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.

• Klíčová slova
• Arabidopsis thaliana, NSE4 δ-kleisin, SMC5/6 complex, meiosis, mitosis, nucleus, phylogeny, super-resolution microscopy,
• Publikační typ
• časopisecké články 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 metabolismus   MeSH
• DNA metabolismus   MeSH
• jaderné proteiny metabolismus   MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• proteiny buněčného cyklu chemie   metabolismus   MeSH
• rekombinace genetická MeSH
• replikace DNA MeSH
• Schizosaccharomyces pombe - proteiny metabolismus   MeSH
• Schizosaccharomyces genetika   metabolismus   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromatin MeSH
• DNA MeSH
• jaderné proteiny MeSH
• Nse3 protein, S pombe MeSH Prohlížeč
• proteiny buněčného cyklu MeSH
• Schizosaccharomyces pombe - proteiny 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.

• Klíčová slova
• 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
• biologické modely MeSH
• chromatografie kapalinová MeSH
• domény RING-prstů MeSH
• HEK293 buňky MeSH
• imunoprecipitace MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• multiproteinové komplexy metabolismus   MeSH
• nádorové proteiny chemie   metabolismus   MeSH
• peptidové fragmenty chemie   metabolismus   MeSH
• peptidy chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• tandemová hmotnostní spektrometrie MeSH
• techniky dvojhybridového systému MeSH
• transportní proteiny metabolismus   MeSH
• TRIM protein MeSH
• ubikvitinligasy chemie   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• MAGE-A1 protein (278-286), human MeSH Prohlížeč
• multiproteinové komplexy MeSH
• nádorové proteiny MeSH
• NSE4 protein, human MeSH Prohlížeč
• NSMCE1 protein, human MeSH Prohlížeč
• peptidové fragmenty MeSH
• peptidy MeSH
• transportní proteiny MeSH
• TRIM protein MeSH
• TRIM31 protein, human MeSH Prohlížeč
• ubikvitinligasy MeSH

BACKGROUND: The Nse1, Nse3 and Nse4 proteins form a tight sub-complex of the large SMC5-6 protein complex. hNSE3/MAGEG1, the mammalian ortholog of Nse3, is the founding member of the MAGE (melanoma-associated antigen) protein family and the Nse4 kleisin subunit is related to the EID (E1A-like inhibitor of differentiation) family of proteins. We have recently shown that human MAGE proteins can interact with NSE4/EID proteins through their characteristic conserved hydrophobic pocket. METHODOLOGY/PRINCIPAL FINDINGS: Using mutagenesis and protein-protein interaction analyses, we have identified a new Nse3/MAGE-binding domain (NMBD) of the Nse4/EID proteins. This short domain is located next to the Nse4 N-terminal kleisin motif and is conserved in all NSE4/EID proteins. The central amino acid residues of the human NSE4b/EID3 domain were essential for its binding to hNSE3/MAGEG1 in yeast two-hybrid assays suggesting they form the core of the binding domain. PEPSCAN ELISA measurements of the MAGEC2 binding affinity to EID2 mutant peptides showed that similar core residues contribute to the EID2-MAGEC2 interaction. In addition, the N-terminal extension of the EID2 binding domain took part in the EID2-MAGEC2 interaction. Finally, docking and molecular dynamic simulations enabled us to generate a structure model for EID2-MAGEC2. Combination of our experimental data and the structure modeling showed how the core helical region of the NSE4/EID domain binds into the conserved pocket characteristic of the MAGE protein family. CONCLUSIONS/SIGNIFICANCE: We have identified a new Nse4/EID conserved domain and characterized its binding to Nse3/MAGE proteins. The conservation and binding of the interacting surfaces suggest tight co-evolution of both Nse4/EID and Nse3/MAGE protein families.

• MeSH
• chromozomální proteiny, nehistonové MeSH
• inhibitor diferenciace 2 MeSH
• interakční proteinové domény a motivy MeSH
• intracelulární signální peptidy a proteiny chemie   genetika   metabolismus   MeSH
• jaderné proteiny chemie   MeSH
• konzervovaná sekvence MeSH
• lidé MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• mutageneze cílená MeSH
• peptidové fragmenty chemie   genetika   metabolismus   MeSH
• počítačová simulace MeSH
• proteiny buněčného cyklu genetika   metabolismus   MeSH
• rekombinantní proteiny chemie   genetika   metabolismus   MeSH
• Schizosaccharomyces pombe - proteiny chemie   MeSH
• Schizosaccharomyces MeSH
• sekvence aminokyselin MeSH
• substituce aminokyselin MeSH
• techniky dvojhybridového systému MeSH
• transportní proteiny chemie   genetika   metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• chromozomální proteiny, nehistonové MeSH
• EID2 protein, human MeSH Prohlížeč
• EID3 protein, human MeSH Prohlížeč
• inhibitor diferenciace 2 MeSH
• intracelulární signální peptidy a proteiny MeSH
• jaderné proteiny MeSH
• Nse3 protein, S pombe MeSH Prohlížeč
• Nse4 protein, S pombe MeSH Prohlížeč
• NSMCE3 protein, human MeSH Prohlížeč
• peptidové fragmenty MeSH
• proteiny buněčného cyklu MeSH
• rekombinantní proteiny MeSH
• Schizosaccharomyces pombe - proteiny MeSH
• SMC5 protein, human MeSH Prohlížeč
• transportní proteiny MeSH