12 results on '"Palecek JJ"'
Search Results
2. The SAGA histone acetyltransferase module targets SMC5/6 to specific genes.
- Author
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Mahrik L, Stefanovie B, Maresova A, Princova J, Kolesar P, Lelkes E, Faux C, Helmlinger D, Prevorovsky M, and Palecek JJ
- Subjects
- Acetyltransferases genetics, Carrier Proteins metabolism, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Cell Nucleus metabolism, Chromatin metabolism, Chromosomes metabolism, DNA metabolism, Histone Acetyltransferases genetics, Histone Acetyltransferases metabolism, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins genetics, Schizosaccharomyces pombe Proteins metabolism
- Abstract
Background: Structural Maintenance of Chromosomes (SMC) complexes are molecular machines driving chromatin organization at higher levels. In eukaryotes, three SMC complexes (cohesin, condensin and SMC5/6) play key roles in cohesion, condensation, replication, transcription and DNA repair. Their physical binding to DNA requires accessible chromatin., Results: We performed a genetic screen in fission yeast to identify novel factors required for SMC5/6 binding to DNA. We identified 79 genes of which histone acetyltransferases (HATs) were the most represented. Genetic and phenotypic analyses suggested a particularly strong functional relationship between the SMC5/6 and SAGA complexes. Furthermore, several SMC5/6 subunits physically interacted with SAGA HAT module components Gcn5 and Ada2. As Gcn5-dependent acetylation facilitates the accessibility of chromatin to DNA-repair proteins, we first analysed the formation of DNA-damage-induced SMC5/6 foci in the Δgcn5 mutant. The SMC5/6 foci formed normally in Δgcn5, suggesting SAGA-independent SMC5/6 localization to DNA-damaged sites. Next, we used Nse4-FLAG chromatin-immunoprecipitation (ChIP-seq) analysis in unchallenged cells to assess SMC5/6 distribution. A significant portion of SMC5/6 accumulated within gene regions in wild-type cells, which was reduced in Δgcn5 and Δada2 mutants. The drop in SMC5/6 levels was also observed in gcn5-E191Q acetyltransferase-dead mutant., Conclusion: Our data show genetic and physical interactions between SMC5/6 and SAGA complexes. The ChIP-seq analysis suggests that SAGA HAT module targets SMC5/6 to specific gene regions and facilitates their accessibility for SMC5/6 loading., (© 2023. The Author(s).)
- Published
- 2023
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3. Analysis of BRCT5 domain-containing proteins reveals a new component of DNA damage repair in Arabidopsis.
- Author
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Vladejić J, Yang F, Dvořák Tomaštíková E, Doležel J, Palecek JJ, and Pecinka A
- Abstract
The integrity of plant genetic information is constantly challenged by various internal and external factors. Therefore, plants use a sophisticated molecular network to identify, signal and repair damaged DNA. Here, we report on the identification and analysis of four uncharacterized Arabidopsis BRCT5 DOMAIN CONTAINING PROTEINs (BCPs). Proteins with the BRCT5 domain are frequently involved in the maintenance of genome stability across eukaryotes. The screening for sensitivity to induced DNA damage identified BCP1 as the most interesting candidate. We show that BCP1 loss of function mutants are hypersensitive to various types of DNA damage and accumulate an increased number of dead cells in root apical meristems upon DNA damage. Analysis of publicly available sog1 transcriptomic and SOG1 genome-wide DNA binding data revealed that BCP1 is inducible by gamma radiation and is a direct target of this key DNA damage signaling transcription factor. Importantly, bcp1 plants showed a reduced frequency of somatic homologous recombination in response to both endogenous and induced DNA damage. Altogether, we identified a novel plant-specific DNA repair factor that acts downstream of SOG1 in homology-based repair., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Vladejić, Yang, Dvořák Tomaštíková, Doležel, Palecek and Pecinka.)
- Published
- 2022
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4. Role of Nse1 Subunit of SMC5/6 Complex as a Ubiquitin Ligase.
- Author
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Kolesar P, Stejskal K, Potesil D, Murray JM, and Palecek JJ
- Subjects
- Humans, Carrier Proteins metabolism, Cell Cycle Proteins metabolism, Chromosomal Proteins, Non-Histone metabolism, Ligases metabolism, Schizosaccharomyces pombe Proteins metabolism, Ubiquitin metabolism, Ubiquitination immunology
- Abstract
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.
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- 2022
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5. A role of the Nse4 kleisin and Nse1/Nse3 KITE subunits in the ATPase cycle of SMC5/6.
- Author
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Vondrova L, Kolesar P, Adamus M, Nociar M, Oliver AW, and Palecek JJ
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- Carrier Proteins genetics, Cell Cycle Proteins genetics, Macromolecular Substances metabolism, Mutagenesis, Site-Directed, Nuclear Proteins genetics, Protein Binding genetics, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Schizosaccharomyces pombe Proteins genetics, Sequence Alignment, Two-Hybrid System Techniques, Adenosine Triphosphatases metabolism, Carrier Proteins metabolism, Cell Cycle Proteins metabolism, Nuclear Proteins metabolism, Schizosaccharomyces pombe Proteins metabolism
- Abstract
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.
- Published
- 2020
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6. Molecular Insights into the Architecture of the Human SMC5/6 Complex.
- Author
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Adamus M, Lelkes E, Potesil D, Ganji SR, Kolesar P, Zabrady K, Zdrahal Z, and Palecek JJ
- Subjects
- DNA Repair genetics, Humans, Multiprotein Complexes genetics, Mutation, Protein Binding genetics, Protein Domains genetics, Protein Multimerization genetics, Carrier Proteins genetics, Cell Cycle Proteins genetics, Chromosomal Proteins, Non-Histone genetics
- Abstract
A family of Structural Maintenance of Chromosome (SMC) complexes is essential for key cellular processes ensuring proper cohesion, condensation and replication. They share a common SMC-kleisin architecture allowing them to embrace DNA. In SMC5/6, the NSE1 and NSE3 KITE and NSE4 kleisin subunits form a stable subcomplex that binds DNA and regulates essential processes. In addition, NSE5 and NSE6 subunits associate with the core SMC5/6 complex and recruit it to DNA repair sites. The architecture of the SMC5/6 complex is crucial for its proper functioning, and mutations within the human SMC5/6 subunits result in severe syndromes. Therefore, we aimed to analyze interactions within the human SMC5/6 complex and determine its detailed architecture. Firstly, we analyzed different parts of SMC5/6 by crosslinking and MS/MS analysis. Our data suggested domain arrangements of hNSE1-hNSE3 and orientation of hNSE4 within the hNSE1-hNSE3-hNSE4 subcomplex. The crosslinking and electron microscopic analysis of the SMC5/6 core complex showed its rod-like architecture with juxtaposed hSMC5-hSMC6 arms. Additionally, we observed fully or partially opened hSMC5-hSMC6 shapes with the hNSE1-hNSE3-hNSE4 trimer localized in the SMC head domains. To complete mapping of the human SMC5/6 complex architecture, we analyzed positions of hNSE5-hNSE6 at the hSMC5-hSMC6 arms. We showed that hNSE6 binding to hNSE5 and the coiled-coil arm of hSMC6 is mediated by a conserved FAM178 domain, which we therefore renamed CANIN (Coiled-coil SMC6 And NSE5 INteracting) domain. Interestingly, hNSE6 bound both hSMC5 and hSMC6 arms, suggesting that hNSE6 may lock the arms and regulate the dynamics of the human SMC5/6 complex., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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7. Visual Analysis of Protein-Protein Interaction Docking Models Using COZOID Tool.
- Author
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Byska J, Jurcik A, Furmanova K, Kozlikova B, and Palecek JJ
- Subjects
- Databases, Protein, Humans, Protein Binding, Protein Conformation, Protein Interaction Mapping, Structural Homology, Protein, Proteins chemistry
- Abstract
Networks of protein-protein interactions (PPI) constitute either stable or transient complexes in every cell. Most of the cellular complexes keep their function, and therefore stay similar, during evolution. The evolutionary constraints preserve most cellular functions via preservation of protein structures and interactions. The evolutionary conservation information is utilized in template-based approaches, like protein structure modeling or docking. Here we use the combination of the template-free docking method with conservation-based selection of the best docking model using our newly developed COZOID tool.We describe a step-by-step protocol for visual selection of docking models, based on their similarity to the original protein complex structure. Using the COZOID tool, we first analyze contact zones of the original complex structure and select contact amino acids for docking restraints. Then we model and dock the homologous proteins. Finally, we utilize different analytical modes of our COZOID tool to select the docking models most similar to the original complex structure.
- Published
- 2020
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8. SMC5/6: Multifunctional Player in Replication.
- Author
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Palecek JJ
- Abstract
The genome replication process is challenged at many levels. Replication must proceed through different problematic sites and obstacles, some of which can pause or even reverse the replication fork (RF). In addition, replication of DNA within chromosomes must deal with their topological constraints and spatial organization. One of the most important factors organizing DNA into higher-order structures are Structural Maintenance of Chromosome (SMC) complexes. In prokaryotes, SMC complexes ensure proper chromosomal partitioning during replication. In eukaryotes, cohesin and SMC5/6 complexes assist in replication. Interestingly, the SMC5/6 complexes seem to be involved in replication in many ways. They stabilize stalled RFs, restrain RF regression, participate in the restart of collapsed RFs, and buffer topological constraints during RF progression. In this (mini) review, I present an overview of these replication-related functions of SMC5/6.
- Published
- 2018
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9. Destabilized SMC5/6 complex leads to chromosome breakage syndrome with severe lung disease.
- Author
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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, and van Haaften G
- Subjects
- Alleles, B-Lymphocytes cytology, Cell Proliferation, Child, Child, Preschool, Chromosomal Proteins, Non-Histone, Chromosome Segregation, Chromosomes ultrastructure, DNA Damage, DNA Repair, DNA Replication, Family Health, Female, Fibroblasts metabolism, Homozygote, Humans, Infant, Male, Meiosis, Mitosis, Mutation, Missense, Pedigree, Recombination, Genetic, Syndrome, T-Lymphocytes cytology, Abnormalities, Multiple genetics, Cell Cycle Proteins genetics, Chromosome Breakage, Intracellular Signaling Peptides and Proteins genetics, Lung Diseases genetics
- Abstract
The structural maintenance of chromosomes (SMC) family of proteins supports mitotic proliferation, meiosis, and DNA repair to control genomic stability. Impairments in chromosome maintenance are linked to rare chromosome breakage disorders. Here, we have identified a chromosome breakage syndrome associated with severe lung disease in early childhood. Four children from two unrelated kindreds died of severe pulmonary disease during infancy following viral pneumonia with evidence of combined T and B cell immunodeficiency. Whole exome sequencing revealed biallelic missense mutations in the NSMCE3 (also known as NDNL2) gene, which encodes a subunit of the SMC5/6 complex that is essential for DNA damage response and chromosome segregation. The NSMCE3 mutations disrupted interactions within the SMC5/6 complex, leading to destabilization of the complex. Patient cells showed chromosome rearrangements, micronuclei, sensitivity to replication stress and DNA damage, and defective homologous recombination. This work associates missense mutations in NSMCE3 with an autosomal recessive chromosome breakage syndrome that leads to defective T and B cell function and acute respiratory distress syndrome in early childhood.
- Published
- 2016
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10. Chromatin association of the SMC5/6 complex is dependent on binding of its NSE3 subunit to DNA.
- Author
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Zabrady K, Adamus M, Vondrova L, Liao C, Skoupilova H, Novakova M, Jurcisinova L, Alt A, Oliver AW, Lehmann AR, and Palecek JJ
- Subjects
- Amino Acid Sequence, Cell Cycle Proteins chemistry, DNA Replication, Humans, Molecular Sequence Data, Protein Binding, Recombination, Genetic, Schizosaccharomyces genetics, Schizosaccharomyces metabolism, Sequence Homology, Amino Acid, Cell Cycle Proteins metabolism, Chromatin metabolism, DNA metabolism, Nuclear Proteins metabolism, Schizosaccharomyces pombe Proteins metabolism
- Abstract
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., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2016
- Full Text
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11. Kite Proteins: a Superfamily of SMC/Kleisin Partners Conserved Across Bacteria, Archaea, and Eukaryotes.
- Author
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Palecek JJ and Gruber S
- Subjects
- Amino Acid Sequence, Archaeal Proteins genetics, Archaeal Proteins metabolism, Bacterial Proteins genetics, Bacterial Proteins metabolism, Cell Cycle Proteins genetics, Cell Cycle Proteins metabolism, Conserved Sequence, Kinesins chemistry, Kinesins metabolism, Molecular Sequence Data, Archaeal Proteins chemistry, Bacterial Proteins chemistry, Cell Cycle Proteins chemistry
- Abstract
SMC/kleisin complexes form elongated annular structures, which are critical for chromosome segregation, genome maintenance, and the regulation of gene expression. We describe marked structural similarities between bacterial and eukaryotic SMC/kleisin partner proteins (designated here as "kite" proteins for kleisin interacting tandem winged-helix (WH) elements of SMC complexes). Kite proteins are integral parts of all prokaryotic SMC complexes and Smc5/6 but not cohesin and condensin. They are made up of tandem WH domains, form homo- or heterodimers via their amino-terminal WH domain, and they associate with the central part of a kleisin subunit. In placental mammals, the kite subunit NSE3 gave rise to several (>60) kite-related proteins, named MAGE, many of which encode tumor- and testis-specific antigens. Based on architectural rather than sequence similarity, we propose an adapted model for the evolution of the SMC protein complexes and discuss potential functional similarities between bacterial Smc/ScpAB and eukaryotic Smc5/6., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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12. The melanoma-associated antigen 1 (MAGEA1) protein stimulates the E3 ubiquitin-ligase activity of TRIM31 within a TRIM31-MAGEA1-NSE4 complex.
- Author
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Kozakova L, Vondrova L, Stejskal K, Charalabous P, Kolesar P, Lehmann AR, Uldrijan S, Sanderson CM, Zdrahal Z, and Palecek JJ
- Subjects
- Amino Acid Sequence, Chromatography, Liquid, HEK293 Cells, Humans, Immunoprecipitation, Models, Biological, Molecular Sequence Data, Neoplasm Proteins chemistry, Peptide Fragments chemistry, Peptides chemistry, Peptides metabolism, Protein Binding, Protein Multimerization, RING Finger Domains, Tandem Mass Spectrometry, Tripartite Motif Proteins, Two-Hybrid System Techniques, Ubiquitin-Protein Ligases chemistry, Carrier Proteins metabolism, Multiprotein Complexes metabolism, Neoplasm Proteins metabolism, Peptide Fragments metabolism, Ubiquitin-Protein Ligases metabolism
- Abstract
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.
- Published
- 2015
- Full Text
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