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8 záznamů v Medvik Filtry

Článek

Termination of non-coding transcription in yeast relies on both an RNA Pol II CTD interaction domain and a CTD-mimicking region in Sen1

Han, Zhong
Autor Han, Zhong Université de Paris, CNRS, Institut Jacques Monod, Paris, France. Université Paris-Saclay, Yvette, France
Jasnovidova, Olga
Autor Jasnovidova, Olga CEITEC-Central European Institute of Technology, Masaryk University, Brno, Czechia
Haidara, Nouhou
Autor Haidara, Nouhou Université de Paris, CNRS, Institut Jacques Monod, Paris, France. Université Paris-Saclay, Yvette, France
Tudek, Agnieszka
Autor Tudek, Agnieszka Université de Paris, CNRS, Institut Jacques Monod, Paris, France
Kubicek, Karel
Autor Kubicek, Karel CEITEC-Central European Institute of Technology, Masaryk University, Brno, Czechia
Libri, Domenico
Autor Libri, Domenico Université de Paris, CNRS, Institut Jacques Monod, Paris, France
Stefl, Richard
Autor Stefl, Richard CEITEC-Central European Institute of Technology, Masaryk University, Brno, Czechia
Porrua, Odil
Autor Porrua, Odil Université de Paris, CNRS, Institut Jacques Monod, Paris, France

EMBO journal. 2020 ; 39 (7) : e101548. [pub] 20200228

EMBO J
ISSN 1460-2075
Medvik
Zdroj

Pervasive transcription is a widespread phenomenon leading to the production of a plethora of non-coding RNAs (ncRNAs) without apparent function. Pervasive transcription poses a threat to proper gene expression that needs to be controlled. In yeast, the highly conserved helicase Sen1 restricts pervasive transcription by inducing termination of non-coding transcription. However, the mechanisms underlying the specific function of Sen1 at ncRNAs are poorly understood. Here, we identify a motif in an intrinsically disordered region of Sen1 that mimics the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II, and structurally characterize its recognition by the CTD-interacting domain of Nrd1, an RNA-binding protein that binds specific sequences in ncRNAs. In addition, we show that Sen1-dependent termination strictly requires CTD recognition by the N-terminal domain of Sen1. We provide evidence that the Sen1-CTD interaction does not promote initial Sen1 recruitment, but rather enhances Sen1 capacity to induce the release of paused RNAPII from the DNA. Our results shed light on the network of protein-protein interactions that control termination of non-coding transcription by Sen1.

Článek

An inter-subunit protein-peptide interface that stabilizes the specific activity and oligomerization of the AAA+ chaperone Reptin

Journal of proteomics. 2019 ; 199 (-) : 89-101. [pub] 20190309

J Proteomics
ISSN 1876-7737
Medvik
Zdroj

Reptin is a member of the AAA+ superfamily whose members can exist in equilibrium between monomeric apo forms and ligand bound hexamers. Inter-subunit protein-protein interfaces that stabilize Reptin in its oligomeric state are not well-defined. A self-peptide binding assay identified a protein-peptide interface mapping to an inter-subunit "rim" of the hexamer bridged by Tyrosine-340. A Y340A mutation reduced ADP-dependent oligomer formation using a gel filtration assay, suggesting that Y340 forms a dominant oligomer stabilizing side chain. The monomeric ReptinY340A mutant protein exhibited increased activity to its partner protein AGR2 in an ELISA assay, further suggesting that hexamer formation can preclude certain protein interactions. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) demonstrated that the Y340A mutation attenuated deuterium suppression of Reptin in this motif in the presence of ligand. By contrast, the tyrosine motif of Reptin interacts with a shallower pocket in the hetero-oligomeric structure containing Pontin and HDX-MS revealed no obvious role of the Y340 side chain in stabilizing the Reptin-Pontin oligomer. Molecular dynamic simulations (MDS) rationalized how the Y340A mutation impacts upon a normally stabilizing inter-subunit amino acid contact. MDS also revealed how the D299N mutation can, by contrast, remove oligomer de-stabilizing contacts. These data suggest that the Reptin interactome can be regulated by a ligand dependent equilibrium between monomeric and hexameric forms through a hydrophobic inter-subunit protein-protein interaction motif bridged by Tyrosine-340. SIGNIFICANCE: Discovering dynamic protein-protein interactions is a fundamental aim of research in the life sciences. An emerging view of protein-protein interactions in higher eukaryotes is that they are driven by small linear polypeptide sequences; the linear motif. We report on the use of linear-peptide motif screens to discover a relatively high affinity peptide-protein interaction for the AAA+ and pro-oncogenic protein Reptin. This peptide interaction site was shown to form a 'hot-spot' protein-protein interaction site, and validated to be important for ligand-induced oligomerization of the Reptin protein. These biochemical data provide a foundation to understand how single point mutations in Reptin can impact on its oligomerization and protein-protein interaction landscape.

MeSH
AAA doména * MeSH
ATPázy spojené s různými buněčnými aktivitami chemie metabolismus MeSH
DNA-helikasy chemie metabolismus MeSH
interakční proteinové domény a motivy fyziologie MeSH
lidé MeSH
molekulární chaperony chemie metabolismus MeSH
multimerizace proteinu * MeSH
mutace MeSH
simulace molekulární dynamiky MeSH
transportní proteiny chemie metabolismus MeSH
tyrosin genetika MeSH
Check Tag
lidé MeSH
Publikační typ
časopisecké články MeSH
práce podpořená grantem MeSH

Článek

A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I

Journal of molecular modeling. 2018 ; 24 (7) : 176. [pub] 20180626

J Mol Model
ISSN 0948-5023
Medvik
Zdroj

Type I restriction-modification enzymes differ significantly from the type II enzymes commonly used as molecular biology reagents. On hemi-methylated DNAs type I enzymes like the EcoR124I restriction-modification complex act as conventional adenine methylases at their specific target sequences, but unmethylated targets induce them to translocate thousands of base pairs through the stationary enzyme before cleaving distant sites nonspecifically. EcoR124I is a superfamily 2 DEAD-box helicase like eukaryotic double-strand DNA translocase Rad54, with two RecA-like helicase domains and seven characteristic sequence motifs that are implicated in translocation. In Rad54 a so-called extended region adjacent to motif III is involved in ATPase activity. Although the EcoR124I extended region bears sequence and structural similarities with Rad54, it does not influence ATPase or restriction activity as shown in this work, but mutagenesis of the conserved glycine residue of its motif III does alter ATPase and DNA cleavage activity. Through the lens of molecular dynamics, a full model of HsdR of EcoR124I based on available crystal structures allowed interpretation of functional effects of mutants in motif III and its extended region. The results indicate that the conserved glycine residue of motif III has a role in positioning the two helicase domains.

Článek

Mass spectrometry analysis of the oxidation states of the pro-oncogenic protein anterior gradient-2 reveals covalent dimerization via an intermolecular disulphide bond

Biochimica et biophysica acta. 2016 ; 1864 (5) : 551-61. [pub] 20160210

Biochim Biophys Acta
ISSN 0006-3002
Medvik
Zdroj

Anterior Gradient-2 (AGR2) is a component of a pro-oncogenic signalling pathway that can promote p53 inhibition, metastatic cell migration, limb regeneration, and cancer drug-resistance. AGR2 is in the protein-disulphide isomerase superfamily containing a single cysteine (Cys-81) that forms covalent adducts with its client proteins. We have found that mutation of Cysteine-81 attenuates its biochemical activity in its sequence-specific peptide docking function, reduces binding to Reptin, and reduces its stability in cells. As such, we evaluated how chemical oxidation of its cysteine affects its biochemical properties. Recombinant AGR2 spontaneously forms covalent dimers in the absence of reductant whilst DTT promotes dimer to monomer conversion. Mutation of Cysteine-81 to alanine prevents peroxide catalysed dimerization of AGR2 in vitro, suggesting a reactive cysteine is central to covalent dimer formation. Both biochemical assays and ESI mass spectrometry were used to demonstrate that low levels of a chemical oxidant promote an intermolecular disulphide bond through formation of a labile sulfenic acid intermediate. However, higher levels of oxidant promote sulfinic or sulfonic acid formation thus preventing covalent dimerization of AGR2. These data together identify the single cysteine of AGR2 as an oxidant responsive moiety that regulates its propensity for oxidation and its monomeric-dimeric state. This has implications for redox regulation of the pro-oncogenic functions of AGR2 protein in cancer cells.

MeSH
chemorezistence genetika MeSH
cystein genetika metabolismus MeSH
disulfidy chemie metabolismus MeSH
DNA-helikasy chemie genetika metabolismus MeSH
hmotnostní spektrometrie MeSH
kyseliny sulfenové metabolismus MeSH
lidé MeSH
MFC-7 buňky MeSH
multimerizace proteinu genetika MeSH
mutace MeSH
nádory chemie genetika patologie MeSH
oxidace-redukce * MeSH
proteiny chemie genetika metabolismus MeSH
sekvence aminokyselin genetika MeSH
signální transdukce MeSH
transportní proteiny chemie genetika metabolismus MeSH
Check Tag
lidé MeSH
Publikační typ
časopisecké články MeSH
práce podpořená grantem MeSH

Článek

The PCNA interaction protein box sequence in Rad54 is an integral part of its ATPase domain and is required for efficient DNA repair and recombination

PLoS One. 2013 ; 8 (12) : e82630.

ISSN 1932-6203
Medvik
Zdroj

Rad54 is an ATP-driven translocase involved in the genome maintenance pathway of homologous recombination (HR). Although its activity has been implicated in several steps of HR, its exact role(s) at each step are still not fully understood. We have identified a new interaction between Rad54 and the replicative DNA clamp, proliferating cell nuclear antigen (PCNA). This interaction was only mildly weakened by the mutation of two key hydrophobic residues in the highly-conserved PCNA interaction motif (PIP-box) of Rad54 (Rad54-AA). Intriguingly, the rad54-AA mutant cells displayed sensitivity to DNA damage and showed HR defects similar to the null mutant, despite retaining its ability to interact with HR proteins and to be recruited to HR foci in vivo. We therefore surmised that the PCNA interaction might be impaired in vivo and was unable to promote repair synthesis during HR. Indeed, the Rad54-AA mutant was defective in primer extension at the MAT locus as well as in vitro, but additional biochemical analysis revealed that this mutant also had diminished ATPase activity and an inability to promote D-loop formation. Further mutational analysis of the putative PIP-box uncovered that other phenotypically relevant mutants in this domain also resulted in a loss of ATPase activity. Therefore, we have found that although Rad54 interacts with PCNA, the PIP-box motif likely plays only a minor role in stabilizing the PCNA interaction, and rather, this conserved domain is probably an extension of the ATPase domain III.

Článek

Dual roles of the SUMO-interacting motif in the regulation of Srs2 sumoylation

Nucleic acids research. 2012 ; 40 (16) : 7831-43.

Nucleic Acids Res
ISSN 1362-4962
Medvik
Zdroj

The Srs2 DNA helicase of Saccharomyces cerevisiae affects recombination in multiple ways. Srs2 not only inhibits recombination at stalled replication forks but also promotes the synthesis-dependent strand annealing (SDSA) pathway of recombination. Both functions of Srs2 are regulated by sumoylation--sumoylated PCNA recruits Srs2 to the replication fork to disfavor recombination, and sumoylation of Srs2 can be inhibitory to SDSA in certain backgrounds. To understand Srs2 function, we characterize the mechanism of its sumoylation in vitro and in vivo. Our data show that Srs2 is sumoylated at three lysines, and its sumoylation is facilitated by the Siz SUMO ligases. We also show that Srs2 binds to SUMO via a C-terminal SUMO-interacting motif (SIM). The SIM region is required for Srs2 sumoylation, likely by binding to SUMO-charged Ubc9. Srs2's SIM also cooperates with an adjacent PCNA-specific interaction site in binding to sumoylated PCNA to ensure the specificity of the interaction. These two functions of Srs2's SIM exhibit a competitive relationship: sumoylation of Srs2 decreases the interaction between the SIM and SUMO-PCNA, and the SUMO-PCNA-SIM interaction disfavors Srs2 sumoylation. Our findings suggest a potential mechanism for the equilibrium of sumoylated and PCNA-bound pools of Srs2 in cells.

Článek

Srs2: the "Odd-Job Man" in DNA repair

DNA repair. 2010 ; 9 (3) : 268-275. [pub] 20100121

DNA Repair (Amst)
ISSN 1568-7856
Medvik
Zdroj

Homologous recombination plays a key role in the maintenance of genome integrity, especially during DNA replication and the repair of double-stranded DNA breaks (DSBs). Just a single un-repaired break can lead to aneuploidy, genetic aberrations or cell death. DSBs are caused by a vast number of both endogenous and exogenous agents including genotoxic chemicals or ionizing radiation, as well as through replication of a damaged template DNA or the replication fork collapse. It is essential for cell survival to recognise and process DSBs as well as other toxic intermediates and launch most appropriate repair mechanism. Many helicases have been implicated to play role in these processes, however their detail roles, specificities and co-operativity in the complex protein-protein interaction networks remain unclear. In this review we summarize the current knowledge about Saccharomyces cerevisiae helicase Srs2 and its effect on multiple DNA metabolic processes that generally affect genome stability. It would appear that Srs2 functions as an "Odd-Job Man" in these processes to make sure that the jobs proceed when and where they are needed.

MeSH
DNA fungální metabolismus MeSH
DNA-helikasy chemie metabolismus MeSH
lidé MeSH
nestabilita genomu MeSH
oprava DNA MeSH
replikace DNA MeSH
Saccharomyces cerevisiae - proteiny chemie metabolismus MeSH
Saccharomyces cerevisiae enzymologie MeSH
Check Tag
lidé MeSH
Publikační typ
časopisecké články MeSH
práce podpořená grantem MeSH
přehledy MeSH

Článek

The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I

Journal of Molecular Biology. 2008 ; 384 (5) : 1273-1286.

J Mol Biol
Medvik
Zdroj

The type I restriction-modification enzyme EcoR124I comprises three subunits with the stoichiometry HsdR2/HsdM2/HsdS1. The HsdR subunits are archetypical examples of the fusion between nuclease and helicase domains into a single polypeptide, a linkage that is found in a great many other DNA processing enzymes. To explore the interrelationship between these physically linked domains, we examined the DNA translocation properties of EcoR124I complexes in which the HsdR subunits had been mutated in the RecB-like nuclease motif II or III. We found that nuclease mutations can have multiple effects on DNA translocation despite being distinct from the helicase domain. In addition to reductions in DNA cleavage activity, we also observed decreased translocation and ATPase rates, different enzyme populations with different characteristic translocation rates, a tendency to stall during initiation and altered HsdR turnover dynamics. The significance of these observations to our understanding of domain interactions in molecular machines is discussed.

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