The structure-specific endonuclease Mus81–Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks
2006; Springer Nature; Volume: 25; Issue: 20 Linguagem: Inglês
10.1038/sj.emboj.7601344
ISSN1460-2075
AutoresKatsuhiro Hanada, Magda Budzowska, Mauro Modesti, Alex Maas, Claire Wyman, Jeroen Essers, Roland Kanaar,
Tópico(s)Plant Genetic and Mutation Studies
ResumoArticle12 October 2006free access The structure-specific endonuclease Mus81–Eme1 promotes conversion of interstrand DNA crosslinks into double-strands breaks Katsuhiro Hanada Katsuhiro Hanada Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Magda Budzowska Magda Budzowska Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Mauro Modesti Mauro Modesti Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Alex Maas Alex Maas Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Claire Wyman Claire Wyman Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Department of Radiation Oncology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Jeroen Essers Jeroen Essers Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Department of Radiation Oncology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Roland Kanaar Corresponding Author Roland Kanaar Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Department of Radiation Oncology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Katsuhiro Hanada Katsuhiro Hanada Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Magda Budzowska Magda Budzowska Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Mauro Modesti Mauro Modesti Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Alex Maas Alex Maas Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Claire Wyman Claire Wyman Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Department of Radiation Oncology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Jeroen Essers Jeroen Essers Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Department of Radiation Oncology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Roland Kanaar Corresponding Author Roland Kanaar Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands Department of Radiation Oncology, Erasmus MC, Rotterdam, The Netherlands Search for more papers by this author Author Information Katsuhiro Hanada1,‡, Magda Budzowska1,‡, Mauro Modesti1, Alex Maas1, Claire Wyman1,2, Jeroen Essers1,2 and Roland Kanaar 1,2 1Department of Cell Biology & Genetics, Erasmus MC, Rotterdam, The Netherlands 2Department of Radiation Oncology, Erasmus MC, Rotterdam, The Netherlands ‡These authors contributed equally to this work *Corresponding author. Department of Cell Biology & Genetics, Erasmus University, Medical Genetics Center, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands. Tel.: +31 10 408 7186; Fax: +31 10 408 9468; E-mail: [email protected] The EMBO Journal (2006)25:4921-4932https://doi.org/10.1038/sj.emboj.7601344 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Repair of interstrand crosslinks (ICLs) requires multiple-strand incisions to separate the two covalently attached strands of DNA. It is unclear how these incisions are generated. DNA double-strand breaks (DSBs) have been identified as intermediates in ICL repair, but enzymes responsible for producing these intermediates are unknown. Here we show that Mus81, a component of the Mus81–Eme1 structure-specific endonuclease, is involved in generating the ICL-induced DSBs in mouse embryonic stem (ES) cells in S phase. Given the DNA junction cleavage specificity of Mus81–Eme1 in vitro, DNA damage-stalled replication forks are suitable in vivo substrates. Interestingly, generation of DSBs from replication forks stalled due to DNA damage that affects only one of the two DNA strands did not require Mus81. Furthermore, in addition to a physical interaction between Mus81 and the homologous recombination protein Rad54, we show that Mus81−/− Rad54−/− ES cells were as hypersensitive to ICL agents as Mus81−/− cells. We propose that Mus81–Eme1- and Rad54-mediated homologous recombination are involved in the same DNA replication-dependent ICL repair pathway. Introduction A DNA interstrand crosslink (ICL) covalently connects the two complementary strands of the DNA double helix, thereby blocking important DNA transactions, such as transcription and replication, that require unwinding of the two DNA strands. Because they cause such a dramatic block to accessing genetic information, ICL-inducing agents are extremely cytotoxic (Dronkert and Kanaar, 2001). ICL-inducing agents, such as mitomycin C, nitrogen mustards, platinum compounds and psoralens, are more cytotoxic to proliferating cells compared to nondividing cells and therefore they are widely used in chemo- and phototherapy of cancers and skin diseases. Owing to the nature of ICLs, mechanism(s) for their repair are complex. ICLs damage both DNA strands at the same, or very close, nucleotide positions. Therefore, repair mechanisms involving a simple excision followed by templated resynthesis are not sufficient. In Escherichia coli and Saccharomyces cerevisiae, ICL repair requires nucleotide excision repair (NER), homologous recombination and translesion DNA synthesis (Dronkert and Kanaar, 2001; McHugh et al, 2001). In these organisms, NER seems to be involved in generating the incision(s) near the ICL. Homologous recombination on the other hand has several roles in ICL repair (Dronkert and Kanaar, 2001). One important role for homologous recombination is the repair of DNA double-strand breaks (DSBs), which can result from ICL processing in S. cerevisiae cells (McHugh et al, 2001). Another role, documented for E. coli RecA-mediated homologous recombination in vitro, is the generation of the substrate for a second round of strand incisions by NER enzymes (Cheng et al, 1991). Alternative roles proposed for homologous recombination repair or homology-directed repair in ICL repair include mechanisms involving break-induced replication, single-strand annealing or the generation of substrates for translesion DNA synthesis (Dronkert and Kanaar, 2001; Niedernhofer et al, 2005). In vertebrate cells, homologous recombination and translesion DNA synthesis are involved in ICL repair as well (Dronkert and Kanaar, 2001). However, an interesting difference between S. cerevisiae and higher eukaryotes is the role of the NER proteins in ICL repair. Most, if not all, NER proteins in S. cerevisiae cells are involved in ICL repair as deduced from the ICL hypersensitivity of the respective mutants. By contrast, in mammalian cells, mutations in XPF and ERCC1 confer extreme ICL sensitivity, but mutations in other genes essential for NER, including XPA, XPG and CSB, are not dramatically ICL hypersensitive (Dronkert and Kanaar, 2001; De Silva et al, 2002). This suggests that XPF-ERCC1, a heterodimeric structure-specific endonuclease (de Laat et al, 1998), plays a central role in ICL repair that is largely independent of NER. In addition to an NER-independent role in ICL repair, XPF-ERCC1 functions in at least two subpathways of homology-directed DNA repair. Both in S. cerevisiae and mammalian cells, XPF-ERCC1 is involved in DSB repair, through single-strand annealing, and in homologous gene targeting (Paques and Haber, 1999; Adair et al, 2000; Sargent et al, 2000; Niedernhofer et al, 2001; Langston and Symington, 2005). Unraveling the mechanism(s) of ICL repair requires answers to two central questions; what are the intermediates in ICL repair at the DNA level and how are they generated? Recently, it has become clear that one pivotal intermediate that can arise during the repair of an ICL is a DSB (Akkari et al, 2000; De Silva et al, 2000; Niedernhofer et al, 2004; Rothfuss and Grompe, 2004). The formation of this DSB intermediate requires DNA replication, suggesting that a stalled replication forks at the site of the ICL is recognized and processed by a structure-specific endonuclease into a DSB. Owing to the ICL hypersensitivity of cells mutated in the XPF–ERCC1 complex and the biochemical properties of this complex, it has been suggested that XPF–ERCC1 would convert ICLs to DSBs. However, recent studies have demonstrated that this conversion is XPF–ERCC1 independent (De Silva et al, 2000; Niedernhofer et al, 2004), thus leaving the question of how DSBs are generated from ICLs partially unanswered. Recently, a structure-specific endonuclease, Mus81–Eme1, with amino-acid sequence similarity to XPF-ERCC1 has been identified in yeast and mammalian cells (Heyer, 2004). In yeast, mus81 mutants exhibit sensitivity to hydroxyurea, UV-light and methyl methanesulfonate but not to ionizing radiation (Interthal and Heyer, 2000; Doe and Whitby, 2004; Doe et al, 2004). This sensitivity profile is consistent with a role for Mus81–Eme1 in processing stalled DNA replication forks. The biochemical properties of the enzyme complex are also consistent with its involvement in forming DSBs at DNA structures resembling replication forks. Like XPF–ERCC1, Mus81–Eme1 also cleaves branched DNA structures (Boddy et al, 2001; Chen et al, 2001; Constantinou et al, 2002; Kaliraman et al, 2001; Ciccia et al, 2003; Ogrunc and Sancar, 2003; Whitby et al, 2003). However, XPF–ERCC1 prefers three-way branched junctions containing two single-stranded DNA arms, whereas Mus81–Eme1 has a preference for three-way junctions that are more double-stranded in nature, such as 3′-flap and replication fork-like structures (Heyer, 2004). Mus81 and Eme1 mutant embryonic stem (ES) cells, which have recently become available, display hypersensitivity to ICL-inducing agents such as mitomycin C and cisplatin (Abraham et al, 2003; McPherson et al, 2004). Results Mus81 is involved in processing ICLs into DSBs To test whether the Mus81–Eme1 structure-specific endonuclease is involved in processing ICLs into DSBs, we first constructed mouse ES cells lacking Mus81 (Supplementary Figure 1). Next, we analyzed ICL-induced DSB formation in Mus81-proficient and -deficient ES cells. Proliferating ES cells were treated with different doses of mitomycin C for 24 h and ICL-induced DSBs were detected using PFGE (Figure 1A). As we previously demonstrated, mitomycin C treatment resulted in an increase in broken DNA in wild-type ES cells (Niedernhofer et al, 2004). The increase was dose dependent (Figure 1A) and was not owing to DNA fragmentation during apoptosis (Supplementary Figure 2). In ES cells lacking ERCC1, mitomycin C-induced DSBs were observed (Figure 1A). However, cells lacking Mus81 mitomycin C failed to induce DSBs, even at the highest dose of mitomycin C used (Figure 1A). Similarly, in response to another ICL-inducing agent, cisplatin, wild-type ES cells showed a dose-dependent increase in broken DNA that was not observed in Mus81−/− ES cells (Figure 2A). We conclude that the structure-specific endonuclease Mus81–Eme1 is involved in processing ICLs into DSBs. Figure 1.Analysis of mitomycin C-induced DSB formation in wild-type, Ercc1−/− and Mus81−/− ES cells. (A) Using PFGE, DSB formation was analyzed. Cells of the indicated genotype were treated with increasing concentrations of mitomycin C for 24 h, collected into agarose plugs and their DNA was separated by size on an agarose gel. Under the electrophoresis conditions used, high molecular weight genomic DNA remains in the well, whereas lower molecular weight DNA fragments (several Mbp to 500 kbp) migrate into the gel and are compacted into a single band. (B) Cell cycle profiles of wild-type and Mus81−/− ES cells after continuous treatment with mitomycin C for 24 h. Using a FACscan, the cell cycle profile of cells pulse labeled with BrdU was analyzed by total DNA content as determined by propidium iodide (PI) staining (x-axis) and replication status as determined by BrdU incorporation (y-axis). Cells were either untreated or incubated with 2.0 μg/ml mitomycin C. (C) Time course of mitomycin C-induced DSB formation in wild-type and Mus81−/− ES cells. Cells were treated with 1.0 μg/ml mitomycin C, indicated by (+). The untreated control sample is indicated by (−). Download figure Download PowerPoint Figure 2.Analysis of cisplatin-induced DSB formation in wild-type and Mus81−/− ES cells. (A) Cells of the indicated genotype were treated with increasing concentrations of cisplatin for 24 h and their DNA was analyzed by PFGE. (B) Clonogenic survival curve of wild-type and Mus81−/− ES cells in response to increasing doses of cisplatin. (C) Cell cycle profiles of wild-type and Mus81−/− ES cells after continuous treatment with increasing doses of cisplatin for 24 h. Bi-parameter (BrdU and PI) FACscan plots are shown. Download figure Download PowerPoint Mus81 operates in S phase Given the biochemical activity of Mus81–Eme1 on splayed arm DNA substrates and the lack of mitomycin C-induced DSBs in Mus81−/−, the role of Mus81–Eme1 in ICL repair is likely the conversion of ICL-stalled replication forks into DSBs. Thus, Mus81–Eme1 would function during S phase. Consistent with this notion, culturing the cells in the continuous presence of mitomycin C or cisplatin resulted in their accumulation in S phase (Figures 1B and 2C, respectively). If Mus81–Eme1 acts in S phase on stalled replication forks, then the induction of DSBs by mitomycin C should be slow, in contrast to DSB induction by agents that directly act on DNA such as ionizing radiation. Indeed, in wild-type ES cells cultured in the presence of 1.0 μg/ml mitomycin C, DSB induction became apparent after around 12–18 h and subsequently increased over time (Figure 1C). Again, no DSBs were induced in Mus81−/− cells, even after 30 h of incubation with mitomycin C. If Mus81–Eme1 functions on ICL-stalled replication forks, then active replication would be required to detect Mus81-dependent, ICL-dependent DSBs. To test this premise, we blocked replication by the addition of thymidine, which resulted in accumulation of the cells in S phase (Figure 3A and B). When mitomycin C was added, no increase in DSBs was detected, either in wild-type or Mus81−/− ES cells (Figure 3C). In normal growth conditions, without added thymidine to block replication, Mus81-dependent DSBs were detected upon the addition of mitomycin C. Furthermore, when cells treated with mitomycin C, under conditions of thymidine-blocked replication (Figure 4A), were allowed to resume replication by incubating them in media lacking thymidine and mitomycin C, Mus81-dependent DSBs were detected (Figure 4C, compare lanes 7 and 14). Based on the results of the PFGE analysis, the cell cycle analysis and the hypersensitivity of Mus81−/− cells to both mitomycin C and cisplatin (Figures 2B and 7C, respectively), we conclude that Mus81–Eme1 is involved in DSB formation when DNA replication forks are blocked by an ICL. Figure 3.Inhibition of replication suppresses Mus81-dependent DSB formation in response to ICLs. (A) Schematic representation of the experimental protocol. Replication in wild-type and Mus81−/− ES cells was inhibited by incubating them in media containing 20 mM thymidine for 2.5 h. Next, the cells were treated with 2 μg/ml mitomycin C and 20 mM thymidine for 10 h (III). Control cells were either untreated (I), treated with 20 mM thymidine for 2.5 h (II) or 12.5 h (IV), or treated with 2 μg/ml mitomycin C for 10 h (V). (B) Cell cycle profiles of cells treated as described in panel (A) were determined by bi-parameter (BrdU and PI) FACS analysis. (C) Wild-type and Mus81−/− ES cells were treated as described above and the DSB formation was analyzed by PFGE. Download figure Download PowerPoint Figure 4.Mus81-dependent generation of mitomycin C-induced DSBs occurs during S phase. (A) Schematic representation of the experimental protocol. Wild-type and Mus81−/− ES cells were incubated in 5 mM thymidine for 12 h. For the last 6 h of the incubation, mitomycin C was added to a final concentration of 2 μg/ml. Control cells were incubated with thymidine-containing media only. Next, the cells were washed twice with PBS, incubated in fresh medium to allow resumption of replication, and collected at the indicated times. (B) Cell cycle profiles of cells treated as described in panel (A), shown as BrdU incorporation versus PI plots. (C) The relative amount of broken DNA in wild-type and Mus81−/− ES cells treated as described in panel (A) was assessed by PFGE. Download figure Download PowerPoint In the experiments described above, the cells were continuously incubated in the presence of mitomycin C. This resulted in accumulation of the cells in S phase (Figure 1B) and allowed us to observe the involvement of Mus81 in converting ICLs to DSBs. In contrast, when cells were incubated for 1 h in the presence of mitomycin C and subsequently placed in fresh media without mitomycin C, the cells did not accumulate in S phase, irrespective of their genotype (Figure 5A). Instead, 12 h after the mitomycin C pulse most cells were in late S phase, whereas G2, G1 and early S phase cells were detected by 36 h. Under these conditions, DNA breaks were observed in both wild-type and Mus81−/− ES cells (Figure 5B). Figure 5.Analysis of DSB formation after pulse treatment of cells with mitomycin C. (A) ES cells of the indicated genotype were treated for 1 h with mitomycin C and continued to be incubated in media without mitomycin C for the indicated amount of time before their cell cycle profile was determined by FACS analysis. The cell count versus PI staining and BrdU incorporation versus PI profile are shown. (B) Wild-type and Mus81−/− ES cells were treated with mitomycin C for 1 h. Cells were incubated in media without mitomycin C for the indicated amount of time and the amount of broken DNA was determined by PFGE. Download figure Download PowerPoint Mus81-mediated DSB formation is DNA lesion selective Next, we asked whether Mus81–Eme1 is required, in general, to produce DSBs under conditions where DNA damage leads to replication fork stalling. Therefore, instead of using mitomycin C, UV light was used to stall replication through DNA damage induction (Courcelle and Hanawalt, 2001; Branzei and Foiani, 2005). Wild-type, Mus81+/− and Mus81−/− ES cells were treated with increasing doses of UV light and the cells were analyzed for DSB formation after 4 h. After the treatment, the cells accumulated in S phase, just as was observed after treatment with the ICL-inducing agents (Figure 6C). A dose-dependent increase in broken DNA was observed, irrespective of whether Mus81 was functional (Figure 6A). Consistent with this observation, Mus81 was not required for cell survival in response to UV-light treatment (Figure 6B). By contrast, Mus81−/− cells were hypersensitive to ICL-inducing agents (Figures 2B and 7C, and McPherson et al, 2004; Dendouga et al, 2005). We conclude that not all stalled replication forks are equivalent and that the replication fork cleavage activity of Mus81–Eme1 depends on the lesion that causes the stalling. Figure 6.Analysis of UV-light-induced DSB formation in wild-type and Mus81−/− ES cells. (A) UV-light-induced DSB formation in wild-type, Mus81+/− and Mus81−/− ES cells as analyzed by PFGE. (B) Survival curve in response to UV light for wild-type, and Mus81−/− ES cells. Xpa−/− ES cells served as a control. (C) Cell cycle profiles of wild-type and Mus81−/− ES cells 4 h after treatment increasing doses of UV light. The BrdU incorporation versus PI profiles are shown. Download figure Download PowerPoint Figure 7.Analysis of relationship between Mus81 and Rad54 with respect to ICL repair. (A) Immunoprecipitation (IP) of Rad54 and Mus81. Using an anti-HA-antibody, HA-tagged Rad54 protein was precipitated from HA-tagged Rad54 knock-in ES cells. The precipitated material was analyzed by immunoblotting using antibodies against Mus81, HA and Rad54. As a negative control, Rad54+/− cells (cell line #18) were used, because the cell line is isogenic to the Rad54HA/− cell line, except for the HA-tag on Rad54. (B) Identification of the Mus81 and Rad54 proteins in the input material for the immunoprecipitation. Immunoblots using whole-cell extracts representing 1% of the material used for the immunoprecipitation. As controls for the identification of Mus81 and Rad54, extracts from Mus81−/− and Rad54−/− ES cells were used. Signals from nonspecific proteins are indicated by an asterix. (C) Comparison of mitomycin C sensitivity of wild-type, Mus81−/−, Rad54−/− and Mus81−/− Rad54−/− ES cells. ES cells of the indicated genotype were treated with increasing doses of mitomycin C for 1 h after which the medium was refreshed. Colonies were fixed, stained and counted after 5–8 days. Error bars indicate the standard error of the mean. (D) Analysis of mitomycin C-induced DSB formation in wild-type, Rad54−/−, Mus81−/− and Mus81−/− Rad54−/− ES cells using PFGE. Download figure Download PowerPoint Physical and genetic interactions between Mus81 and Rad54 A one ended-DSB such as generated by Mus81–Eme1 from stalled replication forks is an ideal substrate for the initiation of homologous recombination as a next step in ICL repair. Consistent with this notion we observed a reduction of mitomcyin C-induced sister chromatid exchanges (SCEs) in Mus81−/− cells compared to wild-type ES cells. Wild-type cells treated with 0.2 μg/ml mitomycin C displayed 40.8±4.0 SCE per metaphase, whereas Mus81−/− cells showed 31.5±1.9 SCE per metaphase. Interestingly, the spontaneous level of SCEs was already slightly, but significantly (P<0.01) reduced in Mus81−/− cells compared to wild type from 9.9±0.8 to 7.2±0.5 SCEs per metaphase. Possibly, repair of replication forks stalled due to endogenous DNA damage is less likely to proceed through a DSB intermediate in the absence of Mus81. In addition to reduced mitomycin C-induced SCEs level in Mus81−/− ES cells, evidence for a link between Mus81 and homologous recombination is also provided by the interaction between S. cerevisiae Mus81 and the homologous recombination protein Rad54 in a two-hybrid assay and in co-immunoprecipitation experiments (Interthal and Heyer, 2000). We asked whether a physical and genetic interaction exists between mouse Mus81–Eme1 and Rad54. Whole-cell extracts were prepared from an ES cell line that carries a Rad54 knockout allele and an HA-tagged Rad54 knock-in allele, which expresses HA-tagged and fully functional Rad54 protein from the endogenous promoter (Tan et al, 1999). HA-tagged Rad54 protein was precipitated with immobilized anti-HA antibodies. Immunoblotting of the precipitated samples was used to detect the presence of Mus81, the HA epitope and Rad54 (Figure 7A and B). Co-immunoprecipitation of Mus81 with Rad54 was detected. In contrast, Mus81 was not detected when the precipitation was performed using extracts prepared from an isogenic ES cell line in which HA-tagged Rad54 was absent. The interaction is likely protein-mediated, because DNA in the extracts was digested with DNase I before the immunoprecipitation. The physical interaction between Mus81–Eme1 and Rad54 is consistent with a function of these proteins in the same ICL repair pathway. The DNA intermediate from which homologous recombination during ICL repair would be initiated is the DSB generated by Mus81–Eme1. Therefore, inactivating mutations in Mus81 should be epistatic to mutations in Rad54 in the context of ICL repair. To test this premise, we generated Mus81−/− Rad54−/− double knockout ES cells and compared their degree of mitomycin C sensitivity to that of either of the single mutants. Rad54−/− ES cells were about three-fold more sensitive to mitomycin C than wild-type ES cells (Figure 7C and Essers et al, 1997). The Mus81−/− ES cells displayed a seven-fold increase in mitomycin C sensitivity. The Mus81−/− Rad54−/− double knockout ES cells were as sensitive to mitomycin C as the Mus81−/− cells (Figure 7C). We conclude that Mus81 is epistatic to Rad54 with respect to repair of ICLs. Consistent with this notion, mitomycin C-induced DSBs were observed in Rad54−/− ES cells, but not in Mus81−/− Rad54−/− ES cells (Figure 7D). Discussion A DNA ICL covalently links both strands of the DNA double helix and thus its repair requires incisions not only on both sides of the crosslink but also in both DNA strands. Previously, DSBs have been identified as intermediates in ICL repair. Here, we identify Mus81–Eme1 as a structure-specific endonuclease involved in converting ICLs to DSBs in a DNA replication-dependent manner. ICL-inducing agents cause very heterogeneous types of DNA distortions (Dronkert and Kanaar, 2001). Therefore, recognition of ICLs posses a problem because recognition based on chemical and three-dimensional structure would require multiple recognition proteins. Instead, cells probably rely on detection methods that do not require direct recognition of the ICL, such as ICL-induced transcription or replication stalling. For cells in S phase, a replication fork stalled by an ICL can provide a branched DNA structure that triggers the required strand cleavages. However, classical excision repair pathways such as NER or base excision repair alone are not sufficient for ICL repair because these pathways have evolved to cleave only one of the two DNA strands. While it has been established that ICLs are converted into DSBs in a replication-dependent manner (Akkari et al, 2000; De Silva et al, 2000; Niedernhofer et al, 2004; Rothfuss and Grompe, 2004), the identity of nucleases responsible for this conversion had not been determined. Mammalian cells contain at least two structure-specific endonucleases that cleave branched DNA structures: XPF-ERCC1 and Mus81–Eme1 (Heyer et al, 2003; Heyer, 2004). XPF-ERCC1, first identified for its function in NER, prefers three-way branched junctions containing two single-stranded DNA arms, whereas Mus81–Eme1 cleaves three-way junctions with at least two double-stranded arms, such as 3′ flaps and structures resembling replication forks. Ercc1−/− cells are extremely sensitive to ICL-inducing agents, but the XPF–ERCC1 complex is not involved in the generation of ICL-induced DSBs and probably plays a role in another step of ICL repair (Niedernhofer et al, 2004). We have generated Mus81−/− ES cells to address whether Mus81 was part of the structure-specific endonuclease complex responsible for DSB formation after treatment with crosslinking agents. Mus81−/− cells, as well as Eme1−/− cells, are hypersensitive to mitomycin C and cisplatin (Figures 2B and 7C, and Abraham et al, 2003; McPherson et al, 2004; Dendouga et al, 2005). Culturing wild-type ES cells in the continuous presence of an ICL-inducing agent results in the accumulation of the cells in S phase (Figures 1B and 2C) and in DSB formation that increases in a time- and dose-dependent manner (Figures 1C, A and 2A). By contrast, no such increase in DSB formation occurs in the absence of Mus81, even through the cells do accumulate in S phase. Inhibition of DNA replication suppresses Mus81-dependent DSBs in response to mitomycin C (Figure 3), whereas resuming replication resulted in their formation (Figure 4). Taken together, the biochemical activity of the Mus81–Eme1 complex, the mitomycin C hypersensitivity of Mus81−/− and Eme1−/− cells, and the lack of ICL-induced DSB formation in Mus81−/− S phase cells suggest that Mus81–Eme1 is a structure-specific endonuclease involved in cleaving one of the branched arms of replication forks stalled by ICL lesions. While Mus81 is involved in cleaving replication forks stalled at ICLs, it does not seem to be responsible for dealing with DNA damage-associated replication stalling in general. After treatment with UV light, Mus81−/− cells accumulate a similar amount of DSBs as wild-type cells (Figure 6). Encountering DNA damage that affects only one strand, such as induced by UV light, will cause problems for a replicative polymerase but may not stop a replicative helicase. The incomplete replicated regions resulting in this case are apparently not substrates for Mus81. However, the DNA structures created, and the challenges to restarting replication, when a fork encounters an ICL are likely to be much different. Because the integrity and movement of a replication fork is determined by the presence and movement of a replicative helicase, halting this enzyme, as an ICL will, will likely cause complete disruption of fork movement. DNA synthesis will stop on both strands and the complex assemblies of replication proteins may disassociate from each other and their DNA templates. While the results of our experiments reveal the involvement of Mus81–Eme1 in converting ICLs into DSBs, they also demonstrate the importance of the assay to detect these DSBs. When cells are continuously exposed to mitomycin C, they accumulate in S phase and Mus81-dependent DSBs can be detected (Figure 1). On the other hand, when they are treated with a short pulse of mitomycin C, cells do not accumulate in S phase and while DSBs are detected they are not Mus81-dependent (Figure 5), consistent with the results of a previous study (Dendouga et al, 2005). It is possible that unrepaired or partially repaired ICL dama
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