Prot-Ex
Publications
3888256
PROTEX
chicago-author-date
50
date
desc
year
45649
https://www.i2bc.paris-saclay.fr/wp-content/plugins/zotpress/
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Bischerour, Julien, Olivier Arnaiz, Coralie Zangarelli, Vinciane Régnier, Florence Iehl, Virginie Ropars, Jean-Baptiste Charbonnier, and Mireille Bétermier. 2024. “Uncoupling Programmed DNA Cleavage and Repair Scrambles the Paramecium Somatic Genome.” Cell Reports 43 (4): 114001. https://doi.org/10.1016/j.celrep.2024.114001.
Babot, Marion, Yves Boulard, Samira Agouda, Laura Pieri, Sonia Fieulaine, Stéphane Bressanelli, and Virginie Gervais. 2024. “Oligomeric Assembly of the C-Terminal and Transmembrane Region of SARS-CoV-2 Nsp3.” Journal of Virology, March, e0157523. https://doi.org/10.1128/jvi.01575-23.
Chades, Théo, Raphaël Le Fèvre, Imène Chebbi, Karine Blondeau, François Guyot, and Edouard Alphandéry. 2024. “Set-up of a Pharmaceutical Cell Bank of Magnetospirillum Gryphiswaldense MSR1 Magnetotactic Bacteria Producing Highly Pure Magnetosomes.” Microbial Cell Factories 23 (1): 70. https://doi.org/10.1186/s12934-024-02313-4.
Ouasti, Fouad, Maxime Audin, Karine Fréon, Jean-Pierre Quivy, Mehdi Tachekort, Elizabeth Cesard, Aurélien Thureau, et al. 2024. “Disordered Regions and Folded Modules in CAF-1 Promote Histone Deposition in Schizosaccharomyces Pombe.” ELife 12 (February):RP91461. https://doi.org/10.7554/eLife.91461.
Kefala Stavridi, Antonia, Amandine Gontier, Vincent Morin, Philippe Frit, Virginie Ropars, Nadia Barboule, Carine Racca, et al. 2023. “Structural and Functional Basis of Inositol Hexaphosphate Stimulation of NHEJ through Stabilization of Ku-XLF Interaction.” Nucleic Acids Research, October, gkad863. https://doi.org/10.1093/nar/gkad863.
Seif-El-Dahan, Murielle, Antonia Kefala-Stavridi, Philippe Frit, Steven W. Hardwick, Dima Y. Chirgadze, Taiana Maia De Oliviera, Sébastien Britton, et al. 2023. “PAXX Binding to the NHEJ Machinery Explains Functional Redundancy with XLF.” Science Advances 9 (22): eadg2834. https://doi.org/10.1126/sciadv.adg2834.
Hardwick, Steven W., Antonia Kefala Stavridi, Dimitri Y. Chirgadze, Taiana Maia De Oliveira, Jean-Baptiste Charbonnier, Virginie Ropars, Katheryn Meek, Tom L. Blundell, and Amanda K. Chaplin. 2023. “Cryo-EM Structure of a DNA-PK Trimer: Higher Order Oligomerisation in NHEJ.” Structure (London, England: 1993), May, S0969-2126(23)00167-3. https://doi.org/10.1016/j.str.2023.05.013.
Audoynaud, Charlotte, Kamila Schirmeisen, Anissia Ait Saada, Armelle Gesnik, Paloma Fernández-Varela, Virginie Boucherit, Virginie Ropars, et al. 2023. “RNA:DNA Hybrids from Okazaki Fragments Contribute to Establish the Ku-Mediated Barrier to Replication-Fork Degradation.” Molecular Cell, February, S1097-2765(23)00106-5. https://doi.org/10.1016/j.molcel.2023.02.008.
Dieudonné, Thibaud, Christine Jaxel, Maylis Lejeune, Guillaume Lenoir, and Cédric Montigny. 2023. “Expression in Saccharomyces Cerevisiae and Purification of a Human Phospholipid Flippase.” Methods in Molecular Biology (Clifton, N.J.) 2652:231–46. https://doi.org/10.1007/978-1-0716-3147-8_13.
Lazar, Noureddine, Carl H. Mesarich, Yohann Petit-Houdenot, Nacera Talbi, Ines Li de la Sierra-Gallay, Emilie Zélie, Karine Blondeau, et al. 2022. “A New Family of Structurally Conserved Fungal Effectors Displays Epistatic Interactions with Plant Resistance Proteins.” PLOS Pathogens 18 (7): e1010664. https://doi.org/10.1371/journal.ppat.1010664.
Habersetzer, Johann, Mohamed Debbah, Marie-Laure Fogeron, Anja Böckmann, Stéphane Bressanelli, and Sonia Fieulaine. 2020. “In Vitro Translation of Virally-Encoded Replication Polyproteins to Recapitulate Polyprotein Maturation Processes.” Protein Expression and Purification 175 (November):105694. https://doi.org/10.1016/j.pep.2020.105694.
Azouaoui, Hassina, Cédric Montigny, Aurore Jacquot, Raphaëlle Barry, Philippe Champeil, and Guillaume Lenoir. 2016. “Coordinated Overexpression in Yeast of a P4-ATPase and Its Associated Cdc50 Subunit: The Case of the Drs2p/Cdc50p Lipid Flippase Complex.” Methods in Molecular Biology (Clifton, N.J.) 1377:37–55. https://doi.org/10.1007/978-1-4939-3179-8_6.