SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key components of higher-order chromatin structures. The SMC5/6 complex is essential for proliferation in yeast and is involved in replication fork stability and processing. However, the precise mechanism of action of SMC5/6 is not known. Here we present evidence that the NSE1/NSE3/NSE4 sub-complex of SMC5/6 binds to double-stranded DNA without any preference for DNA-replication/recombination intermediates. Mutations of key basic residues within the NSE1/NSE3/NSE4 DNA-binding surface reduce binding to DNA in vitro. Their introduction into the Schizosaccharomyces pombe genome results in cell death or hypersensitivity to DNA damaging agents. Chromatin immunoprecipitation analysis of the hypomorphic nse3 DNA-binding mutant shows a reduced association of fission yeast SMC5/6 with chromatin. Based on our results, we propose a model for loading of the SMC5/6 complex onto the chromatin.

Genome Damage and Stability Centre University of Sussex Falmer Brighton BN1 9RQ United Kingdom

Laboratory of Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Kotlarska 2 Brno 61137 Czech Republic

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Kamenice 5 Brno 62500 Czech Republic

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Kamenice 5 Brno 62500 Czech Republic Laboratory of Functional Genomics and Proteomics National Centre for Biomolecular Research Faculty of Science Masaryk University Kotlarska 2 Brno 61137 Czech Republic

Zobrazit více v PubMed

Losada A., Hirano T. Dynamic molecular linkers of the genome: the first decade of SMC proteins. Genes Dev. 2005;19:1269–1287. PubMed

Carter S.D., Sjögren C. The SMC complexes, DNA and chromosome topology: right or knot. Crit. Rev. Biochem. Mol. Biol. 2012;47:1–16. PubMed

Lehmann A.R. The role of SMC proteins in the responses to DNA damage. DNA Repair. 2005;4:309–314. PubMed

Wu N., Yu H. The Smc complexes in DNA damage response. Cell Biosci. 2012;2:5. PubMed PMC

Lehmann A.R., Walicka M., Griffiths D.J., Murray J.M., Watts F.Z., McCready S., Carr A.M. The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair. Mol. Cell. Biol. 1995;15:7067–7080. PubMed PMC

Ju L., Wing J., Taylor E., Brandt R., Slijepcevic P., Horsch M., Rathkolb B., Rácz I., Becker L., Hans W., et al. SMC6 is an essential gene in mice, but a hypomorphic mutant in the ATPase domain has a mild phenotype with a range of subtle abnormalities. DNA Repair. 2013;12:356–366. PubMed

Irmisch A., Ampatzidou E., Mizuno K., O'Connell M.J., Murray J.M. Smc5/6 maintains stalled replication forks in a recombination-competent conformation. EMBO J. 2009;28:144–155. PubMed PMC

Kegel A., Sjögren C. The Smc5/6 Complex: More Than Repair? Cold Spring Harb. Symp. Quant. Biol. 2010;75:179–187. PubMed

Murray J.M., Carr A.M. Smc5/6: a link between DNA repair and unidirectional replication. Nat. Rev. Mol. Cell Biol. 2008;9:177–182. PubMed

De Piccoli G., Cortes-Ledesma F., Ira G., Torres-Rosell J., Uhle S., Farmer S., Hwang J.-Y., Machin F., Ceschia A., McAleenan A., et al. Smc5–Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination. Nat. Cell Biol. 2006;8:1032–1034. PubMed PMC

Sollier J., Driscoll R., Castellucci F., Foiani M., Jackson S.P., Branzei D. The Saccharomyces cerevisiae Esc2 and Smc5–6 proteins promote sister chromatid junction-mediated intra-S repair. Mol. Biol. Cell. 2009;20:1671–1682. PubMed PMC

Copsey A., Tang S., Jordan P.W., Blitzblau H.G., Newcombe S., Chan A.C.-H., Newnham L., Li Z., Gray S., Herbert A.D., et al. Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions. PLoS Genet. 2013;9:e1004071. PubMed PMC

Xaver M., Huang L., Chen D., Klein F. Smc5/6-Mms21 Prevents and Eliminates Inappropriate Recombination Intermediates in Meiosis. PLoS Genet. 2013;9:e1004067. PubMed PMC

McAleenan A., Cordon-Preciado V., Clemente-Blanco A., Liu I.-C., Sen N., Leonard J., Jarmuz A., Aragón L. SUMOylation of the α-kleisin subunit of cohesin is required for DNA damage-induced cohesion. Curr. Biol. 2012;22:1564–1575. PubMed

Outwin E.A., Irmisch A., Murray J.M., O'Connell M.J. Smc5-Smc6-Dependent Removal of Cohesin from Mitotic Chromosomes. Mol. Cell. Biol. 2009;29:4363–4375. PubMed PMC

Gallego-Paez L.M., Tanaka H., Bando M., Takahashi M., Nozaki N., Nakato R., Shirahige K., Hirota T. Smc5/6-mediated regulation of replication progression contributes to chromosome assembly during mitosis in human cells. Mol. Biol. Cell. 2014;25:302–317. PubMed PMC

Potts P.R., Yu H. The SMC5/6 complex maintains telomere length in ALT cancer cells through SUMOylation of telomere-binding proteins. Nat. Struct. Mol. Biol. 2007;14:581–590. PubMed

Cuylen S., Metz J., Haering C.H. Condensin structures chromosomal DNA through topological links. Nat. Struct. Mol. Biol. 2011;18:894–901. PubMed

Ivanov D., Nasmyth K. A Topological Interaction between Cohesin Rings and a Circular Minichromosome. Cell. 2005;122:849–860. PubMed

Arumugam P., Gruber S., Tanaka K., Haering C.H., Mechtler K., Nasmyth K. ATP Hydrolysis Is Required for Cohesin's Association with Chromosomes. Curr. Biol. 2003;13:1941–1953. PubMed

Gruber S., Arumugam P., Katou Y., Kuglitsch D., Helmhart W., Shirahige K., Nasmyth K. Evidence that Loading of Cohesin Onto Chromosomes Involves Opening of Its SMC Hinge. Cell. 2006;127:523–537. PubMed

Weitzer S., Lehane C., Uhlmann F. A Model for ATP Hydrolysis-Dependent Binding of Cohesin to DNA. Curr. Biol. 2003;13:1930–1940. PubMed

Murayama Y., Uhlmann F. Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature. 2014;505:367–371. PubMed PMC

Piazza I., Rutkowska A., Ori A., Walczak M., Metz J., Pelechano V., Beck M., Haering C.H. Association of condensin with chromosomes depends on DNA binding by its HEAT-repeat subunits. Nat. Struct. Mol. Biol. 2014;21:560–568. PubMed

Griese J.J., Witte G., Hopfner K.-P. Structure and DNA binding activity of the mouse condensin hinge domain highlight common and diverse features of SMC proteins. Nucleic Acids Res. 2010;38:3454–3465. PubMed PMC

Roy M.-A., D'Amours D. DNA-binding properties of Smc6, a core component of the Smc5–6 DNA repair complex. Biochem. Biophys. Res. Commun. 2011;416:80–85. PubMed

Roy M.-A., Siddiqui N., D'Amours D. Dynamic and selective DNA-binding activity of Smc5, a core component of the Smc5-Smc6 complex. Cell Cycle. 2011;10:690–700. PubMed

Fousteri M.I., Lehmann A.R. A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein. EMBO J. 2000;19:1691–1702. PubMed PMC

Palecek J., Vidot S., Feng M., Doherty A.J., Lehmann A.R. The Smc5-Smc6 DNA repair complex. bridging of the Smc5-Smc6 heads by the KLEISIN, Nse4, and non-Kleisin subunits. J. Biol. Chem. 2006;281:36952–36959. PubMed

Duan X., Yang Y., Chen Y.-H., Arenz J., Rangi G.K., Zhao X., Ye H. Architecture of the Smc5/6 Complex of Saccharomyces cerevisiae Reveals a Unique Interaction between the Nse5–6 Subcomplex and the Hinge Regions of Smc5 and Smc6. J. Biol. Chem. 2009;284:8507–8515. PubMed PMC

Sergeant J., Taylor E., Palecek J., Fousteri M., Andrews E.A., Sweeney S., Shinagawa H., Watts F.Z., Lehmann A.R. Composition and Architecture of the Schizosaccharomyces pombe Rad18 (Smc5–6) Complex. Mol. Cell. Biol. 2005;25:172–184. PubMed PMC

Hudson J.J.R., Bednarova K., Kozakova L., Liao C., Guerineau M., Colnaghi R., Vidot S., Marek J., Bathula S.R., Lehmann A.R., et al. Interactions between the Nse3 and Nse4 Components of the SMC5–6 Complex Identify Evolutionarily Conserved Interactions between MAGE and EID Families. PLoS ONE. 2011;6:e17270. PubMed PMC

Doyle J.M., Gao J., Wang J., Yang M., Potts P.R. MAGE-RING Protein Complexes Comprise a Family of E3 Ubiquitin Ligases. Mol. Cell. 2010;39:963–974. PubMed PMC

Roy A., Kucukural A., Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nat. Protoc. 2010;5:725–738. PubMed PMC

Emsley P., Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004;60:2126–2132. PubMed

de Vries S.J., van Dijk M., Bonvin A.M.J.J. The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 2010;5:883–897. PubMed

Wassenaar T.A., van Dijk M., Loureiro-Ferreira N., van der Schot G., de Vries S.J., Schmitz C., van der Zwan J., Boelens R., Giachetti A., Ferella L., et al. WeNMR: Structural Biology on the Grid. J. Grid Comput. 2012;10:743–767.

Studier F.W. Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif. 2005;41:207–234. PubMed

Moreno S., Klar A., Nurse P. Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Methods Enzymol. 1991;194:795–823. PubMed

Morikawa H., Morishita T., Kawane S., Iwasaki H., Carr A.M., Shinagawa H. Rad62 protein functionally and physically associates with the Smc5/Smc6 protein complex and is required for chromosome integrity and recombination repair in fission yeast. Mol. Cell. Biol. 2004;24:9401–9413. PubMed PMC

Watson A.T., Werler P., Carr A.M. Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus. Gene. 2011;484:75–85. PubMed

Pebernard S., Schaffer L., Campbell D., Head S.R., Boddy M.N. Localization of Smc5/6 to centromeres and telomeres requires heterochromatin and SUMO, respectively. EMBO J. 2008;27:3011–3023. PubMed PMC

Guerineau M., Kriz Z., Kozakova L., Bednarova K., Janos P., Palecek J. Analysis of the Nse3/MAGE-binding domain of the Nse4/EID family proteins. PloS One. 2012;7:e35813. PubMed PMC

Kozakova L., Vondrova L., Stejskal K., Charalabous P., Kolesar P., Lehmann A.R., Uldrijan S., Sanderson C.M., Zdrahal Z., Palecek J.J. The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex. Cell Cycle. 2015;14:920–930. PubMed PMC

Palecek J.J., Gruber S. Kite proteins: A superfamily of SMC/kleisin partners conserved across bacteria, archaea and eukaryotes. Structure. 2015 in press. PubMed

Gajiwala K.S., Burley S.K. Winged helix proteins. Curr. Opin. Struct. Biol. 2000;10:110–116. PubMed

Harami G.M., Gyimesi M., Kovács M. From keys to bulldozers: expanding roles for winged helix domains in nucleic-acid-binding proteins. Trends Biochem. Sci. 2013;38:364–371. PubMed

Ke C., Humeniuk M., S-Gracz H., Marszalek P.E. Direct Measurements of Base Stacking Interactions in DNA by Single-Molecule Atomic-Force Spectroscopy. Phys. Rev. Lett. 2007;99:018302. PubMed

Cost G.J., Cozzarelli N.R. Smc5p promotes faithful chromosome transmission and DNA repair in Saccharomyces cerevisiae. Genetics. 2006;172:2185–2200. PubMed PMC

Stephan A.K., Kliszczak M., Dodson H., Cooley C., Morrison C.G. Roles of vertebrate Smc5 in sister chromatid cohesion and homologous recombinational repair. Mol. Cell. Biol. 2011;31:1369–1381. PubMed PMC

Pebernard S., Wohlschlegel J., McDonald W.H., Yates J.R., Boddy M.N. The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex. Mol. Cell. Biol. 2006;26:1617–1630. PubMed PMC

Bass K.L., Murray J.M., O'Connell M.J. Brc1-dependent recovery from replication stress. J. Cell Sci. 2012;125:2753–2764. PubMed PMC

McDonald W.H., Pavlova Y., Yates J.R., Boddy M.N. Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 complex. J. Biol. Chem. 2003;278:45460–45467. PubMed

Pebernard S., McDonald W.H., Pavlova Y., Yates J.R., Boddy M.N. Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis. Mol. Biol. Cell. 2004;15:4866–4876. PubMed PMC

Verkade H.M., Bugg S.J., Lindsay H.D., Carr A.M., O'Connell M.J. Rad18 is required for DNA repair and checkpoint responses in fission yeast. Mol. Biol. Cell. 1999;10:2905–2918. PubMed PMC

Lammens A., Schele A., Hopfner K.-P. Structural biochemistry of ATP-driven dimerization and DNA-stimulated activation of SMC ATPases. Curr. Biol. 2004;14:1778–1782. PubMed

The SAGA histone acetyltransferase module targets SMC5/6 to specific genes

Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase

A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6

The SMC5/6 Complex Subunit NSE4A Is Involved in DNA Damage Repair and Seed Development

Arabidopsis NSE4 Proteins Act in Somatic Nuclei and Meiosis to Ensure Plant Viability and Fertility

SMC5/6: Multifunctional Player in Replication

COZOID: contact zone identifier for visual analysis of protein-protein interactions

Scaffolding for Repair: Understanding Molecular Functions of the SMC5/6 Complex