Structural Maintenance of Chromosomes (SMC) complexes are important for many aspects of the chromosomal organization. Unlike cohesin and condensin, the SMC5/6 complex contains a variant RING domain carried by its Nse1 subunit. RING domains are characteristic for ubiquitin ligases, and human NSE1 has been shown to possess ubiquitin-ligase activity in vitro. However, other studies were unable to show such activity. Here, we confirm Nse1 ubiquitin-ligase activity using purified Schizosaccharomyces pombe proteins. We demonstrate that the Nse1 ligase activity is stimulated by Nse3 and Nse4. We show that Nse1 specifically utilizes Ubc13/Mms2 E2 enzyme and interacts directly with ubiquitin. We identify the Nse1 mutation (R188E) that specifically disrupts its E3 activity and demonstrate that the Nse1-dependent ubiquitination is particularly important under replication stress. Moreover, we determine Nse4 (lysine K181) as the first known SMC5/6-associated Nse1 substrate. Interestingly, abolition of Nse4 modification at K181 leads to suppression of DNA-damage sensitivity of other SMC5/6 mutants. Altogether, this study brings new evidence for Nse1 ubiquitin ligase activity, significantly advancing our understanding of this enigmatic SMC5/6 function.

Central European Institute of Technology Masaryk University 62500 Brno Czech Republic

Genome Damage and Stability Centre School of Life Sciences University of Sussex Falmer Brighton BN1 9RH UK

National Centre for Biomolecular Research Faculty of Science Masaryk University 62500 Brno Czech Republic

Cells. 2024 May 10;13(10):812. doi: 10.3390/cells13100812. PubMed

Zobrazit více v PubMed

Uhlmann F. SMC Complexes: From DNA to Chromosomes. Nat. Rev. Mol. Cell Biol. 2016;17:399–412. doi: 10.1038/nrm.2016.30. PubMed DOI

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. doi: 10.1128/MCB.15.12.7067. PubMed DOI PMC

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. doi: 10.1038/emboj.2008.273. PubMed DOI PMC

Aragón L. The Smc5/6 Complex: New and Old Functions of the Enigmatic Long-Distance Relative. Annu. Rev. Genet. 2018;52:89–107. doi: 10.1146/annurev-genet-120417-031353. PubMed DOI

Palecek J.J. SMC5/6: Multifunctional Player in Replication. Genes. 2018;10:7. doi: 10.3390/genes10010007. PubMed DOI 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. doi: 10.1128/MCB.25.1.172-184.2005. PubMed DOI 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. doi: 10.1074/jbc.M608004200. PubMed DOI

Vondrova L., Kolesar P., Adamus M., Nociar M., Oliver A.W., Palecek J.J. A Role of the Nse4 Kleisin and Nse1/Nse3 KITE Subunits in the ATPase Cycle of SMC5/6. Sci. Rep. 2020;10:9694. doi: 10.1038/s41598-020-66647-w. PubMed DOI PMC

Palecek J.J., Gruber S. Kite Proteins: A Superfamily of SMC/Kleisin Partners Conserved Across Bacteria, Archaea, and Eukaryotes. Structure. 2015;23:2183–2190. doi: 10.1016/j.str.2015.10.004. PubMed DOI

Adamus M., Lelkes E., Potesil D., Ganji S.R., Kolesar P., Zabrady K., Zdrahal Z., Palecek J.J. Molecular Insights into the Architecture of the Human SMC5/6 Complex. J. Mol. Biol. 2020;432:3820–3837. doi: 10.1016/j.jmb.2020.04.024. PubMed DOI

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. doi: 10.1371/journal.pone.0035813. PubMed DOI 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. doi: 10.1371/journal.pone.0017270. PubMed DOI PMC

Jo A., Li S., Shin J.W., Zhao X., Cho Y. Structure Basis for Shaping the Nse4 Protein by the Nse1 and Nse3 Dimer within the Smc5/6 Complex. J. Mol. Biol. 2021;433:166910. doi: 10.1016/j.jmb.2021.166910. PubMed DOI PMC

Solé-Soler R., Torres-Rosell J. Smc5/6, an Atypical SMC Complex with Two RING-Type Subunits. Biochem. Soc. Trans. 2020;48:2159–2171. doi: 10.1042/BST20200389. PubMed DOI

Deshaies R.J., Joazeiro C.A.P. RING Domain E3 Ubiquitin Ligases. Annu. Rev. Biochem. 2009;78:399–434. doi: 10.1146/annurev.biochem.78.101807.093809. PubMed DOI

Pebernard S., Perry J.J.P., Tainer J.A., Boddy M.N. Nse1 RING-like Domain Supports Functions of the Smc5-Smc6 Holocomplex in Genome Stability. Mol. Biol. Cell. 2008;19:4099–4109. doi: 10.1091/mbc.e08-02-0226. PubMed DOI PMC

Tapia-Alveal C., O’Connell M.J. Nse1-Dependent Recruitment of Smc5/6 to Lesion-Containing Loci Contributes to the Repair Defects of Mutant Complexes. Mol. Biol. Cell. 2011;22:4669–4682. doi: 10.1091/mbc.e11-03-0272. PubMed DOI 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. doi: 10.1016/j.molcel.2010.08.029. PubMed DOI 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. doi: 10.1080/15384101.2014.1000112. PubMed DOI PMC

Weon J.L., Yang S.W., Potts P.R. Cytosolic Iron-Sulfur Assembly Is Evolutionarily Tuned by a Cancer-Amplified Ubiquitin Ligase. Mol. Cell. 2018;69:113–125.e6. doi: 10.1016/j.molcel.2017.11.010. PubMed DOI

Olsen S.K., Lima C.D. Structure of a Ubiquitin E1-E2 Complex: Insights to E1-E2 Thioester Transfer. Mol. Cell. 2013;49:884–896. doi: 10.1016/j.molcel.2013.01.013. PubMed DOI PMC

Zabrady K., Adamus M., Vondrova L., Liao C., Skoupilova H., Novakova M., Jurcisinova L., Alt A., Oliver A.W., Lehmann A.R., et al. Chromatin Association of the SMC5/6 Complex Is Dependent on Binding of Its NSE3 Subunit to DNA. Nucleic Acids Res. 2016;44:1064–1079. doi: 10.1093/nar/gkv1021. PubMed DOI PMC

Moreno S., Klar A., Nurse P. Molecular Genetic Analysis of Fission Yeast Schizosaccharomyces Pombe. Methods Enzymol. 1991;194:795–823. doi: 10.1016/0076-6879(91)94059-l. PubMed DOI

Kaiser P., Meierhofer D., Wang X., Huang L. Tandem Affinity Purification Combined with Mass Spectrometry to Identify Components of Protein Complexes. Methods Mol. Biol. Clifton. 2008;439:309–326. doi: 10.1007/978-1-59745-188-8_21. PubMed DOI PMC

Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Žídek A., Potapenko A., et al. Highly Accurate Protein Structure Prediction with AlphaFold. Nature. 2021;596:583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Kiss L., Clift D., Renner N., Neuhaus D., James L.C. RING Domains Act as Both Substrate and Enzyme in a Catalytic Arrangement to Drive Self-Anchored Ubiquitination. Nat. Commun. 2021;12:1220. doi: 10.1038/s41467-021-21443-6. PubMed DOI PMC

Furmanová K., Byška J., Gröller E.M., Viola I., Paleček J.J., Kozlíková B. COZOID: Contact Zone Identifier for Visual Analysis of Protein-Protein Interactions. BMC Bioinformatics. 2018;19:125. doi: 10.1186/s12859-018-2113-6. PubMed DOI PMC

Andrews E.A., Palecek J., Sergeant J., Taylor E., Lehmann A.R., Watts F.Z. Nse2, a Component of the Smc5-6 Complex, Is a SUMO Ligase Required for the Response to DNA Damage. Mol. Cell. Biol. 2005;25:185–196. doi: 10.1128/MCB.25.1.185-196.2005. PubMed DOI PMC

Hou X., Zhang W., Xiao Z., Gan H., Lin X., Liao S., Han C. Mining and Characterization of Ubiquitin E3 Ligases Expressed in the Mouse Testis. BMC Genom. 2012;13:495. doi: 10.1186/1471-2164-13-495. PubMed DOI PMC

Christensen D.E., Brzovic P.S., Klevit R.E. E2-BRCA1 RING Interactions Dictate Synthesis of Mono- or Specific Polyubiquitin Chain Linkages. Nat. Struct. Mol. Biol. 2007;14:941–948. doi: 10.1038/nsmb1295. PubMed DOI

Hoege C., Pfander B., Moldovan G.-L., Pyrowolakis G., Jentsch S. RAD6-Dependent DNA Repair Is Linked to Modification of PCNA by Ubiquitin and SUMO. Nature. 2002;419:135–141. doi: 10.1038/nature00991. PubMed DOI

Dou H., Buetow L., Sibbet G.J., Cameron K., Huang D.T. BIRC7-E2 Ubiquitin Conjugate Structure Reveals the Mechanism of Ubiquitin Transfer by a RING Dimer. Nat. Struct. Mol. Biol. 2012;19:876–883. doi: 10.1038/nsmb.2379. PubMed DOI PMC

Plechanovová A., Jaffray E.G., Tatham M.H., Naismith J.H., Hay R.T. Structure of a RING E3 Ligase and Ubiquitin-Loaded E2 Primed for Catalysis. Nature. 2012;489:115–120. doi: 10.1038/nature11376. PubMed DOI PMC

Pruneda J.N., Littlefield P.J., Soss S.E., Nordquist K.A., Chazin W.J., Brzovic P.S., Klevit R.E. Structure of an E3:E2~Ub Complex Reveals an Allosteric Mechanism Shared among RING/U-Box Ligases. Mol. Cell. 2012;47:933–942. doi: 10.1016/j.molcel.2012.07.001. PubMed DOI PMC

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D.J., Inuganti A., Griss J., Mayer G., Eisenacher M., et al. The PRIDE Database and Related Tools and Resources in 2019: Improving Support for Quantification Data. Nucleic Acids Res. 2019;47:D442–D450. doi: 10.1093/nar/gky1106. PubMed DOI PMC

Correction: Kolesar et al. Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase. Cells 2022, 11, 165

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

Multiple Roles of SMC5/6 Complex during Plant Sexual Reproduction