Eme1 is involved in DNA damage processing and maintenance of genomic stability in mammalian cells
2003; Springer Nature; Volume: 22; Issue: 22 Linguagem: Inglês
10.1093/emboj/cdg580
ISSN1460-2075
Autores Tópico(s)CRISPR and Genetic Engineering
ResumoArticle17 November 2003free access Eme1 is involved in DNA damage processing and maintenance of genomic stability in mammalian cells Jacinth Abraham Jacinth Abraham Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Bénédicte Lemmers Bénédicte Lemmers Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author M.Prakash Hande M.Prakash Hande Faculty of Medicine, National University of Singapore, Singapore, 117597 USA Search for more papers by this author Mary Ellen Moynahan Mary Ellen Moynahan Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA Search for more papers by this author Charly Chahwan Charly Chahwan Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Search for more papers by this author Alberto Ciccia Alberto Ciccia Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Jeroen Essers Jeroen Essers Department of Cell Biology and Genetics, and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Katsuhiro Hanada Katsuhiro Hanada Department of Cell Biology and Genetics, and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Richard Chahwan Richard Chahwan Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Search for more papers by this author Aik Kia Khaw Aik Kia Khaw Faculty of Medicine, National University of Singapore, Singapore, 117597 USA Search for more papers by this author Peter McPherson Peter McPherson Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Search for more papers by this author Amro Shehabeldin Amro Shehabeldin Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Rob Laister Rob Laister Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Cheryl Arrowsmith Cheryl Arrowsmith Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Roland Kanaar Roland Kanaar Department of Cell Biology and Genetics, and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Stephen C. West Stephen C. West Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Maria Jasin Maria Jasin Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA Search for more papers by this author Razqallah Hakem Corresponding Author Razqallah Hakem Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Jacinth Abraham Jacinth Abraham Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Bénédicte Lemmers Bénédicte Lemmers Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author M.Prakash Hande M.Prakash Hande Faculty of Medicine, National University of Singapore, Singapore, 117597 USA Search for more papers by this author Mary Ellen Moynahan Mary Ellen Moynahan Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA Search for more papers by this author Charly Chahwan Charly Chahwan Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Search for more papers by this author Alberto Ciccia Alberto Ciccia Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Jeroen Essers Jeroen Essers Department of Cell Biology and Genetics, and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Katsuhiro Hanada Katsuhiro Hanada Department of Cell Biology and Genetics, and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Richard Chahwan Richard Chahwan Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Search for more papers by this author Aik Kia Khaw Aik Kia Khaw Faculty of Medicine, National University of Singapore, Singapore, 117597 USA Search for more papers by this author Peter McPherson Peter McPherson Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Search for more papers by this author Amro Shehabeldin Amro Shehabeldin Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Rob Laister Rob Laister Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Cheryl Arrowsmith Cheryl Arrowsmith Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Roland Kanaar Roland Kanaar Department of Cell Biology and Genetics, and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands Search for more papers by this author Stephen C. West Stephen C. West Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK Search for more papers by this author Maria Jasin Maria Jasin Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA Search for more papers by this author Razqallah Hakem Corresponding Author Razqallah Hakem Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada Search for more papers by this author Author Information Jacinth Abraham1,2, Bénédicte Lemmers1,2, M.Prakash Hande3, Mary Ellen Moynahan4, Charly Chahwan1, Alberto Ciccia5, Jeroen Essers6, Katsuhiro Hanada6, Richard Chahwan1, Aik Kia Khaw3, Peter McPherson1, Amro Shehabeldin1,2, Rob Laister2, Cheryl Arrowsmith2, Roland Kanaar6, Stephen C. West5, Maria Jasin4 and Razqallah Hakem 1,2 1Advanced Medical Discovery Institute, Ontario Cancer Institute, 620 University Avenue, Suite 706, Toronto, Ontario, M5G 2C1 Canada 2Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada 3Faculty of Medicine, National University of Singapore, Singapore, 117597 USA 4Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, 10021 USA 5Cancer Research UK, London Research Institute, Clare Hall Laboratories, South Mimms, Herts, EN6 3LD UK 6Department of Cell Biology and Genetics, and Department of Radiation Oncology, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands ‡J.Abraham and B.Lemmers contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:6137-6147https://doi.org/10.1093/emboj/cdg580 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Yeast and human Eme1 protein, in complex with Mus81, constitute an endonuclease that cleaves branched DNA structures, especially those arising during stalled DNA replication. We identified mouse Eme1, and show that it interacts with Mus81 to form a complex that preferentially cleaves 3′-flap structures and replication forks rather than Holliday junctions in vitro. We demonstrate that Eme1−/− embryonic stem (ES) cells are hypersensitive to the DNA cross-linking agents mitomycin C and cisplatin, but only mildly sensitive to ionizing radiation, UV radiation and hydroxyurea treatment. Mammalian Eme1 is not required for the resolution of DNA intermediates that arise during homologous recombination processes such as gene targeting, gene conversion and sister chromatid exchange (SCE). Unlike Blm-deficient ES cells, increased SCE was seen only following induced DNA damage in Eme1-deficient cells. Most importantly, Eme1 deficiency led to spontaneous genomic instability. These results reveal that mammalian Eme1 plays a key role in DNA repair and the maintenance of genome integrity. Introduction Eme1 (essential meiotic endonuclease 1) was discovered in Schizosaccharomyces pombe by virtue of its interaction with Mus81 (methyl methanesulfonate-sensitive UV- sensitive 81) (Boddy et al., 2001). The ‘fuss’ about these molecules stemmed from the ability of the Mus81–Eme1 complex to function as a heterodimeric endonuclease, cleaving branched DNA structures such as replication forks (RFs), 3′ DNA flaps and Holliday junctions (HJs) (Haber and Heyer, 2001). Yeast Eme1 by itself has no endonuclease activity; however, its interaction with Mus81 is essential for the endonucleolytic activity of Mus81 (Boddy et al., 2001). Mus81 is related to the XPF family of endonucleases. This family shares a similar active site, constituted by the amino acids VERKxxxD (Boddy et al., 2000; Enzlin and Scharer, 2002). The Mus81–Eme1 complex resembles the Rad1–Rad10 protein complex that is involved in nucleotide excision repair (NER), where only one partner (yeast Rad1, human Xpf) possesses endonucleolytic activity. The functional binding partner of Mus81 in Saccharomyces cerevisiae is Mms4 (methyl methanesulfonate-sensitive 4), which shares weak similarity with S.pombe Eme1 (Boddy et al., 2001; Kaliraman et al., 2001; Mullen et al., 2001). Human Eme1 was subsequently identified and, in complex with human Mus81, cleaves RFs, 3′ DNA flaps and HJs (Ciccia et al., 2003; Ogrunc and Sancar, 2003). The physical interaction of yeast Mus81 with Cds1 (Rad53/Chk2) (Boddy et al., 2000) and Rad54 (Interthal and Heyer, 2000), and a functional link with RecQ helicase Sgs1 (Mullen et al., 2001), indicated potential roles for the Mus81–Eme1 complex in cell cycle checkpoints, as well as DNA repair, recombination and replication processes. mus81, eme1 or mms4 mutants in yeast showed sensitivity to UV radiation, MMS and camptothecin (CPT), but not to ionizing radiation (IR). Since UV, MMS and CPT interfere with yeast DNA replication, the sensitivity of the mutants was attributed to an inability to process DNA intermediates arising at sites of stalled DNA replication (Xiao et al., 1998; Boddy et al., 2000, 2001; Interthal and Heyer, 2000; de los Santos et al., 2001; Kaliraman et al., 2001; Mullen et al., 2001; Bastin-Shanower et al., 2003). Rqh1 and Sgs1 helicases, in their respective fission and budding yeast, are known to play a role in the restart of stalled DNA replication. The requirement for Mus81 for the viability of rqh1/sgs1-null yeast indicated that these molecules are mechanistically distinct, yet functionally related, providing further evidence for the involvement of Mus81–Eme1 in the restart of stalled DNA replication (Kaliraman et al., 2001; Mullen et al., 2001; Doe et al., 2002; Fabre et al., 2002). DNA replication can stall due to lack of nucleotides, lack of replication checkpoints, and obstructions caused by DNA lesions or protein complexes (McGlynn and Lloyd, 2002). That Eme1 could play a role in homologous recombination (HR) was expected from yeast studies, where loss of Mus81, Eme1 or Mms4 led to meiotic failure, as seen by a severe reduction in sporulation and spore viability, though some yeast strains displayed a moderate reduction (Boddy et al., 2001; de los Santos et al., 2001; Kaliraman et al., 2001). This defect was traced to DNA processing after the meiosis-induced double-strand breaks (DSBs) were initiated, resulting in improper segregation of chromosomes (Boddy et al., 2001; de los Santos et al., 2001; Kaliraman et al., 2001). This trend was also observed in vegetative yeast cells, where eliminating HR could restore viability to mus81 sgs1 or mms4 sgs1 double mutants, proving that unprocessed HR-generated DNA intermediates led to toxicity in these mutants (Fabre et al., 2002; Bastin-Shanower et al., 2003). Analysis of various branched substrates showed that the Mus81-associated endonuclease activity from human extracts, and prepared as recombinant proteins in bacteria, could cleave RFs and 3′ DNA flaps more efficiently than HJs (Kaliraman et al., 2001; Constantinou et al., 2002; Doe et al., 2002; Ciccia et al., 2003; Whitby et al., 2003). The replication fork and 3′-flap DNA structures that are cleaved well by Mus81–Eme1 are speculated to form when DNA replication stalls (Whitby et al., 2003), and it is postulated that the 3′-flap can occur during synthesis-dependent strand annealing, also referred to as strand displacement and annealing (de los Santos et al., 2001; Haber and Heyer, 2001). Despite the many studies characterizing the function of Mus81 and Eme1/Mms4 in yeast, the biological function of these molecules in mammals is unknown. In this study, we describe the identification and characterization of mouse Eme1, giving evidence for its role in DNA repair and maintenance of genomic stability. Results Cloning of mouse Eme1 Sequence similarity searches of the DNA databases identified mouse open reading frames (ORFs) with similarity to fission yeast Eme1. The mouse cDNA was predicted to encode a 570 amino acid protein, which is the same size as the human counterpart, but smaller than the 738 amino acid yeast Eme1 (Boddy et al., 2001). The identity of the mouse protein to the S.pombe Eme1 molecule was 19%, and the similarity was 36% (Figure 1A). Comparison of mouse Eme1 with human Eme1 showed 66% identity and 76% similarity. Based on NCBI and Ensembl searches, the mouse Eme1 gene is localized to chromosome 11 band C and is encoded by nine exons spanning a genomic DNA region of ∼9 kb. Figure 1.Identification of mammalian Eme1. (A) Alignment of mouse, human and S.pombe Eme1 proteins with S.cerevisae Mms4. Amino acid identities and similarities are highlighted in black and gray, respectively. (B) Mouse Eme1 is mainly expressed in proliferative tissues. Radiolabeled Eme1 full-length cDNA was used to probe northern blots of mouse ES cells (left panel), mouse embryos at day 7, 11, 15 and 17 of gestation (middle panel) and mouse adult tissues (right panel). Northern blots were subsequently probed with a GADPH cDNA to assess loading. Download figure Download PowerPoint In vivo expression of Eme1 Northern blot analysis of Eme1 expression in embryonic stem (ES) cells, mouse embryos and adult tissues was performed using the full-length mouse Eme1 cDNA as a probe (Figure 1B). A major Eme1 transcript of 2.5 kb was detected in ES cells (Figure 1B, left panel), while two major Eme1 transcripts of 2.5 and 5.5 kb were detected at days 7, 11, 15 and 17 of mouse embryonic development (Figure 1B, middle panel). In adult tissues (Figure 1B, right panel), weak expression of the 5.5 kb Eme1 transcript was detected in all tissues tested, but the 2.5 kb transcript was predominantly seen in skin, testis and thymus. Another smaller Eme1 transcript was also detected in some adult tissues such as heart, liver and skin. Together, these results demonstrate that Eme1 is expressed in many adult tissues and at various stages of embryonic development. Mouse Eme1 and Mus81 interact to form a structure-specific endonuclease Using Flag-tagged mouse Mus81 and hemagglutinin (HA)-tagged mouse Eme1 proteins translated in vitro (Figure 2A), and transiently transfected into 293T human embryonic kidney (HEK) cells (data not shown), we demonstrated that these proteins interact with each other, as expected from their yeast and human counterparts (Figure 2A; lanes 3 and 4). Figure 2.Endonuclease activity of the mouse Mus81–Eme1 complex. (A) Interaction of mouse Eme1 with Mus81. In vitro translation reactions containing the templates Mus81-Flag (lane 1), Eme1-HA (lane 2), both Mus81-Flag and Eme1-HA (lanes 3 and 4) or no template (lane 5) were subject to immunoprecipitation (IP). The IP antibodies were anti-Flag (lanes 1, 3 and 5) and anti-HA (lanes 2 and 4). In co-translation reactions, both Mus81 and Eme1 were immunoprecipitated by anti-Flag and anti-HA antibodies (lanes 3 and 4), demonstrating the physical interaction of these proteins. (B) Mus81–Eme1 protein complex has DNA structure-specific endonuclease activity. Three branched DNA substrates, splayed-arm (lanes 1–4), 3′-flap (lanes 5–8) and HJ (lanes 9–12), were assessed for cleavage by immunocomplex negative control (lanes 1, 5 and 9), human Mus81 fraction (lanes 2, 6 and 10), mouse Eme1 immunocomplex (lanes 3, 7 and 11) and mouse Mus81–Eme1 immunocomplex (lanes 4, 8 and 12). Cleavage reactions were resolved by 10% neutral PAGE and visualized by phosphor imager analysis. Mouse Mus81–Eme1 cleaved the 3′-flap structure well (lane 8), but had only faint endonuclease activity against the HJ substrate tested (lane 12). Download figure Download PowerPoint To determine whether mouse Mus81–Eme1 complex exhibited endonuclease activity, the ability of the Mus81–Eme1 pull-down complexes to cleave splayed-arm (Figure 2B, lanes 1–4), 3′-flap (lanes 5–8) and HJ (lanes 9–12) substrates was analyzed. We found that Mus81–Eme1 efficiently cleaved the 3′-flap structure (lane 8). Similar results were obtained with a structure that mimics an RF (data not shown). In contrast, little or no cleavage by Mus81–Eme1 was seen with the splayed-arm or HJ structure (lanes 4 and 12), as previously observed with the yeast and human complexes (Ciccia et al., 2003; Whitby et al., 2003). As a positive control in these reactions, we used a fraction containing Mus81 that had been prepared from fractionated HeLa cells (Constantinou et al., 2002) (Figure 2B, lane 6). Generation of Eme1-deficient ES cells In order to investigate the cellular function of murine Eme1, we generated Eme1-deficient ES cells. A gene targeting construct (Figure 3A) was designed such that proper integration of the construct in the Eme1 genomic locus should result in the replacement of Eme1 exon 2 (partially) and exons 3–7 (completely) by the neomycin resistance (Neo) gene. Two Eme1+/− clones were reselected in high concentrations of G418 to obtain Eme1−/− clones (Figure 3B). Northern blot analysis confirmed the generation of Eme1-null mutation in these clones, as demonstrated by the complete loss of Eme1 transcript in the homozygous clones (Figure 3C). No difference in viability or growth rate was observed between Eme1−/− and wild-type ES cells (data not shown). Therefore, mammalian Eme1 is dispensable for the viability and growth of ES cells, as was observed for S.pombe and S.cerevisiae. Figure 3.Generation of Eme1+/− and Eme1−/− ES cells. (A) Schematic representations of the Eme1 locus, the gene-targeting construct and the targeted Eme1 allele. Exons are denoted by solid black boxes. SA, short arm; LA, long arm; Neo, neomycin resistance gene; X, XbaI site. (B) Southern blot analysis of wild-type, Eme1+/− and Eme1−/− ES cells. Probing Southern blots of XbaI-digested ES cell DNA with a 5′-flanking Eme1 probe differentiates the wild-type allele (5.5 kb) from the recombined allele (4.3 kb). (C) Northern blot analysis showing loss of Eme1 mRNA in Eme1−/− ES cells. A 20 μg aliquot of total RNA from the indicated Eme1 ES genotypes was northern blotted and probed with a 32P-radiolabeled full-length Eme1 cDNA. The northern blot was subsequently probed with a GAPDH cDNA to assess loading of mRNA. Download figure Download PowerPoint Loss of Eme1 sensitizes ES cells to DNA damage mus81, eme1 and mms4 mutation in yeast sensitized cells to UV radiation and hydroxyurea (HU), with little or no sensitivity to IR (Boddy et al., 2000; Interthal and Heyer, 2000; Mullen et al., 2001). In order to assess the role of the mammalian Eme1 in DNA repair, we performed colony survival assays on Eme1−/− and wild-type ES cells following exposure to DNA-damaging agents [IR, UV, HU, cisplatin and mitomycin C (MMC)]. As depicted in Figure 4, loss of Eme1 function led to a dramatic increase in the sensitivity of ES cells to increasing concentrations of MMC and cisplatin (Figure 4A and B), agents that cause interstrand cross-links (ICLs) (Dronkert and Kanaar, 2001). Eme1−/− ES cells were also found to be sensitive, though to a lesser extent, to IR, UV and HU treatments (Figure 4C–E), in comparison with the wild-type control. The sensitivity of Eme1+/− ES cells to these agents was comparable with that of wild-type cells (data not shown). Figure 4.Loss of Eme1 sensitizes ES cells to DNA damage. (A–E) Wild-type (solid circles) and Eme1−/− (open circles) ES colony survival following DNA damage by MMC, cisplatin, IR, HU and UV treatment. Doses of the individual treatments are plotted on the x-axis, while the y-axis denotes the corresponding fold sensitivity of colonies over untreated controls. Eme1−/− ES cells were extremely sensitive to MMC and cisplatin treatments, and only mildly sensitive to the other agents. (F) MMC sensitivity of Eme1−/− ES clones complemented with HA-tagged Eme1 cDNA (closed triangle and open square). Stable transfection of Eme1-HA cDNA in Eme1-deficient ES cells restores MMC sensitivity to near wild-type levels. Download figure Download PowerPoint Reconstitution of Eme1 in the Eme1−/− ES cells was carried out to determine whether the remarkable sensitivity to MMC was specifically caused by the loss of Eme1. Following electroporation of a plasmid construct expressing HA-tagged Eme1 into Eme1−/− ES cell clones, colonies expressing HA-Eme1 were identified by immunoprecipitation and western blotting (data not shown). Two HA-Eme1 reconstituted Eme1−/− ES clones from two different Eme1−/− ES parental clones showed rescue to near wild-type levels when tested for MMC sensitivity (Figure 4F). This complementation demonstrates that the observed phenotype is caused by the specific loss of Eme1. Thus, mammalian Eme1 is essential for the repair of MMC- and cisplatin-induced DNA damage in ES cells, and marginally involved in the repair of IR, UV and HU damage. DNA damage-activated cell cycle checkpoints are intact in Eme1−/− ES cells Cell cycle checkpoints ensure proper DNA repair, and hence cell survival, by preventing cells with damaged DNA from dividing until the damage has been repaired (Kaufmann, 1995; Zhou and Elledge, 2000). The interaction of yeast and human Mus81 with the cell cycle checkpoint protein Chk2, and the increased abundance of Mus81 in human cells exposed to agents that damage DNA or block replication, raised the possibility of a cell cycle checkpoint role for Mus81–Eme1 (Boddy et al., 2000; Chen et al., 2001). Intra-S-phase checkpoints were analyzed by [3H]thymidine incorporation into the DNA of wild-type and Eme1−/− ES cells following MMC treatment, and compared with untreated controls (Figure 5A). Increasing doses of MMC resulted in decreased DNA synthesis that was equivalent in both wild-type and Eme1−/− ES cells, showing no contribution of Eme1 to the cell cycle replication checkpoint. Moreover, fluorescence-activated cell sorting (FACS) analysis of propidium iodide-stained DNA content for cell cycle distribution of untreated and MMC-treated (0.1, 0.5 and 1 μg/ml) wild-type and Eme1−/− ES cells over a time course (6, 12 and 24 h) was comparable in both genotypes (Figure 5B; data not shown). Also, there was no difference in the cell cycle profile of these cells following 24 h of IR, UV and HU treatments (data not shown). These results, again, showed no role for Eme1 in regulating the cell cycle. Figure 5.Intact intra-S-phase checkpoint and G2–M cell cycle arrest in the absence of Eme1. (A) The MMC-induced intra S-phase checkpoint is not affected in Eme1−/− ES cells. The proportion of [3H]thymidine incorporation in untreated wild-type (black square) and Eme1−/− (gray diamond) cells was set as 100%. The decreased proportion of [3H]thymidine incorporation following increasing doses of MMC treatments was similar for both wild-type and Eme1−/− ES cells. (B) G2–M checkpoint activation of wild-type and Eme1−/− ES cells following MMC treatment. Wild-type (left panel) and Eme1−/− (right panel) ES cells were subjected to 1 μg/ml of MMC for 1 h and returned to regular medium for 6, 12 and 24 h, following which time they were stained with propidium iodide and analyzed by FACS. Both genotypes displayed similar cell cycle profiles, with cells progressively accumulating in the G2/M phase. Download figure Download PowerPoint Eme1 is not essential for gene targeting We sought to determine if mammalian Eme1 would have a role in HR, as was demonstrated for its yeast counterpart, by analyzing HR-mediated gene targeting. Although the precise mechanism for gene targeting has not been established, HR between the exogenous DNA sequence and the chromosome is expected in the regions of flanking homology followed by resolution of DNA intermediates (Niedernhofer et al., 2001). The efficiency of gene targeting between wild-type/Eme1+/− and Eme1−/− ES cells was assessed at two independent genomic loci, Rad54 (J.Essers and R.Kanaar, in preparation) and Pim1 (te Riele et al., 1990; Moynahan et al., 2001). The Rad54 targeting vector was designed with a promoterless human Rad54 cDNA fused to green fluorescent protein (GFP), such that expression could occur only following integration at its specific locus (Figure 6A). Upon puromycin selection, while Rad54−/− ES cells showed reduced GFP+ cells (2.8%), comparable numbers of wild-type (13.1%), Eme1+/− (21.3%) and Eme1−/− (11.6 and 21.1%) ES clones were GFP+ (Figure 6B), showing no loss of HR-mediated gene targeting potential in the absence of Eme1. Southern analysis confirmed that GFP+ cells had proper integration of the targeting construct (data not shown). The use of a high efficiency Pim1 targeting construct (Figure 6C) and the expression of its selection marker hygromycin (hygR), being dependent on specific integration at the Pim1 locus, led to similar numbers (>90% of the hygR) of Eme1+/− and Eme1−/− clones being correctly gene targeted (Figure 6D). These findings indicate that Eme1 is not required for the resolution of DNA intermediates that arise during HR-mediated gene targeting. Figure 6.Eme1 does not impair homologous recombination processes such as gene targeting. (A) Schematic of the gene targeting strategy at the Rad54 locus. The Rad54 targeting construct (11.1hRAD54-GFP) and gene locus are depicted. Expression of Rad54–GFP is dependent on proper gene targeting at the Rad54 locus. (B) FACS analysis of the percentage of GFP+ cells after puromycin selection. Whereas gene targeting is clearly impaired in Rad54−/− ES cells, levels of GFP expression in Eme1+/− and Eme1−/− were similar to the wild-type control. (C) Gene targeting strategy at the Pim1 locus. (D) PCR analysis of gene-targeted clones. Eme1+/− and Eme1−/− ES cells were electroporated with the linear p59XDR-GFP6 targeting construct that favors gene targeting by selecting predominantly for survival of colonies that had integration at the Pim-1 locus. Homologous recombination-mediated gene targeting was tested by PCR, and showed no difference between the two genotypes. Download figure Download PowerPoint Eme1 is not essential for gene conversion repair of DSBs Continuing our investigation into a role for Eme1 in HR, we assessed HR-mediated gene conversion by analyzing the ability of wild-type, Eme1+/− and Eme1−/− ES cells to repair a DSB by this process. Gene conversion is a Rad51-mediated invasion of the 3′-single-stranded tail of the broken DNA into homologous regions present on either the same or different chromosomes (Johnson and Jasin, 2001). A single DSB was induced by transient expression of SceI at the Pim1 locus, targeted with a construct bearing a SceI site and two non-functional GFP ORFs (Figure 7A, upper panel) (Moynahan et al., 2001). Repair of the break using the homologous downstream iGFP gene can occur by gene conversion without crossing-over. A gene conversion event results in the conversion of the I-SceI restriction site to the native BcgI GFP sequence, and restoration of GFP expression. Repair by gene conversion was scored by the number of GFP+ cells using fl
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