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Functional Characterization of the DNA Polymerase Epsilon and Its Involvement in the Maintenance of Genome Integrity in Arabidopsis Jose Antonio Pedroza-Garcia

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Functional Characterization of the DNA Polymerase Epsilon and Its Involvement in the Maintenance of Genome Integrity in Arabidopsis Jose Antonio Pedroza-Garcia Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis Jose Antonio Pedroza-Garcia To cite this version: Jose Antonio Pedroza-Garcia. Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis. Plants genetics. Université Paris Saclay (COmUE), 2016. English. NNT : 2016SACLS248. tel-03092326 HAL Id: tel-03092326 https://tel.archives-ouvertes.fr/tel-03092326 Submitted on 2 Jan 2021 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. NNT : 2016SACLS248 THESE DE DOCTORAT DE L’UNIVERSITE PARIS-SACLAY, préparée à l’Université Paris-Sud ÉCOLE DOCTORALE N° 567 Sciences du Végétal : du Gène à l’Ecosystème Spécialité de doctorat: Biologie Par M. José Antonio Pedroza-Garcia Functional characterization of the DNA Polymerase epsilon and its involvement in the maintenance of genome integrity in Arabidopsis Thèse présentée et soutenue à Orsay, le 22 septembre 2016 : Composition du Jury : M. Frugier, Florian Directeur de Recherche, CNRS Président Mme Gallego, Maria Professeure, Université Blaise Pascal Rapporteur M. Bendahmane, Mohammed Directeur de Recherche, INRA Rapporteur Mme Mézard, Christine Directrice de Recherche, CNRS Examinatrice Mme Chabouté, Marie-Edith Directrice de Recherche, CNRS Examinatrice Mme Raynaud, Cécile Chargée de Recherche, CNRS Directrice de thèse Acknowledgments First, I would like to express my huge gratitude to my supervisor, Cécile Raynaud. I thank you for the opportunity that you gave me to be part of your research group and your help every day. I learned several things of your research topic, now I love more the science than before. I will miss the nice work discussion that we usually had, and all the feedback that you gave me. I will never forget all the help that you gave me when I was new in your beautiful country. My gratitude also goes to Moussa for believing in me at beginning and during my PhD. I am thankful to Christelle, for helping in cloning and genotyping. Your excellent work contributed to completion of my thesis. Overall, thank you for your friendship; I will miss the “cantine” with you, and our “Frenchglish” that we used to understand us. I would like to thank you to Jeannine for your help sowing and harvesting plants I thank members of my thesis committee: Lieven De Veylder, Patricia Kannouche and Federico Ariel for helpful discussions about this work. I also thank all jury members for your time to participate in the defense of my thesis. I would like to thank to other former and current labmates (Catherine, Marianne, Quentin, Séverine, Elodie, Teddy, Charley, Anaïs, Juan, and Natalia) for making a pleasant working environment. Finally yet importantly, I would like to take the opportunity to thank my parents, brothers and friends for continuous encouragement, especially to Fernando for the helping and supporting during all these years. Gracias a todos mis amigos en México por darme su soporte constantemente especialmente Paulina, Manu y Enrique. Résumé de la thèse en Français : Contrairement aux animaux, les plantes ont un développement largement post-embryonnaire et forment continuellement de nouveaux organes et tissus grâce à l’activité de leurs méristèmes. Ces massifs de cellules indifférenciées conservent la capacité à se diviser tout au long de la vie de la plante, et c’est également à partir du méristème caulinaire que se forment les gamètes. Chaque cycle de division peut être la source de mutations, suite par exemple à des erreurs de réplication. De plus, les méristèmes sont relativement exposés aux stress environnementaux qui peuvent également endommager l’ADN des cellules. Les mécanismes impliqués dans la détection des lésions de l’ADN ou des défauts de réplication et l’arrêt de la prolifération cellulaire en réponse à ces dommages jouent donc un rôle fondamental dans le maintien de la stabilité du génome, aussi bien au cours du développement végétatif que lors de la reproduction sexuée. Le développement des plantes repose sur l’activité de cellules souches présentes au sein des méristèmes qui conservent leur capacité proliférative tout au long de leur vie. De plus, contrairement à ce qui est observé chez les Animaux, la lignée germinale n’est pas individualisée à une étape précoce du développement mais se forme à partir des méristèmes de manière relativement tardive. Ce mode de développement pose la question des mécanismes particuliers qui assurent le maintien de l’intégrité du génome au fil des divisions cellulaires chez les plantes. En effet, les différentes étapes du cycle cellulaire sont la source de lésions de l’ADN, par exemple lors d’erreur de réplication, ou de défaut de ségrégation des chromatides sœurs pendant la mitose. L’objectif de ce travail de thèse était donc de mieux comprendre ces processus en utilisant la plante modèle Arabidopsis thaliana, et en étudiant plus particulièrement les mécanismes mis en jeu pendant la phase S (au cours de laquelle a lieu la réplication de l’ADN) pour assurer la duplication fidèle de l’information génétique. L’une des protéines clés impliquées dans la réplication de l’ADN nucléaire chez tous les eucaryotes est l’ADN Polymérase ε (Pol ε) : elle assure la synthèse du brin précoce pendant la réplication, mais est également impliquée dans la perception du stress réplicatif et l’activation de la réponse cellulaire. Elle est constituée de quatre sous-unités conservées chez tous les eucaryotes: une sous-unité catalytique (Pol2A) et trois sous-unités accessoires (DPB2, 3 et 4). L’étude détaillée de sa fonction est cependant rendue difficile chez beaucoup d’organismes par le fait que son inactivation est létale. Dans ce travail, nous avons utilisé des approches de génétique pour étudier le rôle de l’ADN Pol d’Arabidopsis au cours de la progression du cycle cellulaire et dans la réponse au stress réplicatif et aux lésions de l’ADN Au cours de ce travail de thèse nous avons caractérisé la fonction des sous-unités POL2A and DPB2 et leur interaction avec les voies de réponses aux lésions de l’ADN. Les gènes POL2A et DPB2 sont des gènes essentiels, mais des mutants faibles pour la sous-unité POL2A existent. Afin de caractériser ces protéines, nous avons généré des sur-expresseurs de la protéine DPB2 et comparé leurs phénotypes à celui des mutants partiellement déficients pour la sous-unité catalytique. Les sur-expresseurs de DPB2 présentent une forte réduction de croissance, un cycle cellulaire et notamment une phase S très allongée et une activation constitutive des gènes de réponse aux lésions de l’ADN. Des analyses génétiques nous ont permis d’établir que cette activation est largement dépendante de la protéine kinase ATR qui est connue pour son rôle dans signalisation activée par le stress réplicatif. Cependant, les sur-expresseurs de DPB2 présentent également une formation spontanée de cassures double-brin dans les cellules des méristèmes, ce qui active l’activation de la kinase ATM, spécialisée dans la détection de ce type de lésions. L’activation de cette voie confère aux plantes une tolérance accrue aux dommages de l’ADN. Notre modèle de travail est que la suraccumulation de DPB2 déstabilise le réplisome (structure multiprotéique qui assure la réplication de l’ADN), ce qui conduirait à des défauts de progression de la fourche de réplication et à une activation de la réponse au stress réplicatif. Les mutants partiellement déficients pour la sous-unité catalytique POL2A présentent des défauts similaires aux sur-expresseurs de DPB2 (croissance réduite, activation de la réponse aux lésions de l’ADN, tolérance au stress réplicatif). La viabilité de ces mutants dépend strictement de la kinase ATR. Cependant ces plantes sont hypersensibles aux agents induisant des cassures double-brin de l’ADN, et notre analyse génétique révèle que ce phénotype est probablement induit par une interférence entre les voies de signalisation ATR et ATM dépendantes. Enfin, en utilisant une approche de RNAi, nous avons pu montrer que la sous-unité POL2A elle-même est nécessaire à la perception du stress réplicatif. Nous avons ainsi pu montrer que la sous-unité catalytique du complexe Pol ainsi que sa principale sous-unité accessoire DPB2 sont essentielles à la détection des défauts de réplication, et fonctionnent en amont de la kinase ATR pour induire l’arrêt du cycle cellulaire et activer les voies de réparation au cours du développement végétatif. En outre, nous avons découvert un nouveau point de contrôle activé lors de la phase de réplication pré-méiotique qui permet l’activation d’une mort cellulaire programmée en réponse à des défauts survenus pendant cette phase, grâce au facteur de transcription SOG1. Enfin, ces travaux ont mis en évidence l’existence d’un nouveau point de contrôle activé par le stress réplicatif, dépendant de la Pol ε et médié par le facteur de transcription SOG1, un intégrateur central de la réponse aux lésions de l’ADN. L’ensemble de ces résultats fait l’objet d’une publication parue dans la revue Nucleic Acids Research, et d’une seconde soumise pour publication. Tous les stress biotiques ou abiotiques auxquels la plante est soumise pouvant conduire à la formation de lésions au niveau de l’ADN, nos résultats ouvrent des perspectives de recherche pour comprendre la réponse des plantes aux stress environnementaux. En outre, la disponibilité de mutants viables pour différents facteurs impliqués dans la réplication ou la réponse aux lésions de l’ADN nous a permis d’explorer chez un eucaryote pluricellulaire des mécanismes qui sont pour l’instant essentiellement décrits chez la levure, et ainsi d’acquérir des connaissances qui pourront être transférées aux systèmes animaux et notamment à l’Homme.
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