Papers by Katerina Bednarova
<p>(<b>A.</b>) The His-MBP-tagged fragment of yeast Nse3 (aa 200 - 307) was pre... more <p>(<b>A.</b>) The His-MBP-tagged fragment of yeast Nse3 (aa 200 - 307) was pre-bound to amylose-beads (lanes 1–3, 7–9 and 10–12) and then incubated with <i>in vitro</i> translated fragments spanning aa 1 to 110 (lanes 1–3), aa 1 to 77 (lanes 7–9) and/or aa 75 to 104 (lanes 10–12) of the yeast Nse4 protein. The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the His-MBP-tagged protein was analyzed by immunoblotting with anti-His antibody and the <i>in vitro</i> translated proteins were measured by autoradiography. I, input (5% of total); U, unbound (5%); B, bound (40%). Control, no His-MBP-tagged protein present. Alignment of Nse4 (<b>B.</b>) and EID (<b>C.</b>) subfamilies. The orthologs are from <i>Schizosaccharomyces pombe</i> (<i>S.p.</i>), <i>Aspergillus nidulans</i> (<i>A.n.</i>), <i>Neosartorya fischeri</i> (<i>N.f.</i>), <i>Aspergillus terreus</i> (<i>A.t.</i>), <i>Aspergillus clavatus</i> (<i>A.c.</i>), <i>S. cerevisiae</i> (<i>S.c.</i>), <i>Danio rerio</i> (<i>D.r.</i>), <i>Xenopus leavis</i> (<i>X.l.</i>), <i>Galus galus</i> (<i>G.g.</i>), <i>Monodelphis domestica</i> (<i>M.d.</i>), <i>Dasypus novemcinctus</i> (<i>D.n.</i>), <i>Canis lupus familiaris</i> (<i>C.f.</i>), <i>Mus musculus</i> (<i>M.m.</i>), <i>Homo sapiens</i> (<i>H.s.</i>). The EID homologs are present only in some mammals. Red box, Nse3/MAGE-binding domain; blue plus, mutation not affecting interaction; red minus, mutation disrupting the interaction. Amino acid shading represents groups conserved across the family: <i>dark green</i>, hydrophobic and aromatic; <i>light green</i>, polar; <i>pink</i>, acidic; <i>blue</i>, basic; all glycine and proline residues are highlighted in <i>yellow</i>.</p
<p>The GST-His-S-tagged fragment of human NSE4b(106 - 135) was bound to S-protein agarose b... more <p>The GST-His-S-tagged fragment of human NSE4b(106 - 135) was bound to S-protein agarose beads (lanes 1–3) and then incubated with <i>in vitro</i> translated class I (panel <b>A</b> and <b>B</b>, lanes 1–6) and/or class II (panel <b>C</b> and <b>D</b>, lanes 1–6) MAGE proteins: MAGEA1 (aa 1-309; panel <b>A</b>), MAGEC2 (aa 6-373; panel <b>B</b>), MAGED4b (aa 1-741; panel <b>C</b>) and necdin (aa 1-321; panel <b>D</b>). The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the GST-His-S-tagged protein was analyzed by immunoblotting with anti-His antibody and the <i>in vitro</i> translated proteins were measured by autoradiography. Control, GST-His-S-tag protein (lanes 4–6).</p
Analýza Smc5/6 komplexu a MAGE proteinů Naplni teto diplomove prace byla analýza Smc5/6 komplexu ... more Analýza Smc5/6 komplexu a MAGE proteinů Naplni teto diplomove prace byla analýza Smc5/6 komplexu (S. pombe) – konkretně analýza vzajemných interakci jeho tři proteinů v subkomplexu Nse1/3/4 a hloubkova analýza MAGE homologni domeny Nse3 a jeji interakce s proteinem Nse4. K tomuto ucelu byly využity metody cilena mutageneze a kvasinkový dvou- a trojhybridni system. Dosud byla pozorovana pouze silna interakce Nse1-Nse3 a slabsi interakce Nse3-Nse4. Pomoci kvasinkoveho trojhybridniho testu bylo zjistěno, že obě dosud identifikovane interakce Nse1-Nse3 a Nse3-Nse4 byly zesileny za přitomnosti třetiho proteinu ze subkomplexu Nse1/3/4. Ke stejně silne interakci mezi proteiny subkomplexu dochazelo i v připadě, kdy jsme k dvouhybridnim proteinům Nse1 a Nse4 přidavali třeti nehybridni protein Nse3. Interakce celeho proteinu Nse1 s Nse4 bez přitomnosti Nse3 ovsem pozorovana nebyla. Nově jsme vsak pomoci kvasinkoveho dvouhybridniho systemu identifikovali interakci mezi zkraceným Nse1 a celým proteinem Nse4. Nse1 s Nse4 interaguje přes C-konec. Zřejmě je tomu tak i v subkomplexu Nse1/3/4, kdy k interakci celeho Nse1 s Nse4 dochazi pouze za přitomnosti proteinu Nse3. Nse3 s těmito dvěma podjednotkami interaguje a tim měni jejich konformaci a tak umožňuje jejich přime spojeni. Při analýze N- a C-koncove casti Nse1 byla dale in vivo potvrzena interakce jeho N- konce s proteinem Nse3. Hloubkovou analýzou MAGE homologni domeny proteinu Nse3 (S. pombe) byly pomoci cilene mutageneze evolucně konzervovaných aminokyselinových zbytků a kvasinkoveho dvouhybridniho testu identifikovany aminokyseliny, ktere specificky interaguji s proteinem Nse4. Take byly identifikovany aminokyseliny, ktere se nachazeji uvnitř tohoto proteinu a tim jsou důležite pro udrženi jeho spravne konformace. Tyto výsledky koreluji s předpovězeným modelem tercialni struktury proteinu Nse3, který byl navržen na zakladě sekvencni podobnosti s již znamou strukturou MAGE homologni domeny proteinu MAGEA4. Vzhledem k tomu, že se v lidskem organizmu nachazi vice než 30 MAGE proteinů, ktere vsechny obsahuji vysoce konzervativni MAGE homologni domenu stejně jako protein Nse3 (S. pombe), bude možne výsledky těchto testů v budoucnu využit při jejich podrobne analýze.
bioRxiv (Cold Spring Harbor Laboratory), Sep 20, 2018
Topoisomerase II (Top2) is an essential enzyme that decatenates DNA via a transient Top2-DNA cova... more Topoisomerase II (Top2) is an essential enzyme that decatenates DNA via a transient Top2-DNA covalent intermediate. This intermediate can be stabilized by a class of drugs termed Top2 poisons, resulting in massive DNA damage. Thus, Top2 activity is a double-edged sword that needs to be carefully controlled to maintain genome stability. We show that Uls1, an adenosine triphosphate (ATP)-dependent chromatin remodelling (Snf2) enzyme, can alter Top2 chromatin binding and prevent Top2 poisoning in yeast. Deletion mutants of ULS1 are hypersensitive to the Top2 poison acriflavine (ACF), activating the DNA damage checkpoint. We map Uls1 s Top2 interaction domain and show that this, together with its ATPase activity, is essential for Uls1 function. By performing ChIP-seq, we show that ACF leads to a general increase in Top2 binding across the genome. We map Uls1 binding sites and identify tRNA genes as key regions where Uls1 associates after ACF treatment. Importantly, the presence of Uls1 at these sites prevents ACF-dependent Top2 accumulation. Our data reveal the effect of Top2 poisons on the global Top2 binding landscape and highlights the role of Uls1 in antagonizing Top2 function. Remodelling Top2 binding is thus an important new means by which Snf2 enzymes promote genome stability.
SMC5-6 is a highly conserved protein complex related to cohesin and condensin complexes which are... more SMC5-6 is a highly conserved protein complex related to cohesin and condensin complexes which are the key components of higher-order chromatin structures. The SMC5-6 complex is essential for proliferation in yeast and is involved in the homologous recombination-based DNA repair processes, including repair of DNA double strand breaks, restart of stalled replication forks etc. However, the precise mechanism of SMC5-6 function is not known. We will present the evidence for direct physical interaction of its part, Nse1-Nse3-Nse4 sub-complex, to DNA and its essential role for the function of the whole SMC5-6 complex. The Nse1-Nse3-Nse4 sub-complex is rich in winged-helix domain motifs and at least one of them form a putative DNA-binding cleft. The purified Nse1-Nse3-Nse4 sub-complexes shift different DNA substrates in electrophoretic mobility shift assays (EMSA) proving their ability to bind DNA in vitro. Mutations of the key basic residues within the putative DNA-binding cleft reduce in vitro binding to DNA. Introduction of these mutations into Schizosaccharomyces pombe genome results in cell death or hypersensitivity to hydroxyurea. The chromatin immuneprecipitation (ChIP) analysis of the DNA-binding mutant shows reduced association of SMC5-6 with chromatin. The above data and our genetic analysis indicate the essential role of the interaction between Nse1-Nse3-Nse4 and DNA for the loading and/or maintenance of the SMC5-6 complex on chromatin.
Journal of Molecular Biology, Jun 1, 2020
A family of Structural Maintenance of Chromosome (SMC) complexes is essential for key cellular pr... more 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.
We described marked structural similarities between prokaryotic SMC/ScpAB, MukBEF and eukaryotic ... more We described marked structural similarities between prokaryotic SMC/ScpAB, MukBEF and eukaryotic SMC5/6 complexes (Palecek and Gruber, Structure, 2015). They posses short kleisin molecules while condensin and cohesin complexes contain long kleisins. All kleisins bind SMC proteins through their conserved N- and C-terminal domains while their different middle regions associate with different types of subunits: short kleisins interact with KITE (Kleisin-Interacting Tandem winged-helix Element) and long klesins interact with HAWK (HEAT Associated With Kleisin) proteins. The presence of KITE proteins in prokaryotic SMC/ScpAB and eukaryotic SMC5/6 complexes suggests their close evolutionary relation and an evolutionary path from prokaryotic to eukaryotic SMC complexes via SMC5/6-like ancestors. Different structural features of the KITE and HAWK proteins suggest different regulation and mechanics of their respective SMC complexes. We will show KITE structural elements and their dynamic properties that might be involved in shaping of the kleisin molecules. We will propose a new KITE role in regulation of prokaryotic SMC/ScpAB and eukaryotic SMC5/6 complexes.
Nucleic Acids Research, Oct 7, 2015
SMC5/6 is a highly conserved protein complex related to cohesin and condensin, which are the key ... more 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.
Structural Maintenance of Chromosomes (SMC) proteins are highly conserved from bacteria to humans... more Structural Maintenance of Chromosomes (SMC) proteins are highly conserved from bacteria to humans and play fundamental roles in chromosome dynamics, gene regulation and DNA repair. SMC1/3 (cohesin) complex is involved in sister chromatid cohesion, SMC2/4 (condensin) contributes to chromosome condensation and SMC5/6 complex is involved in several DNA repair pathways. SMC5/6 heterodimer associates with four non-SMC elements (Nse). The human Nse3 ortholog MAGEG1, founding member of MAGE (Melanoma Antigen Gene) protein family, interacts with both NSE1 (RING-finger containing protein) and NSE4 subunits. The NSE1-MAGEG1-NSE4 subcomplex bridges the head domains of Smc5-Smc6 heterodimer. In our work we have determined interacting domains of Nse3/MAGEG1 and Nse4/EID proteins. The conservation of interacting surface suggests their tight co-evolution. Functional analysis showed involvement of both protein families in transcription regulation and uncovered one possible function of enigmatic MAGE proteins.
Structural Maintenance of Chromosomes (SMC) complexes are involved in a wide range of cellular pr... more Structural Maintenance of Chromosomes (SMC) complexes are involved in a wide range of cellular processes from cell division to gene regulation and DNA repair. In eukaryotes, three separate SMC protein complexes are conserved. Each contains a heterodimeric core of two SMC proteins interconnected by a kleisin subunit, together with from one to five more non-SMC elements (Nse). The complex containing SMC1 and SMC3, named cohesin, is responsible for sister chromatid cohesion during mitosis and meiosis. The condensin complex with SMC2 and SMC4 at its core is required for proper chromosome condensation and segregation during cell division. Both cohesin and condensin are also involved in gene regulation and DNA repair. The less well characterized SMC5/6 complex is involved in several DNA repair pathways. The SMC5/6 complex is composed of two subcomlexes SMC5-SMC6-Nse2 and Nse1-Nse3-Nse4. Nse3 shows sequence similarity to the Melanoma Antigen Gene (MAGE) family of proteins, which are overexpressed in certain types of cancers and whose function has been linked to cell cycle regulation, apoptosis and neuronal development. Using site-directed mutagenesis, protein-protein interaction analyses and modelling of the Schizosaccharomyces pombe Nse3 onto the crystal structure of the MAGEA4 protein, we have identified surface on the C-terminal domain of Nse3 that interacts with Nse4. The binding site for Nse4 is well conserved within the Nse3/MAGE superfamily of proteins. Nse4 is related to the EID (E1A-like inhibitor of differentiation) family of transcriptional repressors. We show that there is a relatively promiscuous interaction between members of the MAGE family and those of the EID family with some specificity. Our data suggest that the Nse3/MAGE-Nse4/EID interaction is evolutionarily conserved.
Current Opinion in Structural Biology
Bioscience Reports
To pass on genetic information to the next generation, cells must faithfully replicate their geno... more To pass on genetic information to the next generation, cells must faithfully replicate their genomes to provide copies for each daughter cell. To synthesise these duplicates, cells employ specialised enzymes called DNA polymerases, which rapidly and accurately replicate nucleic acid polymers. However, most polymerases lack the ability to directly initiate DNA synthesis and required specialised replicases called primases to make short polynucleotide primers, from which they then extend. Replicative primases (eukaryotes and archaea) belong to a functionally diverse enzyme superfamily known as Primase-Polymerases (Prim-Pols), with orthologues present throughout all domains of life. Characterised by a conserved catalytic Prim-Pol domain, these enzymes have evolved various roles in DNA metabolism, including DNA replication, repair, and damage tolerance. Many of these biological roles are fundamentally underpinned by the ability of Prim-Pols to generate primers de novo. This review examin...
Nucleic Acids Research
The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that g... more The discovery of reverse transcriptases (RTs) challenged the central dogma by establishing that genetic information can also flow from RNA to DNA. Although they act as DNA polymerases, RTs are distantly related to replicases that also possess de novo primase activity. Here we identify that CRISPR associated RTs (CARTs) directly prime DNA synthesis on both RNA and DNA. We demonstrate that RT-dependent priming is utilized by some CRISPR-Cas complexes to synthesise new spacers and integrate these into CRISPR arrays. Expanding our analyses, we show that primer synthesis activity is conserved in representatives of other major RT classes, including group II intron RT, telomerase and retroviruses. Together, these findings establish a conserved innate ability of RTs to catalyse de novo DNA primer synthesis, independently of accessory domains or alternative priming mechanisms, which likely plays important roles in a wide variety of biological pathways.
<p>Homology modelling was used to generate the predicted MAGEC2 (aa133-336) and EID2 (<s... more <p>Homology modelling was used to generate the predicted MAGEC2 (aa133-336) and EID2 (<sup>197</sup>QRNPHRVDLDILTFTIALTASEVINPLIEE<sup>226</sup>) structure. (<b>A.</b>) Ribbon representation of the predicted MAGEC2 3D structure model (blue; helices indicated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone.0035813-Hudson1" target="_blank">[9]</a>). (<b>B.</b>) MAGEC2 surface view (blue) with docked EID2 peptide (yellow; ribbon representation). (<b>C.</b>) Stereoscopic detailed view of the MAGEC2 pocket with bound EID2 peptide. The EID2 residues involved in the binding to MAGEC2 are indicated in red (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone-0035813-g005" target="_blank">Fig. 5A</a>). The central EID2 amino acid residues (L205, D206, I207, L208, F210, I212 and L214) are in physical contact with the MAGEC2 pocket surface (formed by H4, H5 and H8). The N-terminus of the Nse3/MAGE-binding domain makes contact through the essential residues R198 and R202 (only R198 is labeled) to the MAGEC2 loop region (between H5 and H6). The D204 residue protruding to the solvent is black labeled.</p
<p>(<b>A.</b>) The GST-His-S-NSE4b fragments spanning amino acids 106 to 135 (l... more <p>(<b>A.</b>) The GST-His-S-NSE4b fragments spanning amino acids 106 to 135 (lanes 1–3) and/or 113 to 135 (lanes 4–6) were pre-incubated with S-protein agarose beads and then incubated with <i>in vitro</i> translated full-length hNSE3/MAGEG1 protein (lanes 1–9). The amount of the GST-His-S-tagged protein was analyzed by immunoblotting with anti-His antibody and the <i>in vitro</i> translated proteins were measured by autoradiography. Control, GST-His-S-protein present (lanes 7–9). (<b>B.</b>) Yeast-2-hybrid analysis of the interaction between the indicated mutants of NSE4b (aa 1 to 333) and hNSE3/MAGEG1 (aa 55 to 292). Interactions result in growth on -Leu, -Trp, -His plates+5 mM AT. Specificity of the NSE4b mutants was checked against the hSMC5 (aa 4 to 1101) construct (interactions result in growth on -Leu, -Trp, -His plates). Control, plate without Leu and Trp. (<b>C.</b>) Co-immunoprecipitation from HEK293T cells co-transfected with S-tagged wild-type (wt) and/or mutant (L, L267A) hNSE3/MAGEG1 construct and with FLAG-tagged wild-type (wt) and/or mutant (FF, F114A+F124A) NSE4b/EID3 construct. Lysates were immunoprecipitated with S-protein agarose beads and immunoblotted with either S-HRP (top) or anti-FLAG (bottom). The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis.</p
<p>(<b>A.</b>) Quantification of relative binding of the MAGEC2(129-339) protei... more <p>(<b>A.</b>) Quantification of relative binding of the MAGEC2(129-339) protein (red columns) to the EID2 protein-based synthetic mutant peptides (listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone-0035813-t001" target="_blank">Table 1</a>) using the PEPSCAN-ELISA method. Results show mean ± SEM of 3 independent measurements. His-hTRF2 protein (white column) was used in the control experiment. (<b>B. to D.</b>) The short (biotin-SGSG-<sup>201</sup>HRVDLDILTFTIALTAS<sup>217</sup>) and long (biotin-SGSG-<sup>197</sup>QRNPHRVDLDILTFTIALTAS<sup>217</sup>) EID2 peptides were pre-bound to the streptavidin-agarose beads and then incubated with <i>in vitro</i> translated MAGEC2 (aa 6-373; C2 in panel <b>B.</b>), MAGEA1 (aa 1-309; A1 in panel <b>C.</b>) and/or necdin (aa 1-321; nd in panel <b>D.</b>) protein, respectively. (<b>E.</b>) Wild type and selected EID2 mutant peptides (as indicated) were pre-bound to the streptavidin-agarose beads and then incubated with <i>in vitro</i> translated necdin protein. The reaction mixtures were analyzed by 15% SDS–PAGE gel electrophoresis. The amount of the <i>in vitro</i> translated proteins was measured by autoradiography. Control, no peptide.</p
<p>(<b>A.</b>) Alignments of the five members of the human NSE4/EID family: EID... more <p>(<b>A.</b>) Alignments of the five members of the human NSE4/EID family: EID1, EID2, EID2b, EID3/NSE4b and NSE4a. The red box indicates Nse3/MAGE-binding domain; hatched and crosshatched boxes indicate kleisin and kleisin-like motifs, respectively (based on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone-0035813-g001" target="_blank">Fig. 1B and C</a>; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035813#pone.0035813-Palecek1" target="_blank">[10]</a>); grey boxes indicate regions of homology between EID1 and 2, respectively. (<b>B. and C.</b>) GST-His-S-tagged fragments of each of the NSE4/EID family members were pre-bound to S-protein agarose beads and incubated with <i>in vitro</i> translated MAGEA1 (aa 1-309; panel <b>B</b>) and necdin (aa 1-321; panel <b>C</b>). The indicated NSE4/EID proteins correspond to the MAGE-interacting domain of EID1(aa146-177), EID2(aa197-225), EID2b(aa135-161), NSE4b/EID3(aa106-135) and NSE4a(aa150-179). In lanes 8-12, immunoblotting of bound fragments are shown in the upper panel and bound radioactive MAGE proteins (MAGEA1 and necdin) in the lower panel. Control, GST-His-S protein alone.</p
We described marked structural similarities between prokaryotic SMC/ScpAB, MukBEF and eukaryotic ... more We described marked structural similarities between prokaryotic SMC/ScpAB, MukBEF and eukaryotic SMC5/6 complexes (Palecek and Gruber, Structure, 2015). They posses short kleisin molecules while condensin and cohesin complexes contain long kleisins. All kleisins bind SMC proteins through their conserved N- and C-terminal domains while their different middle regions associate with different types of subunits: short kleisins interact with KITE (Kleisin-Interacting Tandem winged-helix Element) and long klesins interact with HAWK (HEAT Associated With Kleisin) proteins. The presence of KITE proteins in prokaryotic SMC/ScpAB and eukaryotic SMC5/6 complexes suggests their close evolutionary relation and an evolutionary path from prokaryotic to eukaryotic SMC complexes via SMC5/6-like ancestors. Different structural features of the KITE and HAWK proteins suggest different regulation and mechanics of their respective SMC complexes. We will show KITE structural elements and their dynamic pro...
Uploads
Papers by Katerina Bednarova