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Thesis Reference Thesis Telomere length homeostasis in the yeast "Saccharomyces cerevisiae" ARNERIC, Milica Abstract La télomérase compense la perte d'ADN télomérique causée par le problème de réplication des extrémités. Nous avons développé un système pour analyser l'élongation d'un télomère dans "Saccharomyces Cerevisae" et nous avons montré que la télomérase permet l'homéostasie de la taille des télomères en allongeant de manière préférentielle les télomères courts. Cette régulation implique différentes protéines dont Rap1-Rif1-Rif2 qui contrôlent de manière négative la longueur des télomères ou les kinases Tel1 et Mec1 qui sont des régulateurs positifs. Nous avons montré que dans les cellules "tel1[delta]", sur les extrémités des chromosomes sauvages, la fréquence d'élongation décroît considérablement, ce qui explique leur phénotype de télomères courts. Nous avons aussi montré que les régions subtélomériques sont impliquées dans la régulation de l'élongation des télomères très courts. Finalement, nous avons confirmé que la télomérase n'est pas processive en terme d'addition de répétitions télomériques, sauf pour les télomères très courts par un processus dépendant de [...] Reference ARNERIC, Milica. Telomere length homeostasis in the yeast "Saccharomyces cerevisiae". Thèse de doctorat : Univ. Genève, 2007, no. Sc. 3939 URN : urn:nbn:ch:unige-5224 DOI : 10.13097/archive-ouverte/unige:522 Available at: http://archive-ouverte.unige.ch/unige:522 Disclaimer: layout of this document may differ from the published version. 1 / 1 UNIVERSITE DE GENEVE Département de Biologie Moléculaire FACULTE DES SCIENCES Professeur David Shore ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE Institute Suisse de Recherche FACULTE DES SCIENCES DE LA VIE Expérimentale sur le Cancer Professeur Joachim Lingner Telomere Length Homeostasis in the Yeast Saccharomyces cerevisiae THESE présentée à la Faculté des sciences de l’Université de Genève pour obtenir la grade de Docteur ès sciences par Milica ARNERIĆ de Split (Croatie) Thèse n° 3939 Lausanne, 2007 Acknowledgments My first big “thank You” goes to Joachim, who made me feel welcome and free during my time in his lab. He was a great support and a calming presence when I was panicking the most. Thanks to Susan and David – my PhD committee, for encouragement and belief in me – which still surprises me sometimes. Since the last few years I found quite challenging in many aspect I felt privileged to have an understanding as well as lively and dynamic lab team to spend most of my days with. I love you ALL! Still, I want to mention a few past-present lab members with whom I shared more intensive moments: ÇIsabel honey - nothing was the same since you left. No one to teach me Swiss customs, no playing elephant, no one to save a woman in danger (me?)… We grew a lot together, and I hope for more to come! ÇNele Gwendolin Ruth - we shared so much…laughs, tears, evil sense of humor, moods, problems, finally even kitchen, bathroom and the living room! And I know there is a lot of fun in front! ÇGaël – you will always be my only lab husband! Thanks for taking all my moods so well; even though you left me - you are the one who was always there when needed! ÇClaus – I’ll never forget crazy nights in Thun! Even though we’ve never organized that Virginia Wolf party, when you are ready just call! I will wear my best Diesel jeans! ÇSophie – for being kind and gentle as you are! Kathi – for almost convincing me that I am way younger then I am! Dr. Luke (ok - Brian!)– for unique humor and warm, enthusiastic nature! ÇSpecial thanks goes to my French-translators! They know who they are ;). Ç…and finally two of my collaborators – Teresa for opening the door to the secret life of STEX, and Mike – for being the best yeast postdoc I could have hoped for! You turned to be “all that” and even more, thank you for you patience and acceptance. You are full of surprises and I didn’t have enough yet! This time would be way harder without the knowledge that I’m loved and supported from the friends back home (including my mum <) and my dancing tribe, which keeps reminding me who I really am. Table of Contents Résumé en français.......................................................................................................... 1 Références ................................................................................................................. 13 CHAPTER 1: Introduction............................................................................................... 17 1 Looking at chromosome ends - historical background.................................. 17 2 Telomere structure........................................................................................... 18 2.1 Telomeric DNA sequences ........................................................................ 18 2.2 Telomere Capping Structure ..................................................................... 20 2.3 Organization of the Subtelomeric Region ................................................ 23 3 Telomere Length Regulation ........................................................................... 24 3.1 The End-Replication Problem.................................................................... 24 3.2 Telomerase................................................................................................. 25 3.3 Cell Cycle Dependent Recruitment of Telomerase.................................. 28 3.4 Telomere Elongation is Coupled to DNA-replication................................ 31 3.5 A “Protein Counting Model” of Telomere Length Control........................ 33 3.6 DNA-damage Response Proteins in Telomere Length Regulation ......... 37 3.6.1 The Ku Heterodimer ........................................................................... 37 3.6.2 The MRX Complex............................................................................... 39 3.6.3 Tel1 and Mec1 Kinases...................................................................... 41 3.7 Other Regulators of Telomere Length ...................................................... 42 3.8 Recombination-Dependent Maintenance of Telomeres ......................... 43 4 Thesis Research Aims...................................................................................... 44 CHAPTER 2: Telomere Length Homeostasis Is Achieved via a Switch between Telomerase-Extendible and -Nonextendible States ..................................................... 47 CHAPTER 3: Tel1 Kinase and Subtelomere-Bound Tbf1 Mediate Preferential Elongation of Short Telomeres by Telomerase in Yeast............................................... 69 CHAPTER 4: Telomerase Repeat Addition Processivity is Increased at Critically Short Telomeres in a Tel1-Dependent Manner in Saccharomyces cerevisiae .................... 85 CHAPTER 5: Discussion and Perspectives.................................................................103 References ....................................................................................................................111 Résumé en français Les télomères chez la levure Saccharomyces cerevisiae Les génomes eucaryotes sont organisés en chromosomes linéaires. Les extrémités de ces chromosomes linéaires sont à l’origine de deux problèmes pour la cellule. Le premier problème concerne la machinerie de réplication de l’ADN qui n’est pas capable de répliquer la totalité de l’ADN linéaire. Sans un mécanisme spécifique pour résoudre ce problème, cela entrainerait le raccourcissement des chromosomes, ainsi que la perte de séquences terminales à chaque cycle de réplication et de division cellulaire. Le deuxième problème porte sur les extrémités chromosomiques, qui, sans une protection spécifique, seraient reconnues comme lésions double brin de l’ADN, et par conséquence soumis à des dégradations exonucléolytiques ou des phénomènes de réparation des cassures double-brin par recombinaison homologue ou par ligature non-homologue (NHEJ: «non-homologous end-joining»). La réparation de type NHEJ est particulièrement dangereuse pour la cellule, puisque ce processus entraîne la création de chromosomes dicentriques, qui posent des problèmes considérables lors de la répartition des chromosomes pendant l’anaphase de la mitose (cassure). Les télomères sont des complexes nucléoprotéiques spécialisés qui ont évolué à l’extrémité des chromosomes pour contribuer à la résolution de ces deux problèmes: ils permettent leur réplication complète et les protègent contre l’instabilité génétique. Les séquences ADN telomériques sont organisées de façon similaire chez la plupart des eucaryotes; elles contiennent des plages de répétitions courtes (Blackburn & Gall, 1978), mais dont la séquence et le nombre varient considérablement selon les espèces. Chez la levure Saccharomyces cerevisiae, les télomères sont composés d’environ 200-300 pb de la répétition irrégulière 5’- (TG)0–6TGGGTGT(G)1–2 -3’ (abrégée TG1-3) (Szostak & Blackburn, 1982; Shampay et al, 1984; Forstemann & Lingner, 2001). Cette séquence dégénérée n’est pas une caractéristique unique des télomères de S. cerevisiae car il a été démontré que la 1 Résumé en français variation des répétitions télomériques est une caractéristique partagée par plusieurs espèces fongiques (pour revue, voir (Lundblad, 2006)). Une autre caractéristique propre aux télomères est la présence d’une extension simple brin 3’ sortante, riche en guanine («G-tail»). Ces «G-tails» sont détectables tout au long du cycle cellulaire et leur élimination est létale (Wellinger et al, 1993). Les régions sous-télomériques de S.
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