The SMC (Structural Maintenance of Chromosomes) complexes are composed of SMC dimers, kleisin and kleisin-interacting (HAWK or KITE) subunits. Mutual interactions of these subunits constitute the basal architecture of the SMC complexes. In addition, binding of ATP molecules to the SMC subunits and their hydrolysis drive dynamics of these complexes. Here, we developed new systems to follow the interactions between SMC5/6 subunits and the relative stability of the complex. First, we show that the N-terminal domain of the Nse4 kleisin molecule binds to the SMC6 neck and bridges it to the SMC5 head. Second, binding of the Nse1 and Nse3 KITE proteins to the Nse4 linker increased stability of the ATP-free SMC5/6 complex. In contrast, binding of ATP to SMC5/6 containing KITE subunits significantly decreased its stability. Elongation of the Nse4 linker partially suppressed instability of the ATP-bound complex, suggesting that the binding of the KITE proteins to the Nse4 linker constrains its limited size. Our data suggest that the KITE proteins may shape the Nse4 linker to fit the ATP-free complex optimally and to facilitate opening of the complex upon ATP binding. This mechanism suggests an important role of the KITE subunits in the dynamics of the SMC5/6 complexes.

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

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

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 62500 Brno Czech Republic

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Schwarzer W, et al. Two independent modes of chromatin organization revealed by cohesin removal. Nature. 2017;551:51–56. doi: 10.1038/nature24281. PubMed DOI PMC

Kschonsak M, Haering CH. Shaping mitotic chromosomes: From classical concepts to molecular mechanisms. Bioessays. 2015;37:755–766. doi: 10.1002/bies.201500020. PubMed DOI PMC

Aragon, L. In Annual Review of Genetics, Vol 52 Vol. 52 Annual Review of Genetics (ed. Bonini, N. M.) 89–107 (Annual Reviews, 2018).

Palecek JJ. SMC5/6: Multifunctional Player in Replication. Genes. 2019;10:E7. doi: 10.3390/genes10010007. PubMed DOI PMC

Bürmann F, Gruber S. SMC condensin: promoting cohesion of replicon arms. Nat. Struct. Mol. Biol. 2015;22:653–655. doi: 10.1038/nsmb.3082. PubMed DOI

van der Crabben SN, et al. Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease. J. Clin. Investigation. 2016;126:2881–2892. doi: 10.1172/jci82890. PubMed DOI PMC

Palecek JJ, 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

Wells JN, Gligoris TG, Nasmyth KA, Marsh JA. Evolution of condensin and cohesin complexes driven by replacement of Kite by Hawk proteins. Curr. Biol. 2017;27:R17–R18. doi: 10.1016/j.cub.2016.11.050. PubMed DOI PMC

Burmann F, et al. Tuned SMC Arms Drive Chromosomal Loading of Prokaryotic Condensin. Mol. Cell. 2017;65:861–+. doi: 10.1016/j.molcel.2017.01.026. PubMed DOI PMC

Diebold-Durand ML, et al. Structure of Full-Length SMC and Rearrangements Required for Chromosome Organization. Mol. Cell. 2017;67:334–347.e335. doi: 10.1016/j.molcel.2017.06.010. PubMed DOI PMC

Nasmyth K, Haering CH. The structure and function of SMC and kleisin complexes. Annu. Rev. Biochem. 2005;74:595–648. doi: 10.1146/annurev.biochem.74.082803.133219. PubMed DOI

Hassler M, Shaltiel IA, Haering CH. Towards a Unified Model of SMC Complex Function. Curr. Biol. 2018;28:R1266–R1281. doi: 10.1016/j.cub.2018.08.034. PubMed DOI PMC

Gligoris T, Löwe J. Structural Insights into Ring Formation of Cohesin and Related Smc Complexes. Trends Cell Biol. 2016;26:680–693. doi: 10.1016/j.tcb.2016.04.002. PubMed DOI PMC

Lammens A, Schele A, Hopfner KP. Structural biochemistry of ATP-driven dimerization and DNA-stimulated activation of SMC ATPases. Curr. Biol. 2004;14:1778–1782. doi: 10.1016/j.cub.2004.09.044. PubMed DOI

Arumugam P, et al. ATP hydrolysis is required for cohesin’s association with chromosomes. Curr. Biol. 2003;13:1941–1953. doi: 10.1016/j.cub.2003.10.036. PubMed DOI

Bürmann F, et al. An asymmetric SMC-kleisin bridge in prokaryotic condensin. Nat. Struct. Mol. Biol. 2013;20:371–379. doi: 10.1038/nsmb.2488. PubMed DOI

Gligoris TG, et al. Closing the cohesin ring: structure and function of its Smc3-kleisin interface. Science. 2014;346:963–967. doi: 10.1126/science.1256917. PubMed DOI PMC

Zawadzka, K. et al. MukB ATPases are regulated independently by the N- and C-terminal domains of MukF kleisin. Elife7, 10.7554/eLife.31522 (2018). PubMed PMC

Murayama Y, Uhlmann F. DNA Entry into and Exit out of the Cohesin Ring by an Interlocking Gate Mechanism. Cell. 2015;163:1628–1640. doi: 10.1016/j.cell.2015.11.030. PubMed DOI PMC

Beckouët F, et al. Releasing Activity Disengages Cohesin’s Smc3/Scc1 Interface in a Process Blocked by Acetylation. Mol. Cell. 2016;61:563–574. doi: 10.1016/j.molcel.2016.01.026. PubMed DOI PMC

Buheitel J, Stemmann O. Prophase pathway-dependent removal of cohesin from human chromosomes requires opening of the Smc3-Scc1 gate. EMBO J. 2013;32:666–676. doi: 10.1038/emboj.2013.7. PubMed DOI PMC

Eichinger CS, Kurze A, Oliveira RA, Nasmyth K. Disengaging the Smc3/kleisin interface releases cohesin from Drosophila chromosomes during interphase and mitosis. EMBO J. 2013;32:656–665. doi: 10.1038/emboj.2012.346. PubMed DOI PMC

Chan KL, et al. Cohesin’s DNA exit gate is distinct from its entrance gate and is regulated by acetylation. Cell. 2012;150:961–974. doi: 10.1016/j.cell.2012.07.028. PubMed DOI PMC

Elbatsh AMO, et al. Cohesin Releases DNA through Asymmetric ATPase-Driven Ring Opening. Mol. Cell. 2016;61:575–588. doi: 10.1016/j.molcel.2016.01.025. PubMed DOI PMC

Hassler M, et al. Structural Basis of an Asymmetric Condensin ATPase Cycle. Mol. Cell. 2019;74:1175–1188.e1179. doi: 10.1016/j.molcel.2019.03.037. PubMed DOI PMC

Minnen A, et al. Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA. Cell Rep. 2016;14:2003–2016. doi: 10.1016/j.celrep.2016.01.066. PubMed DOI PMC

Kamada K, Su’etsugu M, Takada H, Miyata M, Hirano T. Overall Shapes of the SMC-ScpAB Complex Are Determined by Balance between Constraint and Relaxation of Its Structural Parts. Structure. 2017;25:603–616.e604. doi: 10.1016/j.str.2017.02.008. PubMed DOI

Palecek J, Vidot S, Feng M, Doherty AJ, Lehmann AR. The SMC5-6 DNA repair complex: Bridging of the SMC5-6 heads by the Kleisin, NSE4, and non-Kleisin subunits. J. Biol. Chem. 2006;281:36952–36959. doi: 10.1074/jbc.M608004200. PubMed DOI

Guerineau M, et al. 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

Paleček JJ, Vondrová L, Zábrady K, Otočka J. Multicomponent Yeast Two-Hybrid System: Applications to Study Protein-Protein Interactions in SMC Complexes. Methods Mol. Biol. 2019;2004:79–90. doi: 10.1007/978-1-4939-9520-2_7. PubMed DOI

Duan X, et al. 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. doi: 10.1074/jbc.M809139200. PubMed DOI PMC

Diaz, M. et al. SMC5/6 Complex Subunit NSE4A is Involved in DNA Damage Repair and Seed Development in Arabidopsis. Plant Cell, 10.1105/tpc.18.00043 (2019). PubMed PMC

Sergeant J, et al. 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

Hudson, J. J. 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 one6, 10.1371/journal.pone.0017270 (2011). PubMed PMC

Kanno T, Berta DG, Sjögren C. The Smc5/6 Complex Is an ATP-Dependent Intermolecular DNA Linker. Cell Rep. 2015;12:1471–1482. doi: 10.1016/j.celrep.2015.07.048. PubMed DOI

Zabrady K, 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

Fousteri MI, Lehmann AR. A novel SMC protein complex in Schizosaccharomyces pombe contains the Rad18 DNA repair protein. EMBO J. 2000;19:1691–1702. doi: 10.1093/emboj/19.7.1691. PubMed DOI PMC

Kamada K, Miyata M, Hirano T. Molecular basis of SMC ATPase activation: role of internal structural changes of the regulatory subcomplex ScpAB. Structure. 2013;21:581–594. doi: 10.1016/j.str.2013.02.016. PubMed DOI

Woo JS, et al. Structural studies of a bacterial condensin complex reveal ATP-dependent disruption of intersubunit interactions. Cell. 2009;136:85–96. doi: 10.1016/j.cell.2008.10.050. PubMed DOI

Gloyd M, Ghirlando R, Guarné A. The role of MukE in assembling a functional MukBEF complex. J. Mol. Biol. 2011;412:578–590. doi: 10.1016/j.jmb.2011.08.009. PubMed DOI PMC

Pebernard S, Wohlschlegel J, McDonald WH, Yates JR, 3rd, Boddy MN. The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex. Mol. Cell Biol. 2006;26:1617–1630. doi: 10.1128/MCB.26.5.1617-1630.2006. PubMed DOI PMC

Ganji, M. et al. Real-time imaging of DNA loop extrusion by condensin. Science, 10.1126/science.aar7831 (2018). PubMed PMC

Davidson, I. F. et al. DNA loop extrusion by human cohesin. Science, 10.1126/science.aaz3418 (2019). PubMed

Hirano, M. & Hirano, T. Positive and negative regulation of SMC-DNA interactions by ATP and accessory proteins. Embo J. 23, 2664–2673. Epub 2004 Jun 2663. (2004). PubMed PMC

Ouyang, Z. & Yu, H. Releasing the cohesin ring: A rigid scaffold model for opening the DNA exit gate by Pds5 and Wapl. Bioessays39, 10.1002/bies.201600207 (2017). PubMed

Soh YM, et al. Molecular basis for SMC rod formation and its dissolution upon DNA binding. Mol. Cell. 2015;57:290–303. doi: 10.1016/j.molcel.2014.11.023. PubMed DOI PMC

Chapard C, Jones R, van Oepen T, Scheinost JC, Nasmyth K. Sister DNA Entrapment between Juxtaposed Smc Heads and Kleisin of the Cohesin Complex. Mol. Cell. 2019;75:224–237.e225. doi: 10.1016/j.molcel.2019.05.023. PubMed DOI PMC

Bürmann F, et al. A folded conformation of MukBEF and cohesin. Nat. Struct. Mol. Biol. 2019;26:227–236. doi: 10.1038/s41594-019-0196-z. PubMed DOI PMC

Hara K, et al. Structure of cohesin subcomplex pinpoints direct shugoshin-Wapl antagonism in centromeric cohesion. Nat. Struct. Mol. Biol. 2014;21:864–870. doi: 10.1038/nsmb.2880. PubMed DOI PMC

Muir KW, et al. Structure of the Pds5-Scc1 Complex and Implications for Cohesin Function. Cell Rep. 2016;14:2116–2126. doi: 10.1016/j.celrep.2016.01.078. PubMed DOI

Lee BG, et al. Crystal Structure of the Cohesin Gatekeeper Pds5 and in Complex with Kleisin Scc1. Cell Rep. 2016;14:2108–2115. doi: 10.1016/j.celrep.2016.02.020. 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

Kushnirov, V. V. Rapid and reliable protein extraction from yeast. Yeast16, 857-860, 10.1002/1097-0061(20000630)16:9<857::aid-yea561>3.0.co;2-b (2000). PubMed

Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinforma. 2008;9:40. doi: 10.1186/1471-2105-9-40. PubMed DOI PMC

de Vries SJ, van Dijk M, Bonvin AM. The HADDOCK web server for data-driven biomolecular docking. Nat. Protoc. 2010;5:883–897. doi: 10.1038/nprot.2010.32. PubMed DOI

Furmanová K, et al. COZOID: contact zone identifier for visual analysis of protein-protein interactions. BMC Bioinforma. 2018;19:125. doi: 10.1186/s12859-018-2113-6. PubMed DOI PMC

Byska J, Jurcik A, Furmanova K, Kozlikova B, Palecek JJ. Visual Analysis of Protein-Protein Interaction Docking Models Using COZOID Tool. Methods Mol. Biol. 2020;2074:81–94. doi: 10.1007/978-1-4939-9873-9_7. PubMed DOI

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

Kleisin NSE4 of the SMC5/6 complex is necessary for DNA double strand break repair, but not for recovery from DNA damage in Physcomitrella (Physcomitrium patens)