Current Genetics

, Volume 64, Issue 3, pp 599–617 | Cite as

Interaction of the Saccharomyces cerevisiae RING-domain protein Nse1 with Nse3 and the Smc5/6 complex is required for chromosome replication and stability

  • Saima Wani
  • Neelam Maharshi
  • Deepash Kothiwal
  • Lakshmi Mahendrawada
  • Raju Kalaivani
  • Shikha LalorayaEmail author
Original Article


Genomic stability is maintained by the concerted actions of numerous protein complexes that participate in chromosomal duplication, repair, and segregation. The Smc5/6 complex is an essential multi-subunit complex crucial for repair of DNA double-strand breaks. Two of its subunits, Nse1 and Nse3, are homologous to the RING-MAGE complexes recently described in human cells. We investigated the contribution of the budding yeast Nse1 RING-domain by isolating a mutant nse1-103 bearing substitutions in conserved Zinc-coordinating residues of the RING-domain that is hypersensitive to genotoxic stress and temperature. The nse1-103 mutant protein was defective in interaction with Nse3 and other Smc5/6 complex subunits, Nse4 and Smc5. Chromosome loss was enhanced, accompanied by a delay in the completion of replication and a modest defect in sister chromatid cohesion, in nse1-103. The nse1-103 mutant was synthetic sick with rrm3∆ (defective in fork passage through pause sites), this defect was rescued by inactivation of Tof1, a subunit of the fork protection complex that enforces pausing. The temperature sensitivity of nse1-103 was partially suppressed by deletion of MPH1, encoding a DNA-helicase. Homology modeling of the structure of the budding yeast Nse1–Nse3 heterodimer based on the human Nse1–MAGEG1 structure suggests a similar organization and indicates that perturbation of the Zn-coordinating cluster has the potential to allosterically alter structural elements at the Nse1/Nse3 interaction interface that may abrogate their association. Our findings demonstrate that the budding yeast Nse1 RING-domain organization is important for interaction with Nse3, which is crucial for completion of chromosomal replication, cohesion, and maintenance of chromosome stability.


Chromosome stability DNA replication Mitosis Molecular genetics Protein–protein interaction Yeast two-hybrid 



Structural maintenance of chromosomes


Methyl methane sulfonate




MMS-sensitive 21


Small ubiquitin-related modifier


Non-smc element


Double-strand break


Gross chromosomal rearrangements


Glutathione S-transferase


Melanoma antigen G1


Mutator Phenotype


Pulsed-field gel electrophoresis




Ribosomal DNA


Really interesting new gene


rDNA recombination mutation 3


Topoisomerase I-interacting Factor 1


Yeast extract–peptone–dextrose


Synthetic complete medium


Yeast artificial chromosome


5-fluoroorotic acid



This work was supported by investigator specific grants to Shikha Laloraya from the Science and Engineering Research Board (SERB), Department of Biotechnology (DBT) and Council of Scientific and Industrial Research (CSIR), India, and the DBT-IISc partnership program funded by the Department of Biotechnology, India. Fellowship support for Saima Wani was provided by the Indian Institute of Science and the SERB grant to S.L. Deepash Kothiwal was supported by a Department of Biotechnology Senior Research Fellowship, Lakshmi Mahendrawada by the CSIR grant to S.L., and Neelam Maharshi by a fellowship from I.I.Sc. We thank Yves Barral, Kenji Kohno and Doug Koshland for sharing strains and plasmids, Donald Soubam for construction of pDS58 and pDS72, Deepa Balagopal for construction of pDB30, and N. Srinivasan for discussions related to the structure. Technical support from the DBT funded confocal facility of I.I.Sc. is acknowledged. Equipment support for the Department of Biochemistry, I.I.Sc. is provided by the DST-FIST and UGC CAS/SAP programs.

Author contributions

S.M. and S.L. designed the experiments. S.M., N.M., L.M. and D.K. performed the experiments. S.M., N.M., D.K., L.M. and S.L. analyzed the data. S.L. performed the homology modeling and K.R. analyzed and interpreted the Nse1/3 heterodimer model. S.L. designed and supervised the project and wrote the manuscript. All authors reviewed the results and approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Supplementary material

294_2017_776_MOESM1_ESM.tif (620 kb)
Supplementary material 1 (TIFF 620 KB)


  1. Ampatzidou E, Irmisch A, O’Connell MJ, Murray JM (2006) Smc5/6 is required for repair at collapsed replication forks. Mol Cell Biol 26:9387–9401. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Arnold K, Bordoli L, Kopp J, Schwede T (2006) The SWISS-MODEL workspace: a web-based environment for protein structure homology modelling. Bioinformatics 22:195–201. CrossRefPubMedGoogle Scholar
  3. Bahler J, Wu JQ, Longtine MS, Shah NG, McKenzie A 3rd, Steever AB, Wach A, Philippsen P, Pringle JR (1998) Heterologous modules for efficient and versatile PCR-based gene targeting in Schizosaccharomyces pombe. Yeast 14:943–951.<943::AID-YEA292>3.0.CO;2-Y CrossRefPubMedGoogle Scholar
  4. Benkert P, Biasini M, Schwede T (2011) Toward the estimation of the absolute quality of individual protein structure models. Bioinformatics 27:343–350. CrossRefPubMedGoogle Scholar
  5. Bermudez-Lopez M, Aragon L (2017) Smc5/6 complex regulates Sgs1 recombination functions. Curr Genet 63:381–388. CrossRefPubMedGoogle Scholar
  6. Biasini M, Bienert S, Waterhouse A, Arnold K, Studer G, Schmidt T, Kiefer F, Gallo Cassarino T, Bertoni M, Bordoli L, Schwede T (2014) SWISS-MODEL: modelling protein tertiary and quaternary structure using evolutionary information. Nucleic Acids Res 42:W252-258. CrossRefGoogle Scholar
  7. Borden KL, Freemont PS (1996) The RING finger domain: a recent example of a sequence-structure family. Curr Opin Struct Biol 6:395–401 doiCrossRefPubMedGoogle Scholar
  8. Branzei D, Foiani M (2009) The checkpoint response to replication stress. DNA Repair (Amst) 8:1038–1046. CrossRefGoogle Scholar
  9. Branzei D, Foiani M (2010) Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol 11:208–219. CrossRefPubMedGoogle Scholar
  10. Branzei D, Sollier J, Liberi G, Zhao X, Maeda D, Seki M, Enomoto T, Ohta K, Foiani M (2006) Ubc9- and mms21-mediated sumoylation counteracts recombinogenic events at damaged replication forks. Cell 127:509–522. CrossRefPubMedGoogle Scholar
  11. Bustard DE, Menolfi D, Jeppsson K, Ball LG, Dewey SC, Shirahige K, Sjogren C, Branzei D, Cobb JA (2012) During replication stress, non-SMC element 5 (NSE5) is required for Smc5/6 protein complex functionality at stalled forks. J Biol Chem 287:11374–11383. CrossRefPubMedPubMedCentralGoogle Scholar
  12. Bustard DE, Ball LG, Cobb JA (2016) Non-Smc element 5 (Nse5) of the Smc5/6 complex interacts with SUMO pathway components. Biol Open 5:777–785. CrossRefPubMedPubMedCentralGoogle Scholar
  13. De Piccoli G, Cortes-Ledesma F, Ira G, Torres-Rosell J, Uhle S, Farmer S, Hwang JY, Machin F, Ceschia A, McAleenan A, Cordon-Preciado V, Clemente-Blanco A, Vilella-Mitjana F, Ullal P, Jarmuz A, Leitao B, Bressan D, Dotiwala F, Papusha A, Zhao X, Myung K, Haber JE, Aguilera A, Aragon L (2006) Smc5-Smc6 mediate DNA double-strand-break repair by promoting sister-chromatid recombination. Nat Cell Biol 8:1032–1034. CrossRefPubMedPubMedCentralGoogle Scholar
  14. De Piccoli G, Torres-Rosell J, Aragon L (2009) The unnamed complex: what do we know about Smc5-Smc. 6? Chromosome Res 17:251–263. CrossRefGoogle Scholar
  15. Ding DQ, Haraguchi T, Hiraoka Y (2016) A cohesin-based structural platform supporting homologous chromosome pairing in meiosis. Curr Genet 62:499–502. CrossRefPubMedGoogle Scholar
  16. Doyle JM, Gao J, Wang J, Yang M, Potts PR (2010) MAGE-RING protein complexes comprise a family of E3 ubiquitin ligases. Mol Cell 39:963–974. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Duan X, Sarangi P, Liu X, Rangi GK, Zhao X, Ye H (2009a) Structural and functional insights into the roles of the Mms21 subunit of the Smc5/6 complex. Mol Cell 35:657–668. CrossRefPubMedPubMedCentralGoogle Scholar
  18. Duan X, Yang Y, Chen YH, Arenz J, Rangi GK, Zhao X, Ye H (2009b) 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 284:8507–8515. CrossRefPubMedPubMedCentralGoogle Scholar
  19. Fujioka Y, Kimata Y, Nomaguchi K, Watanabe K, Kohno K (2002) Identification of a novel non-structural maintenance of chromosomes (SMC) component of the SMC5-SMC6 complex involved in DNA repair. J Biol Chem 277:21585–21591. CrossRefPubMedGoogle Scholar
  20. Guex N, Peitsch MC, Schwede T (2009) Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: a historical perspective. Electrophoresis 30 Suppl 1: S162-173
  21. Hartwell LH, Kastan MB (1994) Cell cycle control and cancer. Science 266:1821–1828 doiCrossRefPubMedGoogle Scholar
  22. Harvey SH, Sheedy DM, Cuddihy AR, O’Connell MJ (2004) Coordination of DNA damage responses via the Smc5/Smc6 complex. Mol Cell Biol 24:662–674 doiCrossRefPubMedPubMedCentralGoogle Scholar
  23. Hirano T (2016) Condensin-based chromosome organization from bacteria to vertebrates. Cell 164:847–857. CrossRefPubMedGoogle Scholar
  24. Huang D, Koshland D (2003) Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors, elongation factors, and origins. Genes Dev 17:1741–1754. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Iadonato SP, Gnirke A (1996) RARE-cleavage analysis of YACs. Methods Mol Biol 54:75–85 doiPubMedGoogle Scholar
  26. Iwasaki O, Noma KI (2016) Condensin-mediated chromosome organization in fission yeast. Curr Genet 62:739–743. CrossRefPubMedPubMedCentralGoogle Scholar
  27. Janke C, Magiera MM, Rathfelder N, Taxis C, Reber S, Maekawa H, Moreno-Borchart A, Doenges G, Schwob E, Schiebel E, Knop M (2004) A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21:947–962. CrossRefPubMedGoogle Scholar
  28. Jeppsson K, Kanno T, Shirahige K, Sjogren C (2014) The maintenance of chromosome structure: positioning and functioning of SMC complexes. Nat Rev Mol Cell Biol 15:601–614. CrossRefPubMedGoogle Scholar
  29. Kaelin WG Jr, Krek W, Sellers WR, DeCaprio JA, Ajchenbaum F, Fuchs CS, Chittenden T, Li Y, Farnham PJ, Blanar MA et al (1992) Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell 70:351–364 doiCrossRefPubMedGoogle Scholar
  30. Kiefer F, Arnold K, Kunzli M, Bordoli L, Schwede T (2009) The SWISS-MODEL Repository and associated resources. Nucleic Acids Res 37:D387-392. CrossRefGoogle Scholar
  31. Koshland D, Strunnikov A (1996) Mitotic chromosome condensation. Annu Rev Cell Dev Biol 12:305–333. CrossRefPubMedGoogle Scholar
  32. Kushnirov VV (2000) Rapid and reliable protein extraction from yeast. Yeast 16:857–860.<857::AID-YEA561>3.0.CO;2-B CrossRefPubMedGoogle Scholar
  33. Leung GP, Lee L, Schmidt TI, Shirahige K, Kobor MS (2011) Rtt107 is required for recruitment of the SMC5/6 complex to DNA double strand breaks. J Biol Chem 286:26250–26257. CrossRefPubMedPubMedCentralGoogle Scholar
  34. Lindroos HB, Strom L, Itoh T, Katou Y, Shirahige K, Sjogren C (2006) Chromosomal association of the Smc5/6 complex reveals that it functions in differently regulated pathways. Mol Cell 22:755–767. CrossRefPubMedGoogle Scholar
  35. Losada A, Hirano T (2005) Dynamic molecular linkers of the genome: the first decade of SMC proteins. Genes Dev 19:1269–1287. CrossRefPubMedGoogle Scholar
  36. Mahendrawada L, Rai R, Kothiwal D, Laloraya S (2017) Interplay between Top1 and Mms21/Nse2 mediated sumoylation in stable maintenance of long chromosomes. Curr Genet 63:627–645. CrossRefPubMedGoogle Scholar
  37. Matityahu A, Onn I (2017) A new twist in the coil: functions of the coiled-coil domain of structural maintenance of chromosome (SMC) proteins. Curr Genet. PubMedGoogle Scholar
  38. McDonald WH, Pavlova Y, Yates JR 3rd, Boddy MN (2003) Novel essential DNA repair proteins Nse1 and Nse2 are subunits of the fission yeast Smc5-Smc6 complex. J Biol Chem 278:45460–45467. CrossRefPubMedGoogle Scholar
  39. Menolfi D, Delamarre A, Lengronne A, Pasero P, Branzei D (2015) Essential Roles of the Smc5/6 Complex in Replication through Natural Pausing Sites and Endogenous DNA Damage Tolerance. Mol Cell 60:835–846. CrossRefPubMedPubMedCentralGoogle Scholar
  40. Neurohr G, Naegeli A, Titos I, Theler D, Greber B, Diez J, Gabaldon T, Mendoza M, Barral Y (2011) A midzone-based ruler adjusts chromosome compaction to anaphase spindle length. Science 332:465–468. CrossRefPubMedGoogle Scholar
  41. Palecek J, Vidot S, Feng M, Doherty AJ, Lehmann AR (2006) The Smc5-Smc6 DNA repair complex. bridging of the Smc5-Smc6 heads by the KLEISIN, Nse4, and non-Kleisin subunits. J Biol Chem 281:36952–36959. CrossRefPubMedGoogle Scholar
  42. Pebernard S, McDonald WH, Pavlova Y, Yates JR 3rd, Boddy MN (2004) Nse1, Nse2, and a novel subunit of the Smc5-Smc6 complex, Nse3, play a crucial role in meiosis. Mol Biol Cell 15:4866–4876. CrossRefPubMedPubMedCentralGoogle Scholar
  43. Pebernard S, Perry JJ, Tainer JA, Boddy MN (2008) Nse1 RING-like domain supports functions of the Smc5-Smc6 holocomplex in genome stability. Mol Biol Cell 19:4099–4109. CrossRefPubMedPubMedCentralGoogle Scholar
  44. Pfander B, Moldovan GL, Sacher M, Hoege C, Jentsch S (2005) SUMO-modified PCNA recruits Srs2 to prevent recombination during S phase. Nature 436:428–433. CrossRefPubMedGoogle Scholar
  45. Pickart CM (2001) Mechanisms underlying ubiquitination. Annu Rev Biochem 70:503–533. CrossRefPubMedGoogle Scholar
  46. Potts PR, Porteus MH, Yu H (2006) Human SMC5/6 complex promotes sister chromatid homologous recombination by recruiting the SMC1/3 cohesin complex to double-strand breaks. EMBO J 25:3377–3388. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Prakash S, Prakash L (1977) Increased spontaneous mitotic segregation in MMS-sensitive mutants of Saccharomyces cerevisiae. Genetics 87:229–236 doiPubMedPubMedCentralGoogle Scholar
  48. Putnam CD, Srivatsan A, Nene RV, Martinez SL, Clotfelter SP, Bell SN, Somach SB, de Souza JE, Fonseca AF, de Souza SJ, Kolodner RD (2016) A genetic network that suppresses genome rearrangements in Saccharomyces cerevisiae and contains defects in cancers. Nat Commun 7:11256. CrossRefPubMedPubMedCentralGoogle Scholar
  49. Rai R, Laloraya S (2017) Genetic evidence for functional interaction of Smc5/6 complex and Top1 with spatial frequency of replication origins required for maintenance of chromosome stability. Curr Genet 63:765–776. CrossRefPubMedGoogle Scholar
  50. Rai R, Varma SP, Shinde N, Ghosh S, Kumaran SP, Skariah G, Laloraya S (2011) Small ubiquitin-related modifier ligase activity of Mms21 is required for maintenance of chromosome integrity during the unperturbed mitotic cell division cycle in Saccharomyces cerevisiae. J Biol Chem 286:14516–14530. CrossRefPubMedPubMedCentralGoogle Scholar
  51. Rankin S, Dawson DS (2016) Recent advances in cohesin biology. F1000Res. PubMedPubMedCentralGoogle Scholar
  52. Robellet X, Vanoosthuyse V, Bernard P (2016) The loading of condensin in the context of chromatin. Curr Genet. PubMedGoogle Scholar
  53. Santa Maria SR, Gangavarapu V, Johnson RE, Prakash L, Prakash S (2007) Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae. Mol Cell Biol 27:8409–8418. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Skibbens RV (2016) Of Rings and Rods: Regulating Cohesin Entrapment of DNA to Generate Intra- and Intermolecular Tethers. PLoS Genet 12:e1006337. CrossRefPubMedPubMedCentralGoogle Scholar
  55. Tapia-Alveal C, O’Connell MJ (2011) Nse1-dependent recruitment of Smc5/6 to lesion-containing loci contributes to the repair defects of mutant complexes. Mol Biol Cell 22:4669–4682. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Uhlmann F (2016) SMC complexes: from DNA to chromosomes. Nat Rev Mol Cell Biol 17:399–412. CrossRefPubMedGoogle Scholar
  57. van der Crabben SN, Hennus MP, McGregor GA, Ritter DI, Nagamani SC, Wells OS, Harakalova M, Chinn IK, Alt A, Vondrova L, Hochstenbach R, van Montfrans JM, Terheggen-Lagro SW, van Lieshout S, van Roosmalen MJ, Renkens I, Duran K, Nijman IJ, Kloosterman WP, Hennekam E, Orange JS, van Hasselt PM, Wheeler DA, Palecek JJ, Lehmann AR, Oliver AW, Pearl LH, Plon SE, Murray JM, van Haaften G (2016) Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease. J Clin Invest 126:2881–2892. CrossRefPubMedPubMedCentralGoogle Scholar
  58. Wehrkamp-Richter S, Hyppa RW, Prudden J, Smith GR, Boddy MN (2012) Meiotic DNA joint molecule resolution depends on Nse5-Nse6 of the Smc5-Smc6 holocomplex. Nucleic Acids Res 40:9633–9646. CrossRefPubMedPubMedCentralGoogle Scholar
  59. Zhao X, Blobel G (2005) A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proc Natl Acad Sci USA 102:4777–4782. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Saima Wani
    • 1
  • Neelam Maharshi
    • 1
  • Deepash Kothiwal
    • 1
  • Lakshmi Mahendrawada
    • 1
  • Raju Kalaivani
    • 2
  • Shikha Laloraya
    • 1
    Email author
  1. 1.FE13, New Biological Sciences Building, Department of BiochemistryIndian Institute of ScienceBangaloreIndia
  2. 2.Molecular Biophysics UnitIndian Institute of ScienceBangaloreIndia

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