Structural maintenance of chromosomes (SMC) complexes are molecular machines ensuring chromatin organization at higher levels. They play direct roles in cohesion, condensation, replication, transcription, and DNA repair. Their cores are composed of long-armed SMC, kleisin, and kleisin-associated subunits. Additional factors, like NSE6 within SMC5/6, bind to SMC core complexes and regulate their activities. In the human HsNSE6/SLF2, we recently identified a new CANIN domain. Here we tracked down its sequence homology to lower plants, selected the bryophyte Physcomitrium patens, and analyzed PpNSE6 protein-protein interactions to explore its conservation in detail. We identified a previously unrecognized core sequence motif conserved from yeasts to humans within the NSE6 CANIN domain. This motif mediates the interaction between NSE6 and its NSE5 partner in yeasts and plants. In addition, the CANIN domain and its preceding PpNSE6 sequences bind both PpSMC5 and PpSMC6 arms. Interestingly, we mapped the PpNSE6-binding site at the PpSMC5 arm right next to the PpNSE2-binding surface. The position of NSE6 at SMC arms suggests its role in the regulation of SMC5/6 dynamics. Consistent with the regulatory role of NSE6 subunits, Ppnse6 mutant lines were viable and sensitive to the DNA-damaging drug bleomycin and lost a large portion of rDNA copies. These moss mutants also exhibited reduced growth and developmental aberrations. Altogether, our data showed the conserved function of the NSE6 subunit and architecture of the SMC5/6 complex across species.

Centre of Plant Structural and Functional Genomics Institute of Experimental Botany Czech Academy of Sciences Šlechtitelů 31 77900 Olomouc Czech Republic

Institute of Experimental Botany Czech Academy of Sciences Na Karlovce 1 16000 Prague Czech Republic

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

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

Zobrazit více v PubMed

Adamus, M., Lelkes, E., Potesil, D., Ganji, S.R., Kolesar, P., Zabrady, K. et al. (2020) Molecular insights into the architecture of the human SMC5/6 complex. Journal of Molecular Biology, 432, 3820-3837.

Alt, A., Dang, H.Q., Wells, O.S., Polo, L.M., Smith, M.A., McGregor, G.A. et al. (2017) Specialized interfaces of Smc5/6 control hinge stability and DNA association. Nature Communications, 8, 14011.

Altschul, S.F., Madden, T.L., Schäffer, A.A., Zhang, J., Zhang, Z., Miller, W. et al. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 25, 3389-3402.

Andrews, E., Palecek, J., Sergeant, J., Taylor, E., Lehmann, A. & Watts, F. (2005) Nse2, a component of the Smc5-6 complex, is a SUMO ligase required for the response to DNA damage. Molecular and Cellular Biology, 25, 185-196.

Angelis, K.J., Záveská Drábková, L., Vágnerová, R. & Holá, M. (2023) RAD51 and RAD51B play diverse roles in the repair of DNA double strand breaks in Physcomitrium patens. Genes, 14, 305.

Aragón, L. (2018) The Smc5/6 complex: new and old functions of the enigmatic long-distance relative. Annual Review of Genetics, 52, 89-107.

Burmann, F., Funke, L.F.H., Chin, J.W. & Lowe, J. (2021) Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites. Molecular Cell, 81, 4891.

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M. et al. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical Chemistry, 55, 611-622.

Byska, J., Jurcik, A., Furmanova, K., Kozlikova, B. & Palecek, J.J. (2020) Visual analysis of protein-protein interaction docking models using COZOID tool. Methods in Molecular Biology, 2074, 81-94.

Cove, D.J., Perroud, P.F., Charron, A.J., McDaniel, S.F., Khandelwal, A. & Quatrano, R.S. (2009) The moss Physcomitrella patens: a novel model system for plant development and genomic studies. Cold Spring Harbor Protocols, 2009, pdb.emo115.

Davidson, I.F., Bauer, B., Goetz, D., Tang, W., Wutz, G. & Peters, J.M. (2019) DNA loop extrusion by human cohesin. Science, 366, 1338-1345.

Davidson, I.F. & Peters, J.M. (2021) Genome folding through loop extrusion by SMC complexes. Nature Reviews. Molecular Cell Biology, 22, 445-464.

Deep, A., Gu, Y., Gao, Y.Q., Ego, K.M., Herzik, M.A., Zhou, H. et al. (2022) The SMC-family Wadjet complex protects bacteria from plasmid transformation by recognition and cleavage of closed-circular DNA. Molecular Cell, 82, 4145-4159.e7.

Dellaporta, S.L., Wood, J. & Hicks, J.B. (1983) A plant DNA minipreparation: version II. Plant Molecular Biology Reporter, 1, 19-21.

Diaz, M. & Pecinka, A. (2018) Scaffolding for repair: understanding molecular functions of the SMC5/6 complex. Genes (Basel), 9, E36.

Díaz, M., Pečinková, P., Nowicka, A., Baroux, C., Sakamoto, T., Gandha, P.Y. et al. (2019) The SMC5/6 complex subunit NSE4A is involved in DNA damage repair and seed development. Plant Cell, 31, 1579-1597.

Diebold-Durand, M.L., Lee, H., Ruiz Avila, L.B., Noh, H., Shin, H.C., Im, H. et al. (2017) Structure of full-length SMC and rearrangements required for chromosome organization. Molecular Cell, 67, 334-347.e335.

Duan, X., Sarangi, P., Liu, X., Rangi, G.K., Zhao, X. & Ye, H. (2009) Structural and functional insights into the roles of the Mms21 subunit of the Smc5/6 complex. Molecular Cell, 35, 657-668.

Dusek, J., Plchova, H., Cerovska, N., Poborilova, Z., Navratil, O., Kratochvilova, K. et al. (2020) Extended set of GoldenBraid compatible vectors for fast assembly of multigenic constructs and their use to create geminiviral expression vectors. Frontiers in Plant Science, 11, 522059.

Ganji, M., Shaltiel, I.A., Bisht, S., Kim, E., Kalichava, A., Haering, C.H. et al. (2018) Real-time imaging of DNA loop extrusion by condensin. Science, 360, 102-105.

Gligoris, T. & Löwe, J. (2016) Structural insights into ring formation of cohesin and related Smc complexes. Trends in Cell Biology, 26, 680-693.

Goffová, I., Vágnerová, R., Peška, V., Franek, M., Havlová, K., Holá, M. et al. (2019) Roles of RAD51 and RTEL1 in telomere and rDNA stability in Physcomitrella patens. The Plant Journal, 98, 1090-1105.

Grange, L.J., Reynolds, J.J., Ullah, F., Isidor, B., Shearer, R.F., Latypova, X. et al. (2022) Pathogenic variants in SLF2 and SMC5 cause segmented chromosomes and mosaic variegated hyperploidy. Nature Communications, 13, 6664.

Gutierrez-Escribano, P., Hormeno, S., Madariaga-Marcos, J., Sole-Soler, R., O'Reilly, F.J., Morris, K. et al. (2020) Purified Smc5/6 complex exhibits DNA substrate recognition and compaction. Molecular Cell, 80, 1039-1054.

Hallett, S.T., Campbell Harry, I., Schellenberger, P., Zhou, L., Cronin, N.B., Baxter, J. et al. (2022) Cryo-EM structure of the Smc5/6 holo-complex. Nucleic Acids Research, 50, 9505-9520.

Hallett, S.T., Schellenberger, P., Zhou, L.H., Beuron, F., Morris, E., Murray, J.M. et al. (2021) Nse5/6 is a negative regulator of the ATPase activity of the Smc5/6 complex. Nucleic Acids Research, 49, 4534-4549.

Hassler, M., Shaltiel, I.A. & Haering, C.H. (2018) Towards a unified model of SMC complex function. Current Biology, 28, R1266-R1281.

Holá, M., Kozák, J., Vágnerová, R. & Angelis, K.J. (2013) Genotoxin induced mutagenesis in the model plant Physcomitrella patens. BioMed Research International, 2013, 535049.

Holá, M., Vágnerová, R. & Angelis, K.J. (2021) 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). Plant Molecular Biology, 107, 355-364.

Hudson, J.J.R., Bednarova, K., Kozakova, L., Liao, C.Y., Guerineau, M., Colnaghi, R. et al. (2011) Interactions between the Nse3 and Nse4 components of the SMC5-6 complex identify evolutionarily conserved interactions between MAGE and EID families. PLoS One, 6, 14.

Kamisugi, Y., Whitaker, J.W. & Cuming, A.C. (2016) The transcriptional response to DNA-double-strand breaks in Physcomitrella patens. PLoS One, 11, e0161204.

Kozak, J., West, C.E., White, C., da Costa-Nunes, J.A. & Angelis, K.J. (2009) Rapid repair of DNA double strand breaks in Arabidopsis thaliana is dependent on proteins involved in chromosome structure maintenance. DNA Repair (Amst), 8, 413-419.

Lehmann, A.R., Walicka, M., Griffiths, D.J.F., Murray, J.M., Watts, F.Z., McCready, S. et al. (1995) The rad18 gene of Schizosaccharomyces pombe defines a new subgroup of the SMC superfamily involved in DNA repair. Molecular and Cellular Biology, 15, 7067-7080.

Litwin, I. & Wysocki, R. (2018) New insights into cohesin loading. Current Genetics, 64, 53-61.

Liu, H.W., Roisné-Hamelin, F., Beckert, B., Li, Y., Myasnikov, A. & Gruber, S. (2022) DNA-measuring Wadjet SMC ATPases restrict smaller circular plasmids by DNA cleavage. Molecular Cell, 82, 4727-4740.e4726.

Liu, Y.C. & Vidali, L. (2011) Efficient polyethylene glycol (PEG) mediated transformation of the moss Physcomitrella patens. Journal of Visualized Experiments, 50, 2560.

Mahrik, L., Stefanovie, B., Maresova, A., Princova, J., Kolesar, P., Lelkes, E. et al. (2023) The SAGA histone acetyltransferase module targets SMC5/6 to specific genes. Epigenetics & Chromatin, 16, 16.

Mallett, D.R., Chang, M., Cheng, X. & Bezanilla, M. (2019) Efficient and modular CRISPR-Cas9 vector system for Physcomitrella patens. Plant Direct, 3, e00168.

McGuffin, L.J., Bryson, K. & Jones, D.T. (2000) The PSIPRED protein structure prediction server. Bioinformatics, 16, 404-405.

Mirdita, M., Schütze, K., Moriwaki, Y., Heo, L., Ovchinnikov, S. & Steinegger, M. (2022) ColabFold: making protein folding accessible to all. Nature Methods, 19, 679-682.

Mistry, J., Chuguransky, S., Williams, L., Qureshi, M., Salazar, G.A., Sonnhammer, E.L.L. et al. (2021) Pfam: the protein families database in 2021. Nucleic Acids Research, 49, D412-D419.

Murray, J.M. & Carr, A.M. (2007) Smc5/6: a link between DNA repair and unidirectional replication? Nature Reviews. Molecular Cell Biology, 9, 177-182.

Nasmyth, K. & Haering, C.H. (2005) The structure and function of SMC and kleisin complexes. Annual Review of Biochemistry, 74, 595-648.

Oravcová, M. & Boddy, M.N. (2019) Recruitment, loading, and activation of the Smc5-Smc6 SUMO ligase. Current Genetics, 65, 669-676.

Oravcova, M., Nie, M.H., Zilio, N., Maeda, S., Jami-Alahmadi, Y., Lazzerini-Denchi, E. et al. (2022) The Nse5/6-like SIMC1-SLF2 complex localizes SMC5/6 to viral replication centers. eLife, 11, 37.

Palecek, J., Vidot, S., Feng, M., Doherty, A.J. & Lehmann, A.R. (2006) The SMC5-6 DNA repair complex: bridging of the SMC5-6 heads by the Kleisin, NSE4, and non-Kleisin subunits. The Journal of Biological Chemistry, 281, 36952-36959.

Palecek, J.J. (2019) SMC5/6: multifunctional player in replication. Genes, 10, E7.

Palecek, J.J. & Gruber, S. (2015) Kite proteins: a superfamily of SMC/Kleisin partners conserved across bacteria, archaea, and eukaryotes. Structure, 23, 2183-2190.

Pebernard, S., Wohlschlegel, J., McDonald, W.H., Yates, J.R., 3rd & Boddy, M.N. (2006) The Nse5-Nse6 dimer mediates DNA repair roles of the Smc5-Smc6 complex. Molecular and Cellular Biology, 26, 1617-1630.

Peng, X.P., Lim, S., Li, S.B., Marjavaara, L., Chabes, A. & Zhao, X.L. (2018) Acute Smc5/6 depletion reveals its primary role in rDNA replication by restraining recombination at fork pausing sites. PLoS Genetics, 14, 20.

Petrushenko, Z.M., She, W. & Rybenkov, V.V. (2011) A new family of bacterial condensins. Molecular Microbiology, 81, 881-896.

Pfaffl, M.W. (2004) Quantification strategies in real-time PC. La Jolla, CA: International University Line.

Pradhan, B., Kanno, T., Umeda Igarashi, M., Loke, M.S., Baaske, M.D., Wong, J.S.K. et al. (2023) The Smc5/6 complex is a DNA loop-extruding motor. Nature, 616, 843-848.

Prigge, M.J. & Bezanilla, M. (2010) Evolutionary crossroads in developmental biology: Physcomitrella patens. Development, 137, 3535-3543.

Räschle, M., Smeenk, G., Hansen, R.K., Temu, T., Oka, Y., Hein, M.Y. et al. (2015) DNA repair. Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links. Science, 348, 1253671.

Rensing, S.A., Lang, D., Zimmer, A.D., Terry, A., Salamov, A., Shapiro, H. et al. (2008) The Physcomitrella genome reveals evolutionary insights into the conquest of land by plants. Science, 319, 64-69.

Schaefer, D.G. & Zrÿd, J.P. (1997) Efficient gene targeting in the moss Physcomitrella patens. The Plant Journal, 11, 1195-1206.

Sergeant, J., Taylor, E., Palecek, J., Fousteri, M., Andrews, E., Sweeney, S. et al. (2005) Composition and architecture of the Schizosaccharomyces pombe Rad18 (Smc5-6) complex. Molecular and Cellular Biology, 25, 172-184.

Taschner, M., Basquin, J., Steigenberger, B., Schafer, I.B., Soh, Y.M., Basquin, C. et al. (2021) Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding. EMBO Journal, 40, 23.

Torres-Rosell, J., Sunjevaric, I., De Piccoli, G., Sacher, M., Eckert-Boulet, N., Reid, R. et al. (2007) The Smc5-Smc6 complex and SUMO modification of Rad52 regulates recombinational repair at the ribosomal gene locus. Nature Cell Biology, 9, 923-931.

Uhlmann, F. (2016) SMC complexes: from DNA to chromosomes. Nature Reviews. Molecular Cell Biology, 17, 399-412.

Varadi, M., Anyango, S., Deshpande, M., Nair, S., Natassia, C., Yordanova, G. et al. (2022) AlphaFold Protein Structure Database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acids Research, 50, D439-D444.

Vladejić, J., Yang, F., Dvořák Tomaštíková, E., Doležel, J., Palecek, J.J. & Pecinka, A. (2022) Analysis of BRCT5 domain-containing proteins reveals a new component of DNA damage repair in Arabidopsis. Frontiers in Plant Science, 13, 1023358.

Vondrova, L., Kolesar, P., Adamus, M., Nociar, M., Oliver, A.W. & Palecek, J.J. (2020) A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6. Scientific Reports, 10, 13.

Wang, X.D., Hughes, A.C., Brandao, H.B., Walker, B., Lierz, C., Cochran, J.C. et al. (2018) In vivo evidence for ATPase-dependent DNA translocation by the Bacillus subtilis SMC condensin complex. Molecular Cell, 71, 841-847.

Watanabe, K., Pacher, M., Dukowic, S., Schubert, V., Puchta, H. & Schubert, I. (2009) The STRUCTURAL MAINTENANCE OF CHROMOSOMES 5/6 complex promotes sister chromatid alignment and homologous recombination after DNA damage in Arabidopsis thaliana. The Plant Cell, 21, 2688-2699.

Yan, S., Wang, W., Marqués, J., Mohan, R., Saleh, A., Durrant, W.E. et al. (2013) Salicylic acid activates DNA damage responses to potentiate plant immunity. Molecular Cell, 52, 602-610.

Yang, F. & Pecinka, A. (2022) Multiple roles of SMC5/6 complex during plant sexual reproduction. International Journal of Molecular Sciences, 23, 4503.

Yoshinaga, M. & Inagaki, Y. (2021) Ubiquity and origins of structural maintenance of chromosomes (SMC) proteins in eukaryotes. Genome Biology and Evolution, 13, 17.

Yu, Y., Li, S., Ser, Z., Kuang, H., Than, T., Guan, D. et al. (2022) Cryo-EM structure of DNA-bound Smc5/6 reveals DNA clamping enabled by multi-subunit conformational changes. Proceedings of the National Academy of Sciences of the United States of America, 119, e2202799119.

Yu, Y., Li, S.B., Ser, Z., Sanyal, T., Choi, K., Wan, B.B. et al. (2021) Integrative analysis reveals unique structural and functional features of the Smc5/6 complex. Proceedings of the National Academy of Sciences of the United States of America, 118, 11.

Zabrady, K., Adamus, M., Vondrova, L., Liao, C., Skoupilova, H., Novakova, M. et al. (2016) Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA. Nucleic Acids Research, 44, 1064-1079.

Zelkowski, M., Zelkowska, K., Conrad, U., Hesse, S., Lermontova, I., Marzec, M. et al. (2019) NSE4 proteins act in somatic nuclei and meiosis to ensure plant viability and fertility. Frontiers in Plant Science, 10, 774.

Zhao, X. & Blobel, G. (2005) A SUMO ligase is part of a nuclear multiprotein complex that affects DNA repair and chromosomal organization. Proceedings of the National Academy of Sciences of the United States of America, 102, 4777-47782.

The Phenotype of Physcomitrium patens SMC6 Mutant with Interrupted Hinge Interactions