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.

• Keywords
• Nse1, Nse4 kleisin, SMC5/6, Ubc13/Mms2, ubiquitin ligase, ubiquitination,
• MeSH
• Chromosomal Proteins, Non-Histone metabolism   MeSH
• Humans MeSH
• Ligases metabolism   MeSH
• Cell Cycle Proteins metabolism   MeSH
• Schizosaccharomyces pombe Proteins metabolism   MeSH
• Carrier Proteins metabolism   MeSH
• Ubiquitin metabolism   MeSH
• Ubiquitination immunology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromosomal Proteins, Non-Histone MeSH
• Ligases MeSH
• NSMCE1 protein, human MeSH Browser
• Cell Cycle Proteins MeSH
• Schizosaccharomyces pombe Proteins MeSH
• SMC5 protein, human MeSH Browser
• Carrier Proteins MeSH
• Ubiquitin MeSH

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.

• MeSH
• Adenosine Triphosphatases metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Macromolecular Substances metabolism   MeSH
• Mutagenesis, Site-Directed MeSH
• Cell Cycle Proteins genetics   metabolism   MeSH
• Schizosaccharomyces pombe Proteins genetics   metabolism   MeSH
• Schizosaccharomyces genetics   metabolism   MeSH
• Sequence Alignment MeSH
• Two-Hybrid System Techniques MeSH
• Carrier Proteins genetics   metabolism   MeSH
• Protein Binding genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Adenosine Triphosphatases MeSH
• Nuclear Proteins MeSH
• Macromolecular Substances MeSH
• Nse1 protein, S pombe MeSH Browser
• Nse3 protein, S pombe MeSH Browser
• Nse4 protein, S pombe MeSH Browser
• Cell Cycle Proteins MeSH
• Schizosaccharomyces pombe Proteins MeSH
• Smc5 protein, S pombe MeSH Browser
• smc6 protein, S pombe MeSH Browser
• Carrier Proteins MeSH

The SMC 5/6 complex together with cohesin and condensin is a member of the structural maintenance of chromosome (SMC) protein family. In non-plant organisms SMC5/6 is engaged in DNA repair, meiotic synapsis, genome organization and stability. In plants, the function of SMC5/6 is still enigmatic. Therefore, we analyzed the crucial δ-kleisin component NSE4 of the SMC5/6 complex in the model plant Arabidopsis thaliana. Two functional conserved Nse4 paralogs (Nse4A and Nse4B) are present in A. thaliana, which may have evolved via gene subfunctionalization. Due to its high expression level, Nse4A seems to be the more essential gene, whereas Nse4B appears to be involved mainly in seed development. The morphological characterization of A. thaliana T-DNA mutants suggests that the NSE4 proteins are essential for plant growth and fertility. Detailed investigations in wild-type and the mutants based on live cell imaging of transgenic GFP lines, fluorescence in situ hybridization (FISH), immunolabeling and super-resolution microscopy suggest that NSE4A acts in several processes during plant development, such as mitosis, meiosis and chromatin organization of differentiated nuclei, and that NSE4A operates in a cell cycle-dependent manner. Differential response of NSE4A and NSE4B mutants after induced DNA double strand breaks (DSBs) suggests their involvement in DNA repair processes.

• Keywords
• Arabidopsis thaliana, NSE4 δ-kleisin, SMC5/6 complex, meiosis, mitosis, nucleus, phylogeny, super-resolution microscopy,
• Publication type
• Journal Article MeSH

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.

• MeSH
• Chromatin metabolism   MeSH
• DNA metabolism   MeSH
• Nuclear Proteins metabolism   MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Cell Cycle Proteins chemistry   metabolism   MeSH
• Recombination, Genetic MeSH
• DNA Replication MeSH
• Schizosaccharomyces pombe Proteins metabolism   MeSH
• Schizosaccharomyces genetics   metabolism   MeSH
• Amino Acid Sequence MeSH
• Sequence Homology, Amino Acid MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Chromatin MeSH
• DNA MeSH
• Nuclear Proteins MeSH
• Nse3 protein, S pombe MeSH Browser
• Cell Cycle Proteins MeSH
• Schizosaccharomyces pombe Proteins MeSH

The MAGE (Melanoma-associated antigen) protein family members are structurally related to each other by a MAGE-homology domain comprised of 2 winged helix motifs WH/A and WH/B. This family specifically evolved in placental mammals although single homologs designated NSE3 (non-SMC element) exist in most eukaryotes. NSE3, together with its partner proteins NSE1 and NSE4 form a tight subcomplex of the structural maintenance of chromosomes SMC5-6 complex. Previously, we showed that interactions of the WH/B motif of the MAGE proteins with their NSE4/EID partners are evolutionarily conserved (including the MAGEA1-NSE4 interaction). In contrast, the interaction of the WH/A motif of NSE3 with NSE1 diverged in the MAGE paralogs. We hypothesized that the MAGE paralogs acquired new RING-finger-containing partners through their evolution and form MAGE complexes reminiscent of NSE1-NSE3-NSE4 trimers. In this work, we employed the yeast 2-hybrid system to screen a human RING-finger protein library against several MAGE baits. We identified a number of potential MAGE-RING interactions and confirmed several of them (MDM4, PCGF6, RNF166, TRAF6, TRIM8, TRIM31, TRIM41) in co-immunoprecipitation experiments. Among these MAGE-RING pairs, we chose to examine MAGEA1-TRIM31 in detail and showed that both WH/A and WH/B motifs of MAGEA1 bind to the coiled-coil domain of TRIM31 and that MAGEA1 interaction stimulates TRIM31 ubiquitin-ligase activity. In addition, TRIM31 directly binds to NSE4, suggesting the existence of a TRIM31-MAGEA1-NSE4 complex reminiscent of the NSE1-NSE3-NSE4 trimer. These results suggest that MAGEA1 functions as a co-factor of TRIM31 ubiquitin-ligase and that the TRIM31-MAGEA1-NSE4 complex may have evolved from an ancestral NSE1-NSE3-NSE4 complex.

• Keywords
• E3 ubiquitin ligase, MAGE, melanoma-associated antigen, MAGEA1, MDM4, MHD, MAGE homology domain, NSE, non-SMC element, NSE1-NSE3-NSE4 complex, NSE4/EID family, PCGF6, RING-finger proteins, RNF166, SMC, structure maintenance of chromosomes, TRAF6, TRIM family, TRIM, tripartite motif, TRIM31, TRIM41, TRIM8, WH, winged helix, Y2H, yeast 2-hybrid., melanoma-associated antigen family, protein evolution, ubiquitination,
• MeSH
• Models, Biological MeSH
• Chromatography, Liquid MeSH
• RING Finger Domains MeSH
• HEK293 Cells MeSH
• Immunoprecipitation MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Protein Multimerization MeSH
• Multiprotein Complexes metabolism   MeSH
• Neoplasm Proteins chemistry   metabolism   MeSH
• Peptide Fragments chemistry   metabolism   MeSH
• Peptides chemistry   metabolism   MeSH
• Amino Acid Sequence MeSH
• Tandem Mass Spectrometry MeSH
• Two-Hybrid System Techniques MeSH
• Carrier Proteins metabolism   MeSH
• Tripartite Motif Proteins MeSH
• Ubiquitin-Protein Ligases chemistry   metabolism   MeSH
• Protein Binding MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• MAGE-A1 protein (278-286), human MeSH Browser
• Multiprotein Complexes MeSH
• Neoplasm Proteins MeSH
• NSE4 protein, human MeSH Browser
• NSMCE1 protein, human MeSH Browser
• Peptide Fragments MeSH
• Peptides MeSH
• Carrier Proteins MeSH
• Tripartite Motif Proteins MeSH
• TRIM31 protein, human MeSH Browser
• Ubiquitin-Protein Ligases MeSH