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• W1980830652 abstract "•Resolution of Holliday junctions in mouse cells requires the SLX1 nuclease•SLX1 acts cooperatively with MUS81-EME1 in HJ resolution and ICL repair•Mutations in SLX4 that prevent it binding to SLX1 and MUS81-EME1 abolish HJ resolution•DNA substrates of SLX1 and MUS81-EME1 in ICL repair appear to be different from HJs Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability. Holliday junctions (HJs) are X-shaped DNA structures that arise during homologous recombination, which must be removed to enable chromosome segregation. The SLX1 and MUS81-EME1 nucleases can both process HJs in vitro, and they bind in close proximity on the SLX4 scaffold, hinting at possible cooperation. However, the cellular roles of mammalian SLX1 are not yet known. Here, we use mouse genetics and structure function analysis to investigate SLX1 function. Disrupting the murine Slx1 and Slx4 genes revealed that they are essential for HJ resolution in mitotic cells. Moreover, SLX1 and MUS81-EME1 act together to resolve HJs in a manner that requires tethering to SLX4. We also show that SLX1, like MUS81-EME1, is required for repair of DNA interstrand crosslinks, but this role appears to be independent of HJ cleavage, at least in mouse cells. These findings shed light on HJ resolution in mammals and on maintenance of genome stability. SLX4 coordinates a multiprotein complex that is important for DNA repair. In metazoans, this complex includes three structure-selective nucleases: XPF-ERCC1, MUS81-EME1, and SLX1 (Andersen et al., 2009Andersen S.L. Bergstralh D.T. Kohl K.P. LaRocque J.R. Moore C.B. Sekelsky J. Drosophila MUS312 and the vertebrate ortholog BTBD12 interact with DNA structure-specific endonucleases in DNA repair and recombination.Mol. Cell. 2009; 35: 128-135Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar, Fekairi et al., 2009Fekairi S. Scaglione S. Chahwan C. Taylor E.R. Tissier A. Coulon S. Dong M.Q. Ruse C. Yates 3rd, J.R. Russell P. et al.Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.Cell. 2009; 138: 78-89Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, Saito et al., 2009Saito T.T. Youds J.L. Boulton S.J. Colaiácovo M.P. Caenorhabditis elegans HIM-18/SLX-4 interacts with SLX-1 and XPF-1 and maintains genomic integrity in the germline by processing recombination intermediates.PLoS Genet. 2009; 5: e1000735Crossref PubMed Scopus (84) Google Scholar, Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). Together these nucleases confer the complex with the ability to cleave a wide range of branched DNA structures in vitro, which mimic DNA intermediates that occur during the repair of damaged DNA and broken DNA replication forks. Both MUS81-EME1 and XPF-ERCC1 are required for the repair of DNA interstrand crosslinks (ICLs) in mammalian cells, and the latter is also required for repair of UV-induced lesions (Ciccia et al., 2008Ciccia A. McDonald N. West S.C. Structural and functional relationships of the XPF/MUS81 family of proteins.Annu. Rev. Biochem. 2008; 77: 259-287Crossref PubMed Scopus (228) Google Scholar). Depletion of SLX4 from human cells using siRNA duplexes does not affect UV repair but causes pronounced hypersensitivity to agents that induce DNA ICLs (Fekairi et al., 2009Fekairi S. Scaglione S. Chahwan C. Taylor E.R. Tissier A. Coulon S. Dong M.Q. Ruse C. Yates 3rd, J.R. Russell P. et al.Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.Cell. 2009; 138: 78-89Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). The importance of SLX4 in ICL repair in humans is underscored by the observation that biallelic mutations in SLX4/FANCP cause Fanconi anemia (FA) (Kim et al., 2011Kim Y. Lach F.P. Desetty R. Hanenberg H. Auerbach A.D. Smogorzewska A. Mutations of the SLX4 gene in Fanconi anemia.Nat. Genet. 2011; 43: 142-146Crossref PubMed Scopus (261) Google Scholar, Stoepker et al., 2011Stoepker C. Hain K. Schuster B. Hilhorst-Hofstee Y. Rooimans M.A. Steltenpool J. Oostra A.B. Eirich K. Korthof E.T. Nieuwint A.W. et al.SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype.Nat. Genet. 2011; 43: 138-141Crossref PubMed Scopus (231) Google Scholar), a cancer predisposition syndrome accompanied by developmental, skeletal, and hematological defects (Auerbach, 2009Auerbach A.D. Fanconi anemia and its diagnosis.Mutat. Res. 2009; 668: 4-10Crossref PubMed Scopus (392) Google Scholar). Despite the clear importance of the SLX4 complex in DNA repair, little is known about the underlying molecular mechanisms. For example, it is not yet known if SLX1 is involved in ICL repair, partly because depletion of SLX1 from human cells destabilized SLX4, preventing the functional analysis of SLX1 (Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Furthermore, it is not yet known how SLX4 affects the associated nucleases, and there has been some debate about whether the exquisite hypersensitivity of SLX4-defective cells reflects the loss of regulation of one or more of these nucleases. In this light, a recent study concluded that the role of SLX4 in ICL repair involves XPF-ERCC1 only, because a fragment of SLX4 lacking amino acids 1–499, that did not interact with XPF-ERCC1, did not rescue the mitomycin-C (MMC) sensitivity of Slx4 hypomorphic MEFs (Crossan et al., 2011Crossan G.P. van der Weyden L. Rosado I.V. Langevin F. Gaillard P.H. McIntyre R.E. Gallagher F. Kettunen M.I. Lewis D.Y. Brindle K. et al.Sanger Mouse Genetics ProjectDisruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia.Nat. Genet. 2011; 43: 147-152Crossref PubMed Scopus (166) Google Scholar). However, the first 499 amino acids of SLX4 also contain two ubiquitin-binding domains that are vital for ICL repair but that are not required for SLX4 to interact with XPF (Kim et al., 2011Kim Y. Lach F.P. Desetty R. Hanenberg H. Auerbach A.D. Smogorzewska A. Mutations of the SLX4 gene in Fanconi anemia.Nat. Genet. 2011; 43: 142-146Crossref PubMed Scopus (261) Google Scholar, Stoepker et al., 2011Stoepker C. Hain K. Schuster B. Hilhorst-Hofstee Y. Rooimans M.A. Steltenpool J. Oostra A.B. Eirich K. Korthof E.T. Nieuwint A.W. et al.SLX4, a coordinator of structure-specific endonucleases, is mutated in a new Fanconi anemia subtype.Nat. Genet. 2011; 43: 138-141Crossref PubMed Scopus (231) Google Scholar). Two studies used SLX4 deletion mutants lacking the C-terminal helix-turn-helix (HtH) domain to investigate the importance of SLX1 binding for ICL repair. One study found that the HtH deletion mutant fully rescues the sensitivity of Slx4 hypomorphic MEFs (Crossan et al., 2011Crossan G.P. van der Weyden L. Rosado I.V. Langevin F. Gaillard P.H. McIntyre R.E. Gallagher F. Kettunen M.I. Lewis D.Y. Brindle K. et al.Sanger Mouse Genetics ProjectDisruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia.Nat. Genet. 2011; 43: 147-152Crossref PubMed Scopus (166) Google Scholar), whereas the other showed that this mutant only partly rescued the MMC sensitivity of SLX4-defective FA cells (Kim et al., 2013Kim Y. Spitz G.S. Veturi U. Lach F.P. Auerbach A.D. Smogorzewska A. Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4.Blood. 2013; 121: 54-63Crossref PubMed Scopus (127) Google Scholar). Therefore, the functional relevance of the binding of nucleases to SLX4 in ICL repair remains unclear. The SLX4 complex is capable of processing Holliday junctions (HJs) in vitro (Fekairi et al., 2009Fekairi S. Scaglione S. Chahwan C. Taylor E.R. Tissier A. Coulon S. Dong M.Q. Ruse C. Yates 3rd, J.R. Russell P. et al.Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.Cell. 2009; 138: 78-89Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). HJs are four-way DNA junctions at which two chromatids are topologically intertwined. These structures arise during homologous recombination (HR), a process required for repairing unscheduled double-strand breaks (DSBs) or damaged replication forks in mitotic cells. HJs are also key intermediates during meiotic recombination (Schwartz and Heyer, 2011Schwartz E.K. Heyer W.D. Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes.Chromosoma. 2011; 120: 109-127Crossref PubMed Scopus (143) Google Scholar, West, 2009West S.C. The search for a human Holliday junction resolvase.Biochem. Soc. Trans. 2009; 37: 519-526Crossref PubMed Scopus (36) Google Scholar). Ultimately HJs must be removed to enable chromosome segregation, and two distinct modes of HJ removal have been identified in mammalian cells (Schwartz and Heyer, 2011Schwartz E.K. Heyer W.D. Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes.Chromosoma. 2011; 120: 109-127Crossref PubMed Scopus (143) Google Scholar). The first pathway involves the dissolution of double HJs (dHJs) by the BTR complex (BLM-TOPIII-RMI1-RMI2). The coupled helicase and topoisomerase activities of BLM and TOPIII, respectively, disassemble HJs, resulting exclusively in noncrossover products (Chaganti et al., 1974Chaganti R.S. Schonberg S. German J. A manyfold increase in sister chromatid exchanges in Bloom’s syndrome lymphocytes.Proc. Natl. Acad. Sci. USA. 1974; 71: 4508-4512Crossref PubMed Scopus (785) Google Scholar, Wu and Hickson, 2003Wu L. Hickson I.D. The Bloom’s syndrome helicase suppresses crossing over during homologous recombination.Nature. 2003; 426: 870-874Crossref PubMed Scopus (875) Google Scholar, Wu and Hickson, 2006Wu L. Hickson I.D. DNA helicases required for homologous recombination and repair of damaged replication forks.Annu. Rev. Genet. 2006; 40: 279-306Crossref PubMed Scopus (145) Google Scholar). This pathway dominates in mitotic cells, possibly because minimizing crossovers lowers the incidence of loss of heterozygosity (LOH) that would increase disease risk and impair organism fitness (LaRocque et al., 2011LaRocque J.R. Stark J.M. Oh J. Bojilova E. Yusa K. Horie K. Takeda J. Jasin M. Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells.Proc. Natl. Acad. Sci. USA. 2011; 108: 11971-11976Crossref PubMed Scopus (57) Google Scholar). Alternatively, HJs can be resolved by nucleases (Schwartz and Heyer, 2011Schwartz E.K. Heyer W.D. Processing of joint molecule intermediates by structure-selective endonucleases during homologous recombination in eukaryotes.Chromosoma. 2011; 120: 109-127Crossref PubMed Scopus (143) Google Scholar, West, 2009West S.C. The search for a human Holliday junction resolvase.Biochem. Soc. Trans. 2009; 37: 519-526Crossref PubMed Scopus (36) Google Scholar). Depending on the symmetry of the cleavage, crossover or noncrossover products may occur. Cells from Bloom syndrome (BS) patients lacking BLM show a large increase in the frequency of sister chromatid exchanges (SCEs), which are thought to result from the crossovers generated by nucleolytic resolution of dHJs that escape dissolution (Chaganti et al., 1974Chaganti R.S. Schonberg S. German J. A manyfold increase in sister chromatid exchanges in Bloom’s syndrome lymphocytes.Proc. Natl. Acad. Sci. USA. 1974; 71: 4508-4512Crossref PubMed Scopus (785) Google Scholar, Wechsler et al., 2011Wechsler T. Newman S. West S.C. Aberrant chromosome morphology in human cells defective for Holliday junction resolution.Nature. 2011; 471: 642-646Crossref PubMed Scopus (165) Google Scholar). To date, three nuclear HJ resolving activities have been identified in mammalian cells: MUS81-EME1, SLX1, and GEN1 (Bailly et al., 2010Bailly A.P. Freeman A. Hall J. Déclais A.C. Alpi A. Lilley D.M. Ahmed S. Gartner A. The Caenorhabditis elegans homolog of Gen1/Yen1 resolvases links DNA damage signaling to DNA double-strand break repair.PLoS Genet. 2010; 6: e1001025Crossref PubMed Scopus (74) Google Scholar, Fekairi et al., 2009Fekairi S. Scaglione S. Chahwan C. Taylor E.R. Tissier A. Coulon S. Dong M.Q. Ruse C. Yates 3rd, J.R. Russell P. et al.Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.Cell. 2009; 138: 78-89Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Ip et al., 2008Ip S.C.Y. Rass U. Blanco M.G. Flynn H.R. Skehel J.M. West S.C. Identification of Holliday junction resolvases from humans and yeast.Nature. 2008; 456: 357-361Crossref PubMed Scopus (298) Google Scholar, Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, Taylor and McGowan, 2008Taylor E.R. McGowan C.H. Cleavage mechanism of human Mus81-Eme1 acting on Holliday-junction structures.Proc. Natl. Acad. Sci. USA. 2008; 105: 3757-3762Crossref PubMed Scopus (59) Google Scholar, Wechsler et al., 2011Wechsler T. Newman S. West S.C. Aberrant chromosome morphology in human cells defective for Holliday junction resolution.Nature. 2011; 471: 642-646Crossref PubMed Scopus (165) Google Scholar, West, 2009West S.C. The search for a human Holliday junction resolvase.Biochem. Soc. Trans. 2009; 37: 519-526Crossref PubMed Scopus (36) Google Scholar). GEN1 cleaves HJs symmetrically to produce nicked linear duplex products (Ip et al., 2008Ip S.C.Y. Rass U. Blanco M.G. Flynn H.R. Skehel J.M. West S.C. Identification of Holliday junction resolvases from humans and yeast.Nature. 2008; 456: 357-361Crossref PubMed Scopus (298) Google Scholar, Rass et al., 2010Rass U. Compton S.A. Matos J. Singleton M.R. Ip S.C. Blanco M.G. Griffith J.D. West S.C. Mechanism of Holliday junction resolution by the human GEN1 protein.Genes Dev. 2010; 24: 1559-1569Crossref PubMed Scopus (113) Google Scholar), whereas SLX1 introduces a mixture of symmetric and asymmetric cuts across the junction (Fekairi et al., 2009Fekairi S. Scaglione S. Chahwan C. Taylor E.R. Tissier A. Coulon S. Dong M.Q. Ruse C. Yates 3rd, J.R. Russell P. et al.Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.Cell. 2009; 138: 78-89Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). In contrast to GEN1 and SLX1, MUS81-EME1 does not cleave intact HJs efficiently, but prefers nicked junctions and recombination intermediates such as extended D-loop structures (Boddy et al., 2001Boddy M.N. Gaillard P.H. McDonald W.H. Shanahan P. Yates 3rd, J.R. Russell P. Mus81-Eme1 are essential components of a Holliday junction resolvase.Cell. 2001; 107: 537-548Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar, Ciccia et al., 2003Ciccia A. Constantinou A. West S.C. Identification and characterization of the human mus81-eme1 endonuclease.J. Biol. Chem. 2003; 278: 25172-25178Crossref PubMed Scopus (167) Google Scholar, Doe et al., 2002Doe C.L. Ahn J.S. Dixon J. Whitby M.C. Mus81-Eme1 and Rqh1 involvement in processing stalled and collapsed replication forks.J. Biol. Chem. 2002; 277: 32753-32759Crossref PubMed Scopus (204) Google Scholar, Gaillard et al., 2003Gaillard P.H.L. Noguchi E. Shanahan P. Russell P. The endogenous Mus81-Eme1 complex resolves Holliday junctions by a nick and counternick mechanism.Mol. Cell. 2003; 12: 747-759Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, Whitby et al., 2003Whitby M.C. Osman F. Dixon J. Cleavage of model replication forks by fission yeast Mus81-Eme1 and budding yeast Mus81-Mms4.J. Biol. Chem. 2003; 278: 6928-6935Crossref PubMed Scopus (102) Google Scholar). The elevated SCE frequency in BS cells requires GEN1 and MUS81-EME1 (Wechsler et al., 2011Wechsler T. Newman S. West S.C. Aberrant chromosome morphology in human cells defective for Holliday junction resolution.Nature. 2011; 471: 642-646Crossref PubMed Scopus (165) Google Scholar). However, although GEN1 cleaves intact HJs efficiently in vitro, MUS81-EME1 does not. Instead MUS81-EME1 shows a strong preference for nicked HJs, suggesting that it might act on junctions that are subjected to prior nicking by a different nuclease. One possible candidate is SLX1 because it can process intact HJs efficiently in vitro. Furthermore, SLX1 and MUS81-EME1 bind to the HtH motif and the SAP domain of SLX4, respectively (Fekairi et al., 2009Fekairi S. Scaglione S. Chahwan C. Taylor E.R. Tissier A. Coulon S. Dong M.Q. Ruse C. Yates 3rd, J.R. Russell P. et al.Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.Cell. 2009; 138: 78-89Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Kim et al., 2013Kim Y. Spitz G.S. Veturi U. Lach F.P. Auerbach A.D. Smogorzewska A. Regulation of multiple DNA repair pathways by the Fanconi anemia protein SLX4.Blood. 2013; 121: 54-63Crossref PubMed Scopus (127) Google Scholar, Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). The close proximity of the SLX4 SAP and HtH domains places SLX1 close enough to MUS81-EME1 on SLX4 to suggest the possibility of cooperation between the two nucleases. In this sense, one function of SLX4 in its capacity as a scaffold would be to facilitate serial processing of HJs, a possibility raised previously (Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). In this study, we investigated whether SLX1 is involved in HJ resolution and ICL repair, and we tested potential cooperation with MUS81-EME1 and the functional significance of binding of these nucleases to SLX4. There is currently no information on SLX1 function in mammals. To study the in vivo roles of SLX1, we disrupted the murine Giyd2 (Slx1) gene by eliminating the transcription start site and the remainder of the first exon (Figure S1A). Gene disruption was confirmed by Southern blotting and PCR (Figures S1B and S1C). Both Slx1+/− and Slx1−/− mice were born at Mendelian frequencies (Table S1) without overt morphological, developmental, or hematological defects. Adult mice were fertile (data not shown). SLX1 protein was undetectable in extracts of Slx1−/− mouse embryonic fibroblast extracts (MEFs) (Figure 1A) or testis extracts (Figure S1D) by western blotting. The expression levels of SLX4, ERCC1, and MUS81 proteins, however, were normal in Slx1−/− MEFs (Figure 1A). We also disrupted the murine Btbd12 (Slx4) gene (Figure S2A); gene disruption was confirmed by Southern blotting, PCR (Figures S2B and S2C), and western blotting (Figure S2D). The expression levels of SLX4-associated proteins ERCC1 and MUS81 were normal in MEFs from the Slx4−/− mice but SLX1 protein was undetectable by western blotting (Figure 1A). This suggests that SLX4 regulates SLX1 protein stability, and therefore SLX4 null mice lack both SLX1 and SLX4 proteins. Initially, no viable Slx4−/− offspring were obtained from crossing Slx4 heterozygotes. However, after backcrossing the heterozygotes five times, we obtained viable Slx4−/− mice, albeit at sub-Mendelian frequencies (Table S2). Slx4−/− mice were on average around 10%–15% smaller than heterozygotes or wild-type mice at the age of 6 weeks (data not shown). No overt developmental or morphological defects were observed. Although mating Slx4−/− males with Slx4−/− females resulted in viable progeny, testes in males were on average 47% smaller than in wild-type mice at 10 weeks of age (data not shown). Smaller testis size is in line with two previous reports describing a hypomorphic Slx4 mouse strain made by the European Conditional Mouse Mutagenesis Program (EUCOMM) (Crossan et al., 2011Crossan G.P. van der Weyden L. Rosado I.V. Langevin F. Gaillard P.H. McIntyre R.E. Gallagher F. Kettunen M.I. Lewis D.Y. Brindle K. et al.Sanger Mouse Genetics ProjectDisruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia.Nat. Genet. 2011; 43: 147-152Crossref PubMed Scopus (166) Google Scholar, Holloway et al., 2011Holloway J.K. Mohan S. Balmus G. Sun X. Modzelewski A. Borst P.L. Freire R. Weiss R.S. Cohen P.E. Mammalian BTBD12 (SLX4) protects against genomic instability during mammalian spermatogenesis.PLoS Genet. 2011; 7: e1002094Crossref PubMed Scopus (55) Google Scholar). It is worth noting that the EUCOMM mice were reported to exhibit phenotypes reminiscent of those seen in FA patients such as aplastic anemia (Crossan et al., 2011Crossan G.P. van der Weyden L. Rosado I.V. Langevin F. Gaillard P.H. McIntyre R.E. Gallagher F. Kettunen M.I. Lewis D.Y. Brindle K. et al.Sanger Mouse Genetics ProjectDisruption of mouse Slx4, a regulator of structure-specific nucleases, phenocopies Fanconi anemia.Nat. Genet. 2011; 43: 147-152Crossref PubMed Scopus (166) Google Scholar), even though mouse knockouts of other FA genes did not recapitulate most of these phenotypes (Bakker et al., 2013Bakker S.T. de Winter J.P. te Riele H. Learning from a paradox: recent insights into Fanconi anaemia through studying mouse models.Dis. Model. Mech. 2013; 6: 40-47Crossref PubMed Scopus (48) Google Scholar). However, we saw no evidence of an FA-like syndrome in the Slx4−/− or Slx1−/− mice we generated in this study (data not shown). We next investigated if SLX1 is involved in ICL repair assessed by hypersensitivity to genotoxins that induce ICLs. As shown in Figure 1B, Slx1−/− MEFs are hypersensitive to ICL-inducing agents such as nitrogen mustard (HN-2) and MMC, and embryonic stem cells (ESCs) from Slx1−/− mice were also hypersensitive to MMC (Figure S3A). The sensitivity of Slx1−/− MEFs to agents that induce ICLs was much less pronounced than Slx4−/− MEFs, probably because SLX4 binds to several nucleases involved in ICL repair in addition to SLX1. Slx1−/− MEFs and ESCs were not more sensitive to camptothecin (CPT), ionizing radiation (IR), hydroxyurea (HU), or UV light than wild-type cells (Figures S3A and S3B). Defects in the repair of ICLs often result in chromosome abnormalities (Auerbach, 2009Auerbach A.D. Fanconi anemia and its diagnosis.Mutat. Res. 2009; 668: 4-10Crossref PubMed Scopus (392) Google Scholar). In this light, we observed a slight increase in the number of chromosome abnormalities such as chromatid breaks and radial structures in Slx1−/− MEFs exposed to MMC compared with wild-type cells, and a much larger increase in Slx4−/− MEFs (Figure S3C). We next tested if the nuclease activity of SLX1 is required for cellular resistance to ICL-inducing agents. To this end we mutated a highly conserved residue, Glu79, in the SLX1 URI-type nuclease domain to alanine. This mutation abolished the activity of FLAG-tagged mouse SLX1 immunoprecipitated from HEK293 cells in the cleavage of a radioactively [5′-32P]-labeled HJ with a core that could undergo a number of steps of branch migration, thereby presenting all possible dinucleotides at the point of exchange (Figure 1C) (Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar). Next, Slx1−/− MEFs were infected with viruses expressing untagged SLX1 wild-type or E79A, or with empty virus; mutation of E79 in SLX1 did not affect interaction with SLX4 (Figure 1D). As shown in Figure 1E, wild-type SLX1, but not the E79A mutant, rescued the MMC hypersensitivity of Slx1−/− MEFs. Together, these data provide evidence that SLX1 is involved in repair of DNA ICLs in mammals. SLX1 is capable of processing HJs in vitro (Fekairi et al., 2009Fekairi S. Scaglione S. Chahwan C. Taylor E.R. Tissier A. Coulon S. Dong M.Q. Ruse C. Yates 3rd, J.R. Russell P. et al.Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.Cell. 2009; 138: 78-89Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar, Muñoz et al., 2009Muñoz I.M. Hain K. Déclais A.C. Gardiner M. Toh G.W. Sanchez-Pulido L. Heuckmann J.M. Toth R. Macartney T. Eppink B. et al.Coordination of structure-specific nucleases by human SLX4/BTBD12 is required for DNA repair.Mol. Cell. 2009; 35: 116-127Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar, Svendsen et al., 2009Svendsen J.M. Smogorzewska A. Sowa M.E. O’Connell B.C. Gygi S.P. Elledge S.J. Harper J.W. Mammalian BTBD12/SLX4 assembles a Holliday junction resolvase and is required for DNA repair.Cell. 2009; 138: 63-77Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar). We next investigated whether SLX1 is required for HJ resolution in vivo using the elevated SCE frequency observed in BLM-depleted cells as readout (Figure 2A). To this end, we used shRNA-expressing retroviruses to deplete BLM from Slx1−/− MEFs (Figure S4A) (Sfeir et al., 2009Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). As shown in Figure 2B, BLM protein was undetectable in MEF extracts when cells were infected with retrovirus expressing the BLM shR" @default.
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• W1980830652 title "Cooperative Control of Holliday Junction Resolution and DNA Repair by the SLX1 and MUS81-EME1 Nucleases" @default.
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