induced toxicity. Mus81 is not required for the formation of DSBs after cross-link damage, but it is required for their resolution. Unlike yeast Mus81 mutants, murine cells that lack Mus81 are relatively resistant to camptothecin (CPT), suggesting that alternative or redundant mechanisms ensure replication restart in mammalian cells. Significantly, despite increased sensitivity to agents that induce DNA interstrand cross-links and evidence that Mus81-deficient cells suffer increased chromosomal aberrations in vitro, no increase in the incidence of spontaneously occurring tumors or malignancy is seen in Mus81-deficient mice. In this respect, the data presented here differ significantly from those reported in a different Mus81–/– mouse model (39).
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture. Mouse embryonic fibroblasts (MEFs) were isolated from mixed-genotype littermate embryos at 13.5 days postcoitum. Primary MEFs were harvested and processed for DNA content following 2 days in culture. Twenty-four hours after plating, primary MEFs were infected with LXSN retrovirus containing the coding sequence for human papillomavirus type 16 E6 and cultured in Dulbecco's modified Eagle medium supplemented with 10% calf serum and 100 µg/ml penicillin and streptomycin.
Prior to flow cytometry, cells were cultured in the presence of 10 µM bromodeoxyuridine (Calbiochem) for 30 min and fixed in 70% ethanol. Cells were stained with fluorescein isothiocyanate-anti-bromodeoxyuridine (Becton Dickinson) according to the manufacturer's protocol. After several washes, cells were resuspended in phosphate-buffered saline containing 5 µg/ml propidium iodide and 100 µg/ml RNase for 30 min before fluorescence-activated cell sorter analysis (FACSCalibur; Becton Dickinson). The cell cycle distribution of each sample was calculated using Cell Quest software.
Genomic stability assay. To prepare metaphase spreads, cells were incubated in 500 ng/ml nocodazole (Sigma) for 4 h, and 0.1 µg/ml demecolcine (Sigma) was added to the medium for the last hour. Cells were harvested and resuspended in hypotonic buffer (0.075 M KCl) at 37°C for 10 min. After fixation in methanol:acetic acid (3:1), cells were dropped onto glass slides and stained with Giemsa stain (Sigma) for 10 min. Chromosome aberrations, including chromosome breaks, fragment and chromosomal fusions, and dicentricity, were analyzed by direct visualization using a 100x objective. Twenty-five to 30 metaphases from each cell type were scored.
Colony survival and whole-animal sensitivity assay. To determine cellular sensitivity to DNA-damaging agents, 100 or 500 cells were seeded in six-well plates. Cells were cultured continuously in the concentrations of MMC, CPT and hydroxyurea (HU) indicated below. cis-Platin and nitrogen mustard were added to the culture media for 2 h, and the cells were then cultured in drug-free medium until colonies were visible (10 to 14 days). Where indicated, cells were exposed to UV-C light with a wavelength of 254 nm or were irradiated by use of a cesium 137 source. Following 10 to 14 days in culture, cells were fixed in methanol and stained with Giemsa stain, and colonies were counted. Triplicate cultures were scored for each treatment.
The ability of human Mus81 to complement the MMC sensitivity of Mus81–/– MEFs was assayed by transient transfection. Approximately 0.5 x 106 cells were transfected with pCDNA-3HaMus81WT (wild type); with pCDNA-3HaMus81DD, an endonuclease-dead mutant of Mus81 (12); and with a control vector expressing green fluorescent protein, pcDNAGFP. Effectene from QIAGEN was used for all transfections. Parallel transfection with pCMU-ß-galactosidase was used to determine that 
50% of cells were transfected. Twenty-four h after transfection, 100 or 500 cells were seeded in six-well plates, and an indicated concentration of MMC was added for 2 h. Cells were washed and incubated in fresh media until colonies were visible. The remaining cells were cultured for 72 h and then used to check for the expression of the Mus81 protein by use of Western blotting.
Whole-animal sensitivity studies. Mice (12 to 18 weeks of age) were injected intraperitoneally with a single dose of MMC at 5 mg/kg of body weight and 10 mg/kg. Survival and body weight measurements were recorded daily. The percentages of survival were calculated according to the Kaplan and Meier method (31). Differences in survival between the different groups were tested for significance by the Wilcoxon-Gehan test.
Affinity purification of Mus81 antibody and immunoblotting. Affinity-purified mouse Mus81 antibody was generated by purifying Mus81 serum (12) over GST-mus-Mus81 full-length protein that had been cross-linked to glutathione-Sepharose by use of dimethylpimelimidate (Pierce). For immunoblotting, cells were lysed in 20 mM HEPES, pH 7.4, 150 mM NaCl, 5% glycerol, 0.1% NP-40, 0.1% ß-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 5 µg/ml each of leupeptin, pepstatin, and aprotinin. Lysates were cleared by centrifugation at 10,000 x g for 10 min. Protein concentration was determined using Bradford reagent. One hundred micrograms of cell lysate was resolved on 10% acrylamide-sodium dodecyl sulfate gels. Immunoblots were incubated in phospho-Chk1 (Ser345) (1:1,000; Cell Signaling), Chk1 and tubulin (1:1,000; Santa Cruz), hemagglutinin (1:5,000; Covance), and PCNA (1:5,000; Santa Cruz). The incubations were followed by treatment with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antiserum. Chemiluminescence was used to detect the respective proteins.
Immunofluorescence.
Cells were grown on cover slides overnight before exposure to 3 µM MMC or 1 nM CPT for 1 h. Cells were washed and incubated in fresh media for the amounts of time indicated below. In situ fractionation was performed prior to fixation (40). Cells were fixed in 4% formaldehyde, permeabilized in 0.5% Triton X-100, blocked with 10% calf serum, and then incubated with primary antibody overnight in 4°C and with secondary antibody for 1 h at room temperature. Cells were counterstained with 4',6'-diamidino-2-phenylindole (DAPI) and mounted in 70% glycerol. Primary antibody dilutions were of 
H2AX (Trevigen) at 1:150 and Rad51 (Abcam) at 1:100. Alexa-fluor 488- or 546-conjugated anti-mouse or anti-rabbit immunoglobulin G (Molecular Probes) was used at a dilution of 1:1,000. Images were captured using a charge-coupled-device camera (Photometrics). Gray-scale images were processed using Adobe Photoshop 7.0. Three hundred cells were counted for each sample. Cells containing two or more distinct foci were scored as positive.
Pulsed-field gel electrophoresis. Cells were either not treated or treated with 3 µM MMC for 1 h and then incubated in fresh media for an additional 24 h. Approximately 1 x 106 cells were collected and embedded in 0.85% agarose insert prepared with a contour-clamped homogeneous electric field disposable plug mold (Bio-Rad). The agarose inserts were incubated in proteinase K (1 mg/ml) at 50°C for 48 h and thereafter washed four times in 10 mM Tris-Cl, pH 7.5, 1 mM EDTA prior to loading onto a 0.85% agarose gel. Pulsed-field gel electrophoresis was carried out with a contour-clamped homogeneous electric field DRIII system (Bio-Rad). The gel was run at 14°C for 24 h at 4 V/cm at a 120° angle with a 60- to 120-s switch time for 24 h. DNA was visualized by ethidium bromide staining.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The slight but constitutive checkpoint activation seen in Mus81–/– cells is suggestive of increased levels of spontaneous DNA damage. In cells with compromised p53 function, DNA damage could lead to increased levels of chromosomal abnormalities; therefore, metaphase spreads were examined for the presence of chromosome breaks, fusions, and triradials (Fig. 2D). An approximately threefold increase in the incidence of spontaneous chromosomal abnormalities was detected in cells from Mus81–/– mice compared to wild-type controls (P < 0.05). No significant difference in the incidence of chromosomal aberrations was seen in Mus81+/– MEFs compared to that in Mus81+/+ MEFs (Fig. 2E).
Damage sensitivity of Mus81-deficient MEFs. Mus81-deficient cells and animals have previously been shown to be hypersensitive to the DNA cross-linking agent MMC (39). How Mus81-Eme1 acts to repair MMC-induced damage has not been determined. The sensitivity of Mus81–/– MEFs to MMC and the role of Mus81 in the generation and resolution of MMC-induced lesions were examined. Mus81-deficient MEFs were found to be hypersensitive to MMC, whereas Mus81+/– MEFs had the same level of resistance as wild-type cells did (Fig. 3A). Expression of wild-type human Mus81, but not of a point mutant of Mus81 that lacks endonuclease activity, partially restored resistance to MMC (Fig. 3B and C). The relative insensitivity of Mus81+/– cells could be explained if 50% expression of Mus81 protein is sufficient to protect cells from the toxic effects of DNA cross-linking agents or if the heterozygous cells compensate for the reduced gene dosage by expressing increased levels of protein. Western blot analysis was used to distinguish between these possibilities. Mus81 antibodies were purified using full-length murine Mus81 fused to glutathione S-transferase as an affinity matrix. The resulting antibodies recognized both N-terminal and C-terminal portions of Mus81 (data not shown). Western blot analysis showed that the expression of Mus81 in heterozygous cells was very similar (between 80 and 100%) to that of wild-type cells (Fig. 3D). The relative resistance of heterozygous cells to MMC and cis-platin is thus most likely explained by the fact that cells compensate for the reduced gene dosage by increased protein expression. A longer exposure of the entire Western blot did not reveal any novel bands in the lysates from either null or heterozygote cells. A protein of 36 kDa would be expected if the disrupted allele allowed expression of the 5' exons.
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radiation, UV light, or hydroxyurea (Fig. 3H through J).
The repair defect in Mus81–/– cells.
MMC and other agents that generate ICLs are extremely toxic to cells that are undergoing DNA replication, and this toxicity correlates with the generation of DSBs (2, 19, 38). However, the mechanism by which the breaks are generated is not known. The Mus81-Eme1 endonuclease efficiently cleaves 3' flaps in vitro (14). This enzymatic activity predicts a role for Mus81 in the generation of DSBs by the direct cleavage of blocked replication forks (5, 41). To determine if Mus81 is required for the generation of DSBs in MMC-treated cells, we first looked at the kinetics of -H2AX focus formation by immunofluorescence. -H2AX, the phosphorylated form of the histone variant H2AX, is rapidly and extensively phosphorylated in large regions of chromatin adjacent to DSBs (49). The presence of -H2AX is widely used as a marker for DSBs. Consistent with the elevated phosphorylation of Chk1 seen in Mus81–/– MEFs, the number of Mus81-deficient cells that have -H2AX foci was elevated prior to treatment (28.7% ± 1% compared to 20.6% ± 0.6% in wild-type cells [P < 0.05]) (Fig. 4A). Following a 1-h treatment with MMC, the number of wild-type cells with -H2AX foci increased for 16 h (Fig. 4B). The kinetics of -H2AX focus formation is consistent with the need for MMC-exposed cells to pass through S phase to generate DSBs (2, 38). Having reached a peak in which 70% of cells had -H2AX foci, the numbers of wild-type and Mus81+/– cells with -H2AX foci gradually declined to basal levels within 48 h of drug treatment. Mus81–/– MEFs accumulated -H2AX foci at the same rate as wild-type cells did, but the percentage of cells with -H2AX foci remained constant at 70% for the duration of the experiment. Although -H2AX foci are widely viewed as representing DSBs, the possibility that other forms of damage might also induce H2AX phosphorylation cannot be excluded. We therefore used pulsed-field gel electrophoresis to confirm that MMC induces DSBs in Mus81–/– cells. Under the electrophoretic conditions used, intact genomic DNA is too large to enter the gel. The presence of DSBs is revealed as lower-molecular-weight DNA that is able to enter the gel. Low-molecular-weight DNA was not detected in untreated wild-type MEFs (Fig. 4C). However, a small amount of DNA from untreated Mus81–/– cells entered the gel. MMC treatment of wild-type cells resulted in detectable amounts of lower-molecular-weight DNA. MMC treatment of Mus81–/– cells also resulted in increased amounts of low-molecular-weight DNA. Taken together with the -H2AX focus formation data, these data suggest that Mus81 is not required for the generation of DSBs following MMC treatment.
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-H2AX foci in response to DNA damage. However, Rad51 foci are found only in replicative or postreplicative cells. Focus formation is dependent on a number of upstream activities, and the foci are thought to represent sites of homologous recombination (26, 35, 37). A number of mutant cell lines that are extremely sensitive to MMC show delayed or defective Rad51 focus formation (6, 34). A defect in Rad51 focus formation in Mus81-deficient cells would suggest that Mus81 functions upstream of the strand invasion step in the homologous repair pathway. Following exposure to MMC, Rad51 foci were found to accumulate with nearly identical kinetics in wild-type and Mus81-deficient MEFs (Fig. 5B). Twenty-four h after treatment, the number of cells with Rad51 foci peaked in the wild-type culture, and by 48 h the number of cells with Rad51 had decreased almost to pretreatment levels. By contrast, in Mus81–/– samples, the number of cells with Rad51 foci remained elevated for the duration of the experiment. The generation and persistence of Rad51 foci suggest that Mus81–/– cells initiate but fail to complete the repair of MMC-induced damage.
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-H2AX foci were monitored following treatment with 1 nM CPT for 1 h. After CPT exposure, the number of wild-type cells with -H2AX foci increased for approximately 24 h. Having reached a peak in which 60 to 70% of cells had -H2AX foci, the number of cells with foci declined gradually to basal levels within 48 h of drug treatment. No significant difference was seen in the kinetics of -H2AX focus formation and disappearance in Mus81-deficient cells compared to wild-type or heterozygote controls (P = 0.59). Similar results were obtained when the kinetics of Rad51 foci formation was followed (data not shown). Together, these analyses suggest that the majority of CPT-induced DSBs are efficiently and appropriately repaired in Mus81–/– cells.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mus81-deficient cells are specifically sensitive to agents that generate ICLs.
As previously reported for Eme1–/– embryonic stem cells, we find that Mus81–/– fibroblasts are hypersensitive to MMC and other agents that cause cross-links but only mildly sensitive to infrared, UV, or HU (1). We find that -H2AX focus formation after MMC damage occurs with the same kinetics in wild-type and Mus81–/– cells. This and the pulsed-field gel electrophoresis analysis strongly argue that the endonuclease activity of Mus81-Eme1 is not required for ICL-induced DSB formation. A role for mammalian Mus81 at a late step in ICL repair is consistent with a genetic analysis of budding yeast indicating that Mus81 acts late in recombination (22). Niedernhofer et al. showed that there was normal kinetics of DSB formation following MMC treatment of cells that lack Ercc1 (41), and these authors speculated that Mus81-Eme1 endonuclease function might be needed to generate DSBs from replication forks that have stalled because the replication templates could not be separated (41). Since MMC-induced DSB formation is not compromised in Mus81-deficient cells, the question of how they form remains to be addressed. Rothfuss and Grompe recently showed that incision of ICLs (i.e., single-strand nicking on one or both sides of the cross-link) occurs rapidly and is independent of cell cycle position (50). They also showed that subsequent DSB formation is dependent on DNA replication. Thus, in this model, the conversion of the incised ICL site into a DSB is dependent on replication forks encountering a nick; it is not actively catalyzed by an endonuclease acting on the replication fork. Identification of the enzyme(s) that makes the initial incision will be key to determining how ICLs are repaired in mammalian cells.
We also find that damage-induced focus formation of Rad51, a central molecule in homologous recombination, as well as that of -H2AX, is not impaired in the absence of Mus81. These data suggest that Mus81 either acts independently of Rad51 or acts at a later step in the repair process. The persistence of both -H2AX foci and Rad51 foci may indicate that Mus81-deficient cells are unable to process ICL lesions beyond the assembly of Rad51 filaments. However, it is also possible that in the absence of Mus81, novel, nonproductive recombination intermediates accumulate.
Mus81-deficient murine cells are not sensitive to CPT. The mild sensitivity of Mus81-deficient cells to CPT was something of a surprise. Both budding yeast and fission yeast mutants of Mus81-Eme1/Mms4 are extremely sensitive to CPT (20, 36, 57). This sensitivity is rescued by the expression of the bacterial Holliday junction resolvase, RusA (4, 20). Thus, in the absence of Mus81 function, an exogenous Holliday junction resolvase is able to restore viability, presumably through its ability to promote productive replication restart. Therefore, the presence of a second Holliday junction-resolving activity might explain the lack of sensitivity of Mus81–/– murine cells to CPT. Two Holliday junction-resolving activities, one involving Mus81 and an undefined resolvase, known as resolvase A, have previously been shown to be present in a single human cell type (15). Thus, it is possible that murine resolvase A is able to support the repair of CPT-induced damage.
Mus81–/–: a tumor suppressor or not? Our data concerning the longevity and lack of malignancy in Mus81–/– mice differ dramatically from those described in a recent report in which Mus81 deficiency resulted in early-onset tumors and death (39). Of the Toronto group's mice, only 27% of homozygous and 50% of heterozygous animals survived the first year of life (39). By contrast, in this study, more than 95% of the Mus81-deficient mice survived disease free for at least 15 months. The Mus81-deficient mice in this study may eventually develop tumors, but if they do, it will be with greatly delayed onset. The basis for the discrepancies in longevity and tumor susceptibility between the two studies is not clear. Strain differences are often invoked to explain differences in the rates or penetrance of phenotypes in mouse models. The mice in this study were generated from 129 ES cells, and they were initially crossed with C57BL/6 and then back with a 129 strain to allow Cre-mediated excision of the targeting construct (43). Thus, the final strain is a 129/BL6 mix. Although a different 129-derived embryonic stem-cell line was used for disruption, the Toronto group's mice are also 129/BL6 mixes (Razqallah Hakem, personal communication). Thus, although not identical, the two strains are closely related. Further studies will be needed to determine if a modifier or suppressor mutation accounts for differences in these two models of Mus81 deficiency. Given that the Mus81-deficient mice described here do not spontaneously develop tumors, it will be of interest to characterize the genetic interaction between Mus81 and p53. The cellular phenotypes described here predict a role for p53 in protecting Mus81–/– cells from chromosomal aberrations.
The targeting strategy used by the Toronto group replaced exons 3 and 4 with a selectable marker; the marker and its promoter were left in place in the final construct (39). Therefore, it is formally possible that this promoter inadvertently leads to the increased expression of a proto-oncogene. Such off-target effects, although rare, have given rise to dramatic phenotypic differences in closely related mouse models (45). Although there is no obvious proto-oncogene in the vicinity of murine Mus81, this hypothesis would account for the observation that the Mus81 heterozygous mice were found to be nearly as tumor prone as the homozygous disrupted mice, despite the fact that they have wild-type levels of resistance to DNA damage (39).
The phenotypes of Mus81–/– cells and mice are similar to the phenotypes seen following the disruption of several of the Fanconi anemia complementation (FANC) group genes. Fanconi anemia (FA), a rare autosomal recessive disease, is characterized by congenital abnormalities, progressive bone marrow failure, and cancer susceptibility (reviewed in reference [16]). FA cells are hypersensitive to agents that induce cross-links but have, at most, modestly increased sensitivities to agents that induce other forms of nucleotide damage or that induce strand breaks. Thus, the clinical manifestation of FA is perhaps the best evidence that the inability to repair naturally occurring ICLs poses a serious threat to human health.
Like Mus81 deficiency, the disruption of mouse FANC A, C, or G genes results in increased sensitivity to MMC but not to other DNA-damaging agents (11, 13, 32, 42, 58, 59). Remarkably, the disruption of these genes also does not result in increased tumor susceptibility (11, 13, 32, 42, 58, 59). Given that both the FANC and Mus81 functions are required to repair ICL but not to suppress tumors, it is tempting to suggest that ICLs do not occur frequently enough to create serious problems in mice. On the other hand, the profound anemias and cancer susceptibilities of Fanconi anemia patients suggest that humans might be more dependent on pathways that repair ICL for tumor avoidance.
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ACKNOWLEDGMENTS |
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This work was funded by a National Cancer Institute grant awarded to C.H.M.
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FOOTNOTES |
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These authors contributed equally to this work.
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