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      Cooperative Control of Holliday Junction Resolution and DNA Repair by the SLX1 and MUS81-EME1 Nucleases

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          Summary

          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.

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          Highlights

          • 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

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          Most cited references32

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          The Bloom's syndrome helicase suppresses crossing over during homologous recombination.

          Mutations in BLM, which encodes a RecQ helicase, give rise to Bloom's syndrome, a disorder associated with cancer predisposition and genomic instability. A defining feature of Bloom's syndrome is an elevated frequency of sister chromatid exchanges. These arise from crossing over of chromatid arms during homologous recombination, a ubiquitous process that exists to repair DNA double-stranded breaks and damaged replication forks. Whereas crossing over is required in meiosis, in mitotic cells it can be associated with detrimental loss of heterozygosity. BLM forms an evolutionarily conserved complex with human topoisomerase IIIalpha (hTOPO IIIalpha), which can break and rejoin DNA to alter its topology. Inactivation of homologues of either protein leads to hyper-recombination in unicellular organisms. Here, we show that BLM and hTOPO IIIalpha together effect the resolution of a recombination intermediate containing a double Holliday junction. The mechanism, which we term double-junction dissolution, is distinct from classical Holliday junction resolution and prevents exchange of flanking sequences. Loss of such an activity explains many of the cellular phenotypes of Bloom's syndrome. These results have wider implications for our understanding of the process of homologous recombination and the mechanisms that exist to prevent tumorigenesis.
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            Fanconi anemia and its diagnosis.

            Fanconi anemia (FA) is a genetically and phenotypically heterogeneous recessive disorder characterized by diverse congenital malformations, progressive pancytopenia, and predisposition to both hematologic malignancies and solid tumors. Congenital anomalies vary from patient to patient and may affect skeletal morphogenesis as well as any of the major organ systems. Although this highly variable phenotype makes accurate diagnosis on the basis of clinical manifestations difficult in some patients, laboratory study of chromosomal breakage induced by diepoxybutane (DEB) or other crosslinking agents provides a unique cellular marker for the diagnosis of the disorder either prenatally or postnatally. Diagnosis based on abnormal response to DNA crosslinking agents can be used to identify the pre-anemia patient as well as patients with aplastic anemia or leukemia who may or may not have the physical stigmata associated with the syndrome. This overview will present our current knowledge regarding the varied phenotypic manifestations of FA and procedures for diagnosis based upon abnormal DNA damage responses.
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              Human SLX4 is a Holliday junction resolvase subunit that binds multiple DNA repair/recombination endonucleases.

              Structure-specific endonucleases resolve DNA secondary structures generated during DNA repair and recombination. The yeast 5' flap endonuclease Slx1-Slx4 has received particular attention with the finding that Slx4 has Slx1-independent key functions in genome maintenance. Although Slx1 is a highly conserved protein in eukaryotes, no orthologs of Slx4 were reported other than in fungi. Here we report the identification of Slx4 orthologs in metazoa, including fly MUS312, essential for meiotic recombination, and human BTBD12, an ATM/ATR checkpoint kinase substrate. Human SLX1-SLX4 displays robust Holliday junction resolvase activity in addition to 5' flap endonuclease activity. Depletion of SLX1 and SLX4 results in 53BP1 foci accumulation and H2AX phosphorylation as well as cellular hypersensitivity to MMS. Furthermore, we show that SLX4 binds the XPF(ERCC4) and MUS81 subunits of the XPF-ERCC1 and MUS81-EME1 endonucleases and is required for DNA interstrand crosslink repair. We propose that SLX4 acts as a docking platform for multiple structure-specific endonucleases.
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                Author and article information

                Journal
                Mol Cell
                Mol. Cell
                Molecular Cell
                Cell Press
                1097-2765
                1097-4164
                24 October 2013
                24 October 2013
                : 52
                : 2
                : 221-233
                Affiliations
                [1 ]MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK
                [2 ]CR-UK Nucleic Acid Structure Research Group, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK
                [3 ]Division of Cell Signalling and Immunology, College of Life Sciences, University of Dundee, Dundee DD1 5EH, Scotland, UK
                Author notes
                []Corresponding author j.rouse@ 123456dundee.ac.uk
                [4]

                These authors contributed equally to this work

                Article
                S1097-2765(13)00636-9
                10.1016/j.molcel.2013.08.036
                3808987
                24076219
                dc19b73b-6ec0-40a7-8e82-83aa64c35a05
                © 2013 The Authors

                This document may be redistributed and reused, subject to certain conditions.

                History
                : 26 April 2013
                : 19 July 2013
                : 14 August 2013
                Categories
                Article

                Molecular biology
                Molecular biology

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