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Kihara Institute for Biological Research, Graduate School of Integrated Science, Yokohama City University, Yokohama 244-0813, Japan
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Introduction |
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Mus81 and its binding partner, Eme1 (Mms4 in budding yeast), constitute an endonuclease complex (Boddy et al. 2001; Kaliraman et al. 2001; Ciccia et al. 2003). In vitro, the Mus81–Eme1/Mms4 complex efficiently cleaves various DNA structures, including 3' flaps, replication forks, D-loops and nicked (or gapped) Holliday junctions (Boddy et al. 2001; Chen et al. 2001; Kaliraman et al. 2001; Bastin-Shanower et al. 2003; Ciccia et al. 2003; Gaillard et al. 2003; Osman et al. 2003; Gaskell et al. 2007). In yeast, mus81 mutants are hypersensitive to methyl methanesulfonate (MMS) and ultraviolet, but not ionizing radiation (Boddy et al. 2000, 2001; Interthal & Heyer 2000; Mullen et al. 2001; Bastin-Shanower et al. 2003). Yeast Mus81 is important for surviving exposure to agents that prevent replication fork progression (Boddy et al. 2000; Mullen et al. 2001; Doe et al. 2002; Bastin-Shanower et al. 2003; Wu et al. 2004; Kai et al. 2005). Furthermore, in mammals and plants, Mus81 or Eme1 deficiency increases sensitivity to DNA cross-linking agents, such as mitomycin C (MMC) and cisplatin, which are widely used as anticancer agents (Abraham et al. 2003; McPherson et al. 2004; Dendouga et al. 2005; Hanada et al. 2006; Hartung et al. 2006; Hiyama et al. 2006). These observations suggest that the Mus81 complex participates in DNA repair during replication.
Cells derived from Fanconi anemia (FA) patients also exhibit increased sensitivity to cross-linking agents (Kennedy & D’Andrea 2005; Thompson et al. 2005). FA is a rare recessive genetic disorder characterized by congenital abnormalities, progressive bone marrow failure and cancer susceptibility (Joenje & Patel 2001; D’Andrea & Grompe 2003; Thompson et al. 2005). To date, FA has been classified into at least 13 complementation groups (A, B, C, D1, D2, E, F, G, I, J, L, M and N) (Kennedy & D’Andrea 2005; Reid et al. 2007; Xia et al. 2007). Eight proteins (FANCA, B, C, E, F, G, L and M) encoded by the FA causative genes have been demonstrated to form a nuclear core complex (Meetei et al. 2003; Kennedy & D’Andrea 2005), which is responsible for FANCD2 monoubiquitination, a key event in a DNA damage response pathway referred to as the FA pathway (Garcia-Higuera et al. 2001). FANCB has been shown to be essential for this pathway (Garcia-Higuera et al. 2001; Meetei et al. 2004). Monoubiquitinated FANCD2 co-localizes at DNA damage sites with central molecules for HR, including Rad51, BRCA1 and BRCA2/FANCD1 (Garcia-Higuera et al. 2001; Nakanishi et al. 2002; Taniguchi et al. 2002; Hussain et al. 2004; Wang et al. 2004). Human and chicken cells lacking the FA pathway have defects in HR (Yamamoto et al. 2003, 2005; Niedzwiedz et al. 2004; Hirano et al. 2005; Nakanishi et al. 2005). These results suggest a critical role for the FA pathway in promoting HR repair.
Mus81 and the FA pathway have been implicated in the repair of damaged replication forks to prevent chromosomal instability. However, a functional relationship between Mus81 and the FA pathway remains unknown. In this study, we generate MUS81–/–, FANCB– and MUS81–/–/FANCB– cells from the human Nalm-6 cell line. Our results provide the first evidence for a cooperative linkage between the Mus81 endonuclease and the FA pathway with non-overlapping functions in repairing replication-associated DNA damage in human cells.
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Results |
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To disrupt the human MUS81 gene, we constructed two targeting vectors (Fig. 1A). Wild-type (WT) Nalm-6 cells were transfected with pMUS81-Puro, and three heterozygously targeted clones were obtained. Subsequent transfection of one of the heterozygous clones (+/Pur) with pMUS81-Hyg gave four homozygously disrupted clones. Using one of the homozygous clones (Hyg/Pur), both of the drug resistance genes were removed by transient expression of Cre recombinase. One of the resulting MUS81–/– clones was designated MUS and used for further analysis. Correct gene targeting events and excision of the drug resistance genes were verified by Southern (Fig. 1B) and Western blot analyses (Fig. 1C).
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MUS/FB double mutant cells proliferate much more slowly than either single mutant
We monitored growth curves to characterize proliferative properties of the mutant cell lines (Fig. 2A). While MUS cells showed little or no growth defects, FB cells grew more slowly than WT cells. Surprisingly, MUS/FB double mutant cells exhibited a much lower growth rate than either single mutant. Doubling times of WT, MUS, FB and MUS/FB cells were ~21, 22, 25 and 36 h, respectively (Fig. 2C). To examine the cell cycle distribution, we performed flow cytometric analysis of logarithmically growing cells (Fig. 2B). The distribution pattern of MUS cells was not significantly different from that of WT cells. In contrast, FB cells showed an increased (1.8-fold higher) G2/M fraction, a phenomenon commonly seen in human FA cells (Kubbies et al. 1985; D’Andrea & Grompe 2003). The fraction of S phase in WT, MUS, FB and MUS/FB cells were 40%, 37%, 36% and 25%, respectively. In proportion to the decrease in the S phase fraction, MUS/FB cells showed a 7.7-fold increase in a sub-G1 fraction (representing apoptotic cells). The markedly increased sub-G1 fraction in the double mutant may account for the decreased growth rate and plating efficiencies (Fig. 2C). Collectively, these results indicate that Mus81 has an important role in proliferation of cells lacking FANCB, and suggest that Mus81 and the FA pathway function independently in cell growth.
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To investigate a link between Mus81 and the FA pathway in repairing DNA cross-links, we examined the sensitivity of the mutant cell lines to cisplatin and MMC. Clonogenic survival assays revealed that MUS cells were more sensitive to these drugs than WT cells (Fig. 3A,B). By contrast, FB cells were extremely hypersensitive. This hypersensitivity in FB cells was not or only marginally affected by Mus81 deficiency (Fig. 3C,D). These results indicate that the FA pathway plays a major role for DNA cross-link repair, while Mus81 plays a minor role that appears to have functional overlap with the FA pathway. We note that sensitivities to MMS and X-rays were only marginally affected by the lack of Mus81 and/or FANCB (Fig. 3E,F). These results suggest that Mus81 and the FA pathway do not play vital roles in repair of base damage and radiation-induced DSBs.
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To assess the impact of Mus81 and FANCB deficiency on cellular HR capacity, we measured gene targeting efficiencies at the HPRT locus. As shown in Table 1, MUS cells exhibited similar efficiencies to those of WT cells. In contrast, FB and MUS/FB cells showed greatly reduced efficiencies. These results indicate that the FA pathway has a critical function in gene targeting events, while Mus81 does not.
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To investigate the roles of Mus81 and the FA pathway in the repair of DNA damage arising during DNA replication, we performed survival assays following exposure to HU, a ribonucleotide reductase inhibitor that causes replication fork stalls by depleting dNTP pools (Bianchi et al. 1986; Lomonosov et al. 2003; Kai et al. 2005). Accumulation of single-stranded regions at stalled forks activates S-phase checkpoint to prevent fork breakage until the replication can resume (Sogo et al. 2002; Branzei & Foiani 2005). As shown in Fig. 4A, MUS cells did not show increased sensitivity to HU. By contrast, FB and MUS/FB cells exhibited increased sensitivity. These results strongly suggest that resumption of stalled replication forks depends on the FA pathway, and Mus81 has no roles in this process.
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Mus81 and FANCD2 co-localize with Rad51 after CPT treatment
In recent years, evidence has accumulated that HR-mediated repair is the major mechanism responsible for resumption of DSB-containing replication forks (Johnson & Jasin 2001; Cox 2002). Rad51, a protein binding to single-stranded DNA, is a key factor for HR and forms nuclear foci after DNA damage induced by treatment with various genotoxic agents (Haaf et al. 1995; Gudmundsdottir & Ashworth 2006). These Rad51 foci have been shown to be sites undergoing HR repair (Raderschall et al. 1999; Gudmundsdottir & Ashworth 2006). To investigate whether Mus81 and FANCD2, a target molecule for ubiquitination in the FA pathway, co-localize with Rad51 following CPT-induced fork collapse, we performed immunostaining of HeLa S3 cells treated with CPT. After the CPT treatment, Mus81 formed foci and co-localized with Rad51 (Fig. 5A). Some regions with increased intensity of Mus81, presumably corresponding to nucleoli as shown by Gao et al. (2003), were observed in untreated and CPT-treated cells. In untreated cells, a few spontaneous foci of FANCD2 and Rad51 were observed, as shown in previous studies (Taniguchi et al. 2002; Hussain et al. 2004). By contrast, in CPT-treated cells, FANCD2 formed concentrated foci and these foci co-loclized with Rad51 foci (Fig. 5B). These data suggest that Mus81 and the FA pathway function with Rad51 in repairing CPT-induced fork collapse.
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HR-mediated fork repair involves a process of strand exchange between sister chromatids, which can generate a crossover detectable as an SCE event (Sonoda et al. 1999). To examine whether Mus81 and the FA pathway are responsible for the formation of such crossovers, we measured SCE levels in the mutant cell lines. In WT cells, the mean number of spontaneous SCEs per cell was 8.1. The level of spontaneous SCEs in MUS cells was 29% (P < 0.001) less than that in WT cells (Fig. 6A). In contrast, the level in FB cells was essentially the same as the WT level, whereas MUS/FB cells showed a 28% (P < 0.001) decrease compared to FB cells. After CPT treatment, SCE levels in the entire cell lines were increased ~5-fold (Fig. 6B). Interestingly, in MUS cells, similar to the spontaneous SCE level, CPT-induced SCE level was decreased by 28% (P < 0.001). While the CPT-induced level in FB cells was marginally decreased (10%, P < 0.05), MUS/FB cells showed a 19% (P < 0.01) decrease compared to FB cells. These results suggest that Mus81 mediates SCE events occurring during repair of replication fork collapse, but the FA pathway does not.
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Discussion |
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Previous studies using yeast and murine cells suggest that Mus81 deficiency does not confer defects in cell growth (Mullen et al. 2001; McPherson et al. 2004). Consistent with this, human MUS81–/– mutants (MUS cells) grew nearly normally (Fig. 2A–C). Intriguingly, however, we found that Mus81 is required for proliferation of cells lacking FANCB. The fraction of S phase in MUS/FB cells was decreased to 64% of that in WT cells, and MUS/FB cells showed an ~8-fold increased sub-G1 fraction. These observations suggest that Mus81 and the FA pathway act independently to cope with replication-associated DNA damage, thereby suppressing cell death in normal cell growth.
Evidence has accumulated that DNA cross-link repair is a complicated process that involves a wide variety of DNA repair pathways (Dronkert & Kanaar 2001; Hirano et al. 2005; Kennedy & D’Andrea 2005; Nojima et al. 2005; Thompson et al. 2005). In this study, we suggest from drug sensitivity assays that Mus81 and FANCB have an overlapping function in cross-link repair (Fig. 3A–D). It should be noted, however, that FANCB plays a more prominent role in cross-link repair, as evidenced by the extremely high sensitivity of FB cells to cisplatin and MMC (Fig. 3A,B). Our findings are consistent with the idea that the FA pathway plays a critical role in maintaining fork structures to efficiently promote HR-mediated cross-link repair (Kennedy & D’Andrea 2005; Thompson et al. 2005). In the FA pathway, monoubiquitination of FANCD2 is essential for its co-localization at DNA damage sites with central HR proteins such as RAD51, BRCA1 and BRCA2/FANCD1 (Garcia-Higuera et al. 2001; Wang et al. 2004). We showed in the present study that the loss of FANCB did abolish FANCD2 monoubiquitination (Fig. 1H), a result consistent with the notion that FANCB is a member of the FA core complex (Meetei et al. 2004).
Because of its characteristics as an endonuclease, Mus81 has been implicated in HR (Boddy et al. 2001; Chen et al. 2001; Kaliraman et al. 2001; Bastin-Shanower et al. 2003; Ciccia et al. 2003; Gaillard et al. 2003; Osman et al. 2003). However, only marginal effects of Mus81 deficiency on gene targeting (Table 1) suggest that Mus81 does not participate in the gene targeting process. This notion is consistent with previous observations in Mus81–/– or Eme1–/– mouse ES cells (Abraham et al. 2003; McPherson et al. 2004). In contrast, the severe decline in the gene targeting efficiencies in cells lacking FANCB (Table 1) clearly indicates that gene targeting events strongly depend on the FA pathway. Our results are in agreement with decreased targeting efficiencies in DT40 cells lacking FANCC, FANCD2 and FANCG (Yamamoto et al. 2003, 2005; Niedzwiedz et al. 2004; Hirano et al. 2005).
Recent evidence suggests a critical role for HR in repairing replication-associated DNA damage (Cox 2002; Helleday 2003). MUS cells did not show increased sensitivity to HU, while FB and MUS/FB cells were similarly hypersensitive to HU (Fig. 4A). These results strongly suggest that resumption of stalled replication forks depends on the FA pathway, but not Mus81, as shown in Fig. 7. In yeast, it has been proposed that aberrant DNA junctions generated at stalled forks are actively processed by the Mus81 complex to trigger break-induced replication (Whitby et al. 2003). However, Kai et al. (2005) recently reported that yeast Mus81 is not involved in the processing of HU-induced stalled replication forks, in good agreement with our current observations in human cells. We thus conclude that repair of stalled forks does not require Mus81.
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As described above, growth retardation and increased cell death were observed in the double mutant lacking both Mus81 and FANCB (Fig. 2B,C). In this regard, the independent roles for Mus81 and the FA pathway in repairing collapsed replication forks may account for this greater loss of viability in the double mutant. In support of this idea, it has been suggested that, in normal cell proliferation, spontaneous SSBs are frequently generated by free radicals or by specific endonucleases during the repair of damaged bases (Lindahl 1993; Boiteux & Guillet 2004), and that such SSBs give rise to collapsed replication forks (Helleday 2003; Saleh-Gohari et al. 2005). Alternatively, the observed growth retardation of the double mutant could be attributable to spontaneously occurring topoisomerase I-mediated DNA damage, as has been suggested in yeast cells lacking certain repair pathways (Vance & Wilson 2002).
Our findings regarding roles of Mus81 and the FA pathway in cells treated with CPT have additional implications for fork collapse repair pathways. Previous studies have shown that DNA damage-induced Rad51 foci are sites undergoing HR repair (Haaf et al. 1995; Raderschall et al. 1999; Gudmundsdottir & Ashworth 2006). Co-localization of CPT-induced foci of Mus81 and FANCD2 with Rad51 foci (Fig. 5A,B) suggests that both Mus81 and the FA pathway function in Rad51-mediated HR repair of replication fork collapse (Fig. 7). HR events occurring between sister chromatids are shown to be the major mechanism for repair of fork collapse and can undergo strand exchange with crossovers, cytologically detectable as SCEs (Sonoda et al. 1999; Johnson & Jasin 2001; Cox 2002). Our MUS81-deficient cells exhibited significantly decreased levels of spontaneous and CPT-induced SCEs (Fig. 6A,B), suggesting that human Mus81 mediates SCE events during HR repair for restarting replication after fork collapse (Fig. 7). This notion is supported by previous observations that Mus81–/– mouse ES cells show decreased levels of spontaneous and MMC-induced SCEs (Hanada et al. 2006), and that crossovers can occur through cleavage of HR intermediates by yeast Mus81 (Gaillard et al. 2003; Osman et al. 2003; Gaskell et al. 2007). In contrast, FB cells showed essentially normal levels of spontaneous and CPT-induced SCEs (Fig. 6A,B), similar to human FA cells (Thompson et al. 2005), suggesting that the FA pathway is dispensable for SCE events.
In summary, we have provided the first evidence for the functional relationship between the Mus81 endonuclease and the FA pathway in human somatic cells. Clearly, Mus81 and the FA pathway have both overlapping and non-overlapping functions in cell proliferation, DNA repair and HR. In particular, we emphasize that Mus81 and the FA pathway independently contribute to the repair of collapsed, but not stalled replication forks.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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Two targeting vectors for the human MUS81 gene were constructed by using the simple vector construction method based on the MultiSite Gateway® Technology (Invitrogen, Carlsbad, CA) (Iiizumi et al. 2006). Briefly, 3.2- and 2.5-kb genomic fragments of MUS81 were obtained by PCR with ExTaq DNA polymerase (Takara Bio Inc., Otsu, Japan) using Nalm-6 genomic DNA as a template and were used as 5'- and 3'-arms, respectively. A puromycin or hygromycin resistance gene (Puror or Hygr) was then inserted between the 5'- and 3'-arms on a plasmid carrying a diphtheria toxin A (DT-A) gene, yielding pMUS81-Puro and pMUS81-Hyg, respectively. Likewise, a targeting vector for the human FANCB gene containing the Puror gene was constructed with 3.0-kb 5'- and 3.1-kb 3'-arms, yielding pFANCB-Puro.
An HPRT targeting vector, pHPRT-Hyg, was made by inserting the Hygr gene into the XhoI site in exon 3 of an 8.9-kb HPRT fragment (So et al. 2004), and the DT-A gene was added to the 5' terminus of the HPRT fragment.
Cell culture and DNA transfection
The human pre-B cell line Nalm-6, its derivatives and HeLa S3 cells were cultured in ES medium (Nissui Seiyaku Co., Tokyo, Japan) supplemented with 10% calf serum (Hyclone, Logan, UT) and 50 µM 2-mercaptoethanol (growth medium) at 37 °C. DNA transfection and subsequent selection were performed as described previously (So et al. 2004; Adachi et al. 2006; Iiizumi et al. 2006). Resulting drug-resistant clones were subjected to PCR screening using their genomic DNA (Iiizumi et al. 2006).
Southern and Western blot analyses
Southern blotting was performed as described previously (So et al. 2004). Briefly, 20–40 µg of genomic DNA were digested with an appropriate restriction enzyme, electrophoresed in a 0.8% agarose gel and transferred to a Hybond-N+ membrane (GE Healthcare Bio-Sciences, Piscataway, NJ). Probes were labeled with digoxigenin or alkaline phosphatase by using DIG-High Prime (Roche Diagnostics, Basel, Switzerland) or AlkPhos Direct (GE Healthcare Bio-Sciences) and were used for hybridization according to the manufactures’ protocols. Western blotting was performed as described previously (So et al. 2006). Antibodies against human Mus81 (IQ285, ImmuQuest, Cleveland, UK), FANCD2 (NB 100–316, Novus Biologicals, Littleton, CO) and actin (A 2066, Sigma-Aldrich, St. Louis, MO) were used for probing.
RT-PCR and flow cytometric analyses
Total RNA was isolated by using TRIzol® Reagent (Invitrogen) and converted to cDNA by M-MLV reverse transcriptase (Promega, Madison, WI). A part of FANCB or GAPDH cDNA was amplified with ExTaq or rTaq DNA polymerase (Takara Bio Inc.). Flow cytometric analysis was performed as described (So et al. 2004).
Clonogenic assays
To determine sensitivity to genotoxic agents, 0.5 x 102–2 x 105 cells were plated into 60-mm dishes containing 5 mL of ES medium supplemented with 20% calf serum, 50 µM 2-mercaptoethanol, and 0.15% agarose with various concentrations of MMS, cisplatin, HU and CPT (all obtained from Sigma-Aldrich). After 3-week incubation at 37 °C, visible colonies were counted, and the percent survival was determined by comparing the number of surviving colonies to that of untreated controls. For X-ray sensitivity assays, cells were plated as above and exposed to various doses of X-rays. For MMC sensitivity assays, cells were exposed to different concentrations of MMC (Wako Pure Chemical, Osaka, Japan) for 1 h, washed twice with Ca2+- and Mg2+-free phosphate-buffered saline (PBS–) and plated as above. For each assay, at least three independent experiments were performed.
Measurement of gene targeting efficiencies at the HPRT locus
The structure of pHPRT-Hyg is the same as that of pHPRT-Sce (So et al. 2004), except that pHPRT-Hyg has no I-SceI site. Cells were transfected with linearized pHPRT-Hyg. Resulting hygromycin-resistant clones were transferred into growth medium containing either 20 µM 6-thioguanine (6TG) (Sigma-Aldrich) or HAT (0.1 mM hypoxanthine, 0.25 µM amethopterin and 10 µM thymidine) and 6 TG-resistant (or HAT-sensitive) clones were expanded. Correct HPRT targeting events were confirmed by genomic PCR. Gene targeting efficiencies were expressed as the percentage of targeted clones to hygromycin-resistant clones examined.
Immunostaining
Immunostaining was performed essentially as described (Wang et al. 2004). HeLa S3 cells were plated onto glass coverslips in dishes and cultured for 2 days. The cells were then fixed with chilled methanol, permeabilized with 0.2% Triton X-100 in PBS- and treated with 2% bovine serum albumin in PBS-. The coverslips were incubated with a mixture of polyclonal anti-Rad51 antibody (PC130, Calbiochem, San Diego, CA) and monoclonal anti-Mus81 antibody (IQ285, ImmuQuest) or monoclonal anti-FANCD2 antibody (NB 100–316, Novus Biologicals) in Can Get Signal® immunostain Solution A (Toyobo, Osaka, Japan) overnight at 4 °C. Subsequently, they were double-stained with Alexa 488-conjugated anti-mouse IgG (A11029 [GenBank] , Invitrogen) and Alexa 594-conjugated anti-rabbit IgG (A11037 [GenBank] , Invitrogen). Images were captured on a microscope (AX70, Olympus, Tokyo, Japan) equipped with a PXL cooled CCD camera (Photometrics, Tucson, AZ).
SCE analysis
Cells were cultured for 40 h in growth medium containing 1 µM 5'-bromodeoxyuridine (Sigma-Aldrich) with and without 3 nM CPT. The cells were then fixed and stained with Giemsa as described previously (So et al. 2004), except for the use of 0.6% sodium citrate in place of 75 mM KCl. For each cell line, 30–40 metaphases were scored. Significance of differences in the mean number of SCEs was analyzed by t-test.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: koyama{at}yokohama-cu.ac.jp; or nadachi{at}yokohama-cu.ac.jp
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References |
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Received: 15 May 2007
Accepted: 27 June 2007
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