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Telomere clustering and anchoring in budding yeast

SCHOBER, Heiko

Abstract

Organisation spatiale des 32 télomères de la levure "Saccharomyces cerevisiae" dans des foyers périnucléaires. J'ai posé la question: "Quel télomère se situe dans quel foyer?". Un télomère donné reste-t-il toujours dans le même foyer ou existe-t-il des télomères en dehors des foyers? l'objectif de ma thèse était de visualiser ces foyers des télomères et un télomère individuel dans des cellules vivantes pour répondre à ces questions. En outre j'ai pu montrer que l'interaction de la télomerase avec Yku influence le positionnement des télomères près de l'enveloppe nucléaire. Dans ce contexte j'ai également exploré la relation entre la localisation des télomères et des aspects majeurs de leur biologie, à savoir la protection de ceux-ci vis-à-vis des systèmes de réparation et la régulation de leur longueur.

SCHOBER, Heiko. Telomere clustering and anchoring in budding yeast. Thèse de doctorat : Univ. Genève, 2008, no. Sc. 3957

URN : urn:nbn:ch:unige-822

DOI : 10.13097/archive-ouverte/unige:82

Available at:

http://archive-ouverte.unige.ch/unige:82

Disclaimer: layout of this document may differ from the published version.

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DÉPARTEMENT DE BIOLOGIE MOLÉCULAIRE

Telomere Clustering And Anchoring In Budding Yeast

THÈSE

présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention biologique

par

Heiko Schober d’Allemagne

Thèse N° 3957

Professeure Susan Gasser Professeur David Shore FRIEDRICH MIESCHER INSTITUTE

GENÈVE

Atelier de reproduction de la section de physique 2008

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List of Abbreviations ...1

Résumé en français ...2

L’organisation fonctionnelle...2

L’organisation globale des chromosomes ...3

Les associations physiques entre éléments répétitifs ...5

Les télomères chez Saccharomyces cerevisiae...6

Références...9

Introduction ...12

A historical view of the biology of chromosome ends ...12

Telomeric DNA sequence ...14

The telomerase ribonucleoprotein (RNP) complex...15

Accessory proteins that function in the regulation of telomerase ...18

The Ku heterodimer...18

Rap1...19

MRX/N...21

Replication machinery ...22

Tel1/Mec1 ...23

Cdc13/Stn1/Ten1 ...24

The telomere cap ...24

Telomere clustering and recruitment to the NE ...26

Telomere organization in the interphase nucleus ...26

Anchoring pathways ...28

Subtelomeric regions and the formation of telomeric foci...30

Telomerase independent telomere maintenance in yeast ...31

Thesis Research Aims ...33

Results ...43

Controlled exchange of chromosomal arms reveals principles driving telomere interactions in yeast...43

Supplemental information...60

Supplemental Figure legends...60

Supplemental Figure References...63

A role for telomerase in telomere anchoring in budding yeast...71

Supplemental Figures...116

Supplemental strain table ...120

Outlook and Discussion ...123

Long-range chromosomal interactions ...124

Anchoring of telomeric chromatin to the NE ...127

Telomere anchoring in S Phase ...127

How is telomere anchoring mediated in G1? ...129

Acknowledgments/Danksagungen ...134

References ...35

Supplemental Figures ...64

Objectifs de thèse ...7

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m micrometer

DNA /ADN deoxyribonucleic acid, acide désoxyribonucléique RNA / ARN ribonucleic acid, acide ribonucléique

rDNA ribosomal DNA tRNA transfer RNA Pol polymerase

Sir silent information regulator HP1 heterochromatin protein 1 PcG polycomb group

H3, H4 histone H3, histone H4 Su(var) suppressor of variegation PEV position effect variegation TPE telomeric position effect STE subtelomeric elements STR subtelomeric repeats NE nuclear envelope

ARS autonomously replicating sequence CFP cyan fluorescent protein

GFP green fluorescent protein SPB spindle pole body

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Résumé en français

Le matériel génomique des organismes eucaryotes est distribué sur les chromosomes qui sont de longues structures linéaires. Ces longues fibres d’ADN sont enroulées sur elles-mêmes et compactées de façon à ce qu’elles soient contenues dans un volume extrêmement réduit et délimité par une double membrane, le noyau. Le noyau est le site d’activités biologiques très importantes telles que la réplication et la réparation de l’ADN, la transcription et la maturation d’ARN. Il serait de ce fait étonnant que les chromosomes contenus dans le noyau soient empaquetés de façon aléatoire, ce qui compliquerait certainement l’accomplissement des activités biologiques nucléaires. En effet, les chromosomes sont arrangés de façon organisée dans le nucléoplasme.

L’organisation tridimensionnelle des chromosomes paraît être un niveau de régulation additionnel pour de nombreux processus biologiques tels que la transcription de gènes ou la réplication de l’ADN. Il existe plusieurs manières de concevoir l’organisation de l’ADN dans le noyau.

L’organisation fonctionnelle

La première décrit un arrangement des chromosomes basé sur le regroupement d’activités enzymatiques dans des foyers multiples qui sont communément appelés des corps nucléaires (nuclear bodies). Les speckles, les Cajal bodies et les PML bodies ont été majoritairement caractérisés par microscopie. Les protéines faisant partie de ces corps nucléaires sont regroupées dans de petits granules de nature dynamique, capables de bouger,

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de fusionner et de se séparer. La fonction de ces corps nucléaires n’est pas claire mais ils regroupent un grand nombre de protéines impliquées dans la maturation des ARN messagers (pour revue, voir Lamond and Sleeman, 2003).

Le génome est répliqué à partir de multiples sites d’initiation de la réplication qui sont allumés de façon séquentielle à la fois dans le temps et dans l’espace. Il a été démontré que les sites actifs de réplication sont regroupés d’une manière régulée à des endroits caractéristiques dans le nucléoplasme (Dimitrova and Gilbert, 1999; Nakamura et al., 1986).

Une organisation tridimensionnelle très impressionnante est le regroupement des gènes ribosomiques en un corps nucléaire appelé nucléole.

Chez les mammifères, les gènes codant pour les protéines ribosomiques sont localisés sur des chromosomes différents, ce qui implique un regroupement de certaines régions chromosomiques.

Ces quelques exemples illustrent le fait que les cellules ont favorisé une organisation où la plupart des activités enzymatiques nucléaires sont concentrées dans des sous-régions du noyau ce qui implique une contrainte tridimensionnelle sur le comportement des chromosomes.

L’organisation globale des chromosomes

En 1885, Carl Rabl décrivit la disposition particulière des chromosomes dans les cellules de salamandre, où les télomères et les centromères sont regroupés aux pôles opposés du noyau. Cette configuration polarisée des chromosomes est conservée chez certains organismes tels que la mouche de

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vinaigre (Drosophila melanogaster), certaines plantes (blé, seigle, orge,…) et les levures bourgeonnantes (Saccharomyces cerevisiae) et fissipares (Schizosaccharomyces pombe). Elle n’est au contraire pas ou peu conservée dans d’autres plantes (Arabidopsis thaliana, riz, maïs,…) et chez les cellules de mammifères (Dong and Jiang, 1998 ; Funabiki et al., 1993 ; Gotta et al., 1996 ; Hochstrasser et al., 1986 ; Jin et al., 1998 ; Manuelidis and Borden, 1988; Shaw et al., 2002 ).

Un autre type d’organisation des chromosomes a été abondement décrit pour les cellules de mammifères, c’est l’organisation en territoires chromosomiques. Elle décrit une situation où les chromosomes ne sont pas enchevêtrés les uns dans les autres mais sont confinés dans des compartiments distincts. L’observation de ces territoires chromosomiques a été rendue possible par l’hybridation in situ de sondes marquées reconnaissant un chromosome entier, ce qui a permit de cartographier les chromosomes et leur position globale dans le noyau. Des analyses plus poussées ont détecté certaines règles auxquelles semblent être soumis les chromosomes. Les chromosomes comportant en grandes quantités des gènes hautement exprimés sont par exemple préférentiellement situés dans la partie centrale du noyau, alors que les chromosomes pauvres en gènes sont souvent localisés en périphérie du noyau (pour revue, voir Cremer and Cremer, 2001).

Ce type d’organisation où les chromosomes sont confinés dans des compartiments définis est remis en question par certaines observations.

Premièrement, les territoires chromosomiques ne sont pas aussi rigides qu’il n’y

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paraît. Il a été démontré que certaines régions chromosomiques sont situées à l’extérieur de leur territoire, en particulier des régions fortement transcrites (Mahy et al., 2002; Volpi et al., 2000 ). De plus, la chromatine n’est pas une structure totalement statique, mais montre une mobilité beaucoup plus grande que ce qui avait été présagé. Aussi bien chez la levure bourgeonnante, chez la mouche ou chez les cellules humaines, le coefficient de diffusion d’un site chromatique est d’environ 110-4 – 110-3m2/s, ce qui à l’échelle du noyau, permet une mobilité non négligeable. Cette mobilité est d’autant plus frappante chez la levure dont la taille du noyau n’est que de 2m (Chubb et al., 2002; Heun et al., 2001; Marshall et al., 1997).

Une organisation nucléaire en territoires chromosomiques n’est donc pas exclue, mais elle a besoin d’être modulée et il est certain que la chromatine a un comportement dynamique au sein des territoires.

Les associations physiques entre éléments répétitifs

Plusieurs régions du génome sont constituées de séquences d’ADN répétées qui constituent des domaines fonctionnels tels que les centromères et les télomères. Dans certains organismes, les centromères et les télomères sont des domaines du génome riches en hétérochromatine, caractérisée par une compaction de l’ADN plus élevée et un contenu très pauvre en gènes. Le caractère hétérochromatique de ces éléments contribue vraisemblablement à leur organisation particulière. En effet ces éléments répétitifs se regroupent et forment des domaines distincts à l’intérieur du noyau visibles presque

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universellement pendant la méiose et dans plusieurs cas dans les cellules en divisions mitotiques.

Dans les cellules de mammifères, les régions péricentriques (entourant les centromères) sont généralement regroupées pour former des chromocentres qui sont souvent placés autours du nucléole (Haaf and Schmid, 1991; Hilliker and Appels, 1989). Dans la levure, les centromères dépourvus d’hétérochromatine se regroupent également en une région bien précise, autours du spindle pole body, l’élément organisateur du fuseau mitotique (Jin et al., 1998).

Chez plusieurs organismes unicellulaires, les télomères forment des groupes qui sont souvent situées en périphérie du noyau. C’est notamment le cas chez la levure bourgeonnante. Alors que très peu de mécanismes moléculaires permettant les organisations nucléaires susmentionnées ont été décrits, les télomères de la levure constituent un modèle idéal pour étudier les protéines impliquées dans cette structure particulière. Cet organisme permet à la fois une approche par la microscopie fluorescente in vivo, ce qui assure une préservation des structures tridimensionnelles, et une approche génétique très puissante.

Les télomères chez Saccharomyces cerevisiae

Le génome de la levure bourgeonnante est constitué de 16 chromosomes ou 32 dans les cellules diploïdes, ce qui représente 64 télomères. L’analyse de leur localisation par immunofluorescence ou par FISH (Fluorescent In Situ

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Hybridization) a révélé que 6-10 foyers (au lieu de 64) qui se trouvent majoritairement proches de la périphérie du noyau (Gotta et al., 1996). Il apparaît donc que les télomères se regroupent entre eux et ont une tendance à s’associer avec l’enveloppe nucléaire (NE). Quelles seraient les protéines responsables de l’organisation spatiale des télomères?

La séquence terminale des télomères de la levure bourgeonnante est constituée de répétitions TG(1-3) qui recrutent la protéine Rap1. L’extrémité des télomères est liée par l’hétérodimère Yku70/80. Rap1 et Yku70/80 permettent le recrutement d’un complexe protéique impliqué dans la répression transcriptionelle, les protéines Sir (Silent Information Regulator). Au travers de l’interaction de Rap1 et de yKu avec Sir4, le complexe Sir2/3/4 est chargé sur le télosome. Il est ensuite capable de diffuser le long de l’ADN nucléosomal grâce à des interactions avec les extrémités N-terminales des histones. La propagation du complexe Sir induit une répression transcriptionelle des gènes localisés proches des télomères (2-4kb, Telomere Position Effect, TPE). En plus du complexe Sir, Rap1 est aussi capable de recruter Rif1/2. Les protéines Rif sont en compétition avec les protéines Sir pour la liaison à Rap1. Il a été démontré que Rif1/2 ont un rôle dans la régulation de la longueur des télomères (pour revue, voire Gasser et al., 2004).

Objectifs de thèse

Toutes les cellules eucaryotes (y compris les cellules humaines) présentent leur matériel génomique sous forme des molécules de l’ADN linéaires. La nature semi-conservative de la réplication impose une structure

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intacte des télomères afin de maintenir la longueur de leurs chromosomes.

D’autre part, la cellule doit être capable de reconnaître les télomères en tant que tels, et de les différencier d’une cassure double brin pour éviter d’activer la machinerie de réparation.

Au début de mon travail de thèse, Il était clairement établi que les 32 télomères d’une cellule haploïde se regroupe en 2 à 8 foyers à la périphérie du noyau (Gotta et al., 1996). De nombreuses questions restaient cependant sans réponse quant à l’organisation spatiale des télomères dans ces domaines périnuclaires. Nous avons ainsi posé la question : « Quel télomère se situe dans quel foyer ? ». Autrement dit, est-ce qu’un télomère donné reste toujours dans le même foyer ou existe-t-il des télomères en dehors des foyers ? L’objectif de ma thèse était de visualiser ces foyers des télomères et un télomère individuel dans des cellules vivantes pour répondre à ces questions.

En outre une autre question majeure qui restait sans réponse était de savoir où étaient localisés dans le noyau les composants de la télomèrase, l’enzyme qui catalyse l’élongation des télomères. D’autre part, nous avons essayé de savoir si il existait une relation entre le positionnement des télomères et leur longueur. A début de ma thèse il était connu qu’il y a au moins deux systèmes de recrutement des télomères à la périphérie; l’un dépend des protéines Sir4 liées à la chromatine télomèrique tandis que l’autre nécessite l’hétérodimère yKu (Hediger et al., 2002; Taddei et al., 2004). yKu se lie directement à tlc1, la sous-unité ARN de le télomèrase (Stellwagen et al., 2003).

Mon but était de déterminer si cette interaction influe sur le positionnement des

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télomères près de l’enveloppe du noyau. Dans ce contexte j’ai également exploré la relation entre la localisation des télomères et des aspects majeurs de la biologie des télomères, à savoir leur protection vis-à-vis des systèmes de réparation et la régulation de leur longueur.

Références

Chubb, J.R., S. Boyle, P. Perry, and W.A. Bickmore. 2002. Chromatin motion is constrained by association with nuclear compartments in human cells.

Curr Biol. 12:439-45.

Cremer, T., and C. Cremer. 2001. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet. 2:292-301.

Dimitrova, D.S., and D.M. Gilbert. 1999. The spatial position and replication timing of chromosomal domains are both established in early G1. Mol Cell. 4:983-993.

Dong, F., and J. Jiang. 1998. Non-Rabl patterns of centromere and telomere distribution in the interphase nuclei of plant cells. Chromosome Res.

6:551-8.

Funabiki, H., I. Hagan, S. Uzawa, and M. Yanagida. 1993. Cell cycle-dependent specific positioning and clustering of centromeres and telomeres in fission yeast.J Cell Biol. 121:961-76.

Gasser, S.M., F. Hediger, A. Taddei, F.R. Neumann, and M.R. Gartenberg.

2004. The function of telomere clustering in yeast: the circe effect. Cold Spring Harb Symp Quant Biol. 69:327-37.

Gotta, M., T. Laroche, A. Formenton, L. Maillet, H. Scherthan, and S.M. Gasser.

1996. The clustering of telomeres and colocalization with Rap1, Sir3, and Sir4 proteins in wild-type Saccharomyces cerevisiae. J Cell Biol.

134:1349-63.

Haaf, T., and M. Schmid. 1991. Chromosome topology in mammalian interphase nuclei. Exp Cell Res. 192:325-32.

Hediger, F., F.R. Neumann, G. Van Houwe, K. Dubrana, and S.M. Gasser. 2002.

Live imaging of telomeres: yKu and Sir proteins define redundant telomere-anchoring pathways in yeast. Curr Biol. 12:2076-89.

Heun, P., T. Laroche, K. Shimada, P. Furrer, and S.M. Gasser. 2001.

Chromosome dynamics in the yeast interphase nucleus. Science.

294:2181-6.

Hilliker, A.J., and R. Appels. 1989. The arrangement of interphase chromosomes: structural and functional aspects. Exp Cell Res. 185:267- 318.

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Hochstrasser, M., D. Mathog, Y. Gruenbaum, H. Saumweber, and J.W. Sedat.

1986. Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster. J Cell Biol. 102:112-23.

Jin, Q., E. Trelles-Sticken, H. Scherthan, and J. Loidl. 1998. Yeast nuclei display prominent centromere clustering that is reduced in nondividing cells and in meiotic prophase. J Cell Biol. 141:21-9.

Lamond, A.I., and J.E. Sleeman. 2003. Nuclear substructure and dynamics. Curr Biol. 13:R825-8.

Mahy, N.L., P.E. Perry, and W.A. Bickmore. 2002. Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH. J Cell Biol. 159:753-63.

Manuelidis, L., and J. Borden. 1988. Reproducible compartmentalization of individual chromosome domains in human CNS cells revealed by in situ hybridization and three-dimensional reconstruction. Chromosoma. 96:397- 410.

Marshall, W.F., A. Straight, J.F. Marko, J. Swedlow, A. Dernburg, A. Belmont, A.W. Murray, D.A. Agard, and J.W. Sedat. 1997. Interphase chromosomes undergo constrained diffusional motion in living cells. Curr Biol. 7:930-9.

Nakamura, H., T. Morita, and C. Sato. 1986. Structural organizations of replicon domains during DNA synthetic phase in the mammalian nucleus.

Experimental Cell Research. 165:291-7.

Shaw, P.J., R. Abranches, A. Paula Santos, A.F. Beven, E. Stoger, E. Wegel, and P. Gonzalez-Melendi. 2002. The architecture of interphase chromosomes and nucleolar transcription sites in plants. J Struct Biol.

140:31-8.

Stellwagen, A.E., Z.W. Haimberger, J.R. Veatch, and D.E. Gottschling. 2003. Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes Dev. 17:2384-95.

Taddei, A., F. Hediger, F.R. Neumann, C. Bauer, and S.M. Gasser. 2004.

Separation of silencing from perinuclear anchoring functions in yeast Ku80, Sir4 and Esc1 proteins. EMBO J. 23:1301-12.

Volpi, E.V., E. Chevret, T. Jones, R. Vatcheva, J. Williamson, S. Beck, R.D.

Campbell, M. Goldsworthy, S.H. Powis, J. Ragoussis, J. Trowsdale, and D. Sheer. 2000. Large-scale chromatin organization of the major histocompatibility complex and other regions of human chromosome 6 and its response to interferon in interphase nuclei. J Cell Sci. 113 ( Pt 9):1565-76.

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Introduction

A historical view of the biology of chromosome ends

As chromosomes became visible by microscopy in the late 1800s, cytogeneticists soon realized that they carry the hereditary unit discovered by Gregor Mendel (mid 1800s), and were envisaged as “beads on a string” along the chromosome by Thomas Hunt Morgan. Morgan’s alumnus Hermann Muller applied Roentgen-X-rays to fruit flies and could recover the products of broken chromosomes using new genetic tools. He found inversions, translocations and deficiencies. Suspiciously underrepresented were terminal deficiencies, leading Muller to hypothesize that the recovered chromosomes were the result of reattachment of two broken ends. These reattachments did not occur between “free ends” or between “free ends” and broken ends. In 1938 Muller baptized the “free end” as the telomere (Muller, 1938).

At around the same time starting in the 1920s Barbara McClintock pioneered new methods for microscopy, and was consequently able to recognize individual chromosomes in maize. Like Muller, McClintock found that inversions, translocations and deficiencies were frequent outcomes of Roentgen X-ray irradiation, all of which could be explained as a result of fusion between broken ends. A report dating from 1931 states that she had already recognized that the natural end of an intact chromosome would not attach to a broken end induced by X-rays (McClintock, 1931).

In 1953, Watson and Crick heralded the new era of Molecular Biology with their discovery of the structure of DNA (Watson and Crick, 1953). In the early 1970's, the mechanisms behind DNA replication were unveiled, and it

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became clear that DNA polymerase, the enzyme responsible for DNA replication, could not fully synthesize the 3' end of linear DNA. James Watson called this the end-replication problem (Watson, 1972). Ideas how to circumvent the end replication problem and to reconcile it with the earlier findings of McClintock and Muller were plentiful. A popular hypothesis was that telomeres consist of palindromic and repetitive sequences allowing them to fold back and base pair with themselves to fill in the sequence that was missing after a round of replication (Cavalier-Smith, 1974). Not as complex was the model from Bateman (Bateman, 1975) in which he suggested that the telomere would form a self-paired hairpin structure, a method that is actually used by some viruses and eubacteria. Others models proposed included the transient fusion of telomeres, or the covalent attachment of a protein to the end of chromosomes.

From the human point of view all of these models were much more logical than the way evolution has solved the problem. Today it is clear that the ends of eukaryotic chromosome do look much like double strand breaks in DNA (DSB). Telomeres do have free 3’ and 5’ ends, biochemically indistinguishable from DSBs generated by x-ray irradiation. And even more paradoxically, proteins found at DSBs are also found at telomeres. However, distinguishing them from DSBs is that the telomeric sequences of most eukaryotes consist of tandem repeats rich in guanine and end in a 3’

overhang (discussed in the following paragraph). The mechanism used by most eukaryotes to overcome the end-replication problem was finally elucidated by Greider and Blackburn (Greider and Blackburn, 1985), with the discovery of a novel activity in Tetrahymena cell free extracts that added

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tandem TTGGGG repeats onto synthetic telomere primers. Nowadays it is known that repeat synthesis onto DNA ends is accomplished by telomerase, a reverse transcriptase, in which an RNA moiety provides the template for the synthesis of the repeat (Greider and Blackburn 1987, Lingner 1997).

Telomeric DNA sequence

The first telomeric DNA sequence was determined in the somatic nucleus of the ciliated protozoan Tetrahymena thermophila (Blackburn and Gall, 1978). This organism propagates its rDNA in the form of minichromosomes which also carry 20-70 tandem repeats of 5’TTGGGG3’

telomeric sequence at each end. Telomeric sequences of most eukaryotes have a tandem repeat rich in guanine that ends in a 3’ overhang, however the number of tandem repeats varies significantly between species and cell (tissue) type.

All vertebrates studied so far have telomeres made out of tandem TTAGGG repeats. In mouse and rats this sequence can be repeated up to 25,000 times whereas in humans TTAGGG is found typically up to 1500 times.

Budding yeast S. cerevisiae telomeres consist of 250-350 base pairs (bp) of irregular tandem repeats with the consensus sequence of T(G)1-3 (Shampay et al., 1984). Similarly, in fission yeast S. pombe, each telomere contains approximately 300 bp of a repeat motif that is degenerate in nature, the consensus sequence of which is TTAC(A)(C)G(2-8) (reviewed in Cooper, 2000).

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Ironically, although telomeres were first defined in Drosophila following Muller’s experiments with X-rays, this organism is an exception in that its telomeres are not maintained by telomerase. Instead the telomeres contain retrotransposon elements that encode among other proteins a reverse transcriptase, and the probable mechanism of telomere maintenance is the addition of retrotransposable elements (reviewed in Pardue and DeBaryshe, 2003)

A crucial feature of telomeric DNA is the 3’overhang of the G-rich strand and this has been intensively studied. In budding yeast telomeres, this G-rich 3’-overhang is about 10-15 bp in length (Larrivee et al., 2004). Toward the end of S phase after completion of replication, the length of this overhang substantially extends (Dionne and Wellinger, 1998; Wellinger et al., 1993).

Semi-conservative replication at telomeres will give rise to one blunt end from leading strand replication and one 3’ overhang end from lagging strand replication where the RNA primer is removed. It has been shown that the blunt end is attacked by exonucleases to generate a 3’-overhang directly after completion of replication. In two independent studies it has been shown that the activity of the S phase cyclin dependent kinase Cdc28 (S-Cdk1) is needed for the resection to occur (Frank et al., 2006; Vodenicharov and Wellinger, 2006).

The telomerase ribonucleoprotein (RNP) complex

S. cerevisiae telomerase RNP is composed of three protein subunits and one RNA moiety (Figure 1). The protein subunits Est1, Est2, and Est3 were identified in a screen for “ever shorter telomeres”, the so called Est

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phenotype defined by continuous telomere shortening and an accompanying decline in cell viability, more commonly termed senescence. Though TLC1, the RNA moiety, was identified in a high copy screen for disruption of telomeric silencing (Singer and Gottschling, 1994), it also deserves the name Est since its deletion results in the same senescence phenotype. The catalytic core of budding yeast telomerase comprises Est2 and TLC1.TLC1 templates for the telomeric repeat addition to the single strand G rich 3’ overhang ends of chromosomes, and Est2 is the reverse transcriptase subunit. Est1 and Est3 are telomere associated proteins that are not essential for in vitro activity (Lingner et al., 1997). The RNA moiety serves as a scaffold and coordinates binding of Est1 and a protein heterodimer yKu70/80 to the telomerase core enzyme (Seto et al., 2002; Stellwagen et al., 2003; Zappulla and Cech, 2004).

Although Est2 is telomere associated already in G1 phase cells, telomerase is not active until late S phase. The presence of Est1 in the cell is regulated through the stability of its mRNA which is unstable in G1 and stable in S phase (Larose et al., 2007). It has been proposed that Est1 is important for Est3 recruitment and hence for the assembly of the RNP telomerase complex (Osterhage et al., 2006). Est3, the smallest subunit of telomerase with 19 kDa, has as yet not found to possess any enzymatic activity. In addition to binding telomerase, Est1 has two more biochemical activities, it binds directly to single stranded telomeric DNA (Virta-Pearlman et al., 1996) and to another telomeric protein Cdc13 (Evans and Lundblad, 1999) that also binds the 3’

single strand telomere tails.

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hTERT is the human homologue of Est2 and together with hTR, the RNA moiety, forms the catalytic core of telomerase. Putative human telomerase accessory proteins hEST1A, hEST1B and hEST1C contain weak sequence similarity with yeast Est1, and were identified in silico (Reichenbach et al., 2003; Snow et al., 2003). The role for these proteins in human telomere function, however, is unknown. Nonetheless hEST1A and B can co- immunoprecipitate with telomerase activity and bind to hTERT independently of hTR (Snow et al., 2003)

In fission yeast, Trt1 has been identified as the homologue of budding yeast Est2 and human hTERT. Est1 is also present in fission yeast, and recently the RNA moiety of S. pombe, TER1, has been identified (Leonardi et al., 2007; Webb and Zakian, 2007). Binding between S. pombe Est1 and Trt1, the two known protein components of fission yeast telomerase, have been shown to be dependent on TER1, arguing for a conservation of the RNA

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moiety to serve as a scaffold for the assembly of telomerase subunits. All eukaryotes that use telomerase to encounter the “end replication problem”

use a conserved set of proteins with an RNA moiety for reverse transcription and coordination of binding of protein subunits.

Accessory proteins that function in the regulation of telomerase.

The Ku heterodimer

As seen in Figure 1, another protein called Ku is found at the telomeres. Ku, or in budding yeast yKu, is a heterodimer composed of a 70kDa and an 80kDa subunit, and is evolutionary conserved from yeast to human and it has been proposed that both subunits have evolved from a common ancestor (Gell and Jackson, 1999). Ku binds with high-affinity to DNA ends in a sequence-independent manner, whether it is a DSB or a telomere. A ‘two-face’ model has been proposed, in which Ku has two modes of binding to a DNA end, with one distinct function for DNA DSB repair and another for telomeric functions (Ribes-Zamora et al., 2007). One important function of yKu at budding yeast telomeres is as a positive regulator of telomerase. It is required to recruit the catalytic core protein Est2 to telomeres in G1 (Fisher et al., 2004) (see Figure 1) through a direct interaction with a stem loop of TLC1, an interaction conserved from yeast to human (Stellwagen et al., 2003; Ting et al., 2005). Cells expressing TLC1 alleles that are unable to interact with yKu display short telomeres (Stellwagen et al., 2003) and reduced Est2 binding to telomeres (Fisher et al., 2004).

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Rap1

The protein Rap1 is a central player at budding yeast telomeres (Figure 2). Rap1 binds directly to the telomeric repeats through two tandem myb-like DNA-binding domains (Konig et al., 1996; Konig and Rhodes, 1997), and importantly inhibits telomerase activity by its interaction with the carboxy- terminal domains of Rif1/2 (Grossi et al., 2001; Krauskopf and Blackburn, 1996; Kyrion et al., 1993; Marcand et al., 1997; Ray and Runge, 1999). This inhibitory activity of Rap1 led to the postulation of the so-called “counting model” (Marcand et al., 1997). Tethering different amounts of the carboxy terminal domain (CTD) of Rap1 immediately adjacent to the telomeric repeat tract has been shown to affect telomere length: the more Rap1-CTD tethered, the shorter the TG tract added by telomerase. S. pombe and vertebrates also possess a RAP1 homologue, however only in budding yeast contains the Myb domain that binds to telomeric DNA repeats. The same CTD of Rap1 that binds Rif1/2 is responsible for recruitment of the Sir proteins, which are known to nucleate silencing at yeast telomeres. In addition, Rap1 has been shown to suppress non homologous end joining (NHEJ) between telomeres (Pardo and Marcand, 2005), one of two major repair mechanisms for DSBs, and consistent with this, Rap1 is required for end concealment at long telomeric tracts (Negrini et al., 2007). Paradoxically, Rap1 possesses a BRCA-1 carboxy-terminal motif (BRCT) in its N’ which is found in numerous proteins involved in DNA repair where they appear to serve as adaptors for protein- protein interactions. Apart from its functions at telomeres, Rap1 plays a major role in transcriptional regulation, most notably of ribosomal protein genes.

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MRX/N (Mre11, Rad50, Xrs2 or Nbs1)

The MRX/N complex is evolutionary conserved (in vertebrates MRN (Nbs1)) and contributes to repair of DSBs and meiotic recombination as well as telomere function. It comprises three proteins, Mre11 is an endonuclease, Rad50 is an SMC family member and Xrs2p binds DNA in a structure-specific manner and has been shown to be important for targeting the MRX complex to DNA ends (Trujillo et al., 2003).

It is possible that the Mre11-Rad50-Xrs2 (MRX) complex performs the 5’ resection activity to generate a 3’-overhang at the telomere directly after completion of replication, since its presence has been shown to be essential for 3’overhang generation at an artificially created short telomere (Diede and Gottschling, 2001).

Mre11, Rad50 and Xrs2 are epistatic towards one another (reviewed in D'Amours and Jackson, 2002). The MRX complex has been shown to possess single-stranded endonuclease, single-stranded and double-stranded 3’ to 5’ exonuclease, and DNA unwinding activities in vitro. In vivo, however, the MRX complex, is required for the 5’ to 3’ resection of DSBs in yeast and for 5’ to 3’ resection of blunt telomeric ends (Diede and Gottschling, 2001).

This activity has been shown to be crucial for Cdc13 loading and telomerase mediated TG repeat addition. The Rad50 dimer is flexible and has been proposed to adopt the general architecture of ‘structural maintenance of chromosome’ (SMC) proteins.

In a recent study it has been shown that the MRX complex binds preferentially to G-quadruplex DNA in G4 DNA (Ghosal and Muniyappa, 2007). However it should be noted that it has never been formally shown that

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G-quadruplex DNA actually exists apart from a study in ciliates by Paeschke and colleagues (Paeschke et al., 2005). Yet it is noteworthy that all eukaryotic telomeres have G-rich strands. G-quadruplex DNA could be the differentiating feature between telomeres and DSB. In mammals, the Mre11 complex has been shown to bind directly to Trf2, an important regulator of telomere function. The Trf2–Mre11 complex interacts with telomeres specifically during S phase (Zhu et al., 2000).

Replication machinery

Telomere elongation via telomerase is tightly coupled to lagging strand DNA synthesis and it has been shown that the essential DNA polymerase- and - and DNA primase are required for telomerase function, indicating that telomeric DNA synthesis by telomerase is tightly coregulated with the production of the opposite strand (Diede and Gottschling, 1999). Cdc13 has been demonstrated to interact with the catalytic subunit of DNA polymerase , Pol1p and point mutations in either Cdc13 or Pol1 that reduced their interaction resulted in telomerase mediated telomere lengthening (Qi and Zakian, 2000). Pol12, the B subunit of the DNA polymerase (Pol1)–primase complex, has been identified as a factor involved in telomere length control as well (Grossi et al., 2004) underlining the importance of replication and telomere maintenance.

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Tel1/Mec1

Interestingly, the central DNA damage response kinases, Tel1 and Mec1, play essential roles in telomere length maintenance. Like their human homologues, called ATM and ATR, they mediate the cascade of events that occurs in response to DNA damage. Yeast cells that lack either of the two kinases have short telomeres, albeit the shortening in tel1 cells is clearly more pronounced. Double mutants display a senescence phenotype (Ritchie et al., 1999) that can be overcome by deletion of the inhibitory proteins Rif1 and Rif2 (Chan et al., 2001). Tel1 and Mec1 have been demonstrated to phosphorylate Cdc13, and this phosphorylation has been shown to be crucial for telomerase activity (Tseng et al., 2006). Tel1 preferentially interacts with short telomeres and stimulates their elongation (Hector et al., 2007; Sabourin et al., 2007). It has been shown that the frequency of elongation decreases in tel1 cells and that at an artificial telomere, without subtelomeric elements, telomere elongation no longer increases as telomeric repeat sequences reach critical short lengths (Arneric and Lingner, 2007). The regulation of telomere length via Tel1 depends on the presence of Rif1 and Rif21 (Craven and Petes, 1999;

Ray and Runge, 1999). Usually yeast telomerase adds repeats in a nonprocessive manner. Nevertheless Tel1 has recently been shown to be required to increase repeat addition processivity at extremely short telomeres (Chang et al., 2007).

Furthermore it has been shown that Tel1 and Mre11 are required for normal levels of Est1 and Est2 telomere association (Goudsouzian et al., 2006).

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Cdc13/Stn1/Ten1

As mentioned earlier, Tel1 and Mec1 phophorylate Cdc13, which is one of three essential budding yeast proteins that form a trimer: Stn1 and Ten1. This trimeric complex associates with the telomeric single stranded overhang in budding yeast, a single protein known as Pot1 (protection of telomeres-1) performs this function in fission yeast, and a two-subunit complex consisting of POT1 and TPP1 associates with telomeric ssDNA in mammals. Cdc13 and Pot1 have related oligonucleotide/oligosaccharide- binding fold (OB-fold) domains that facilitate binding to the telomeric ssDNA overhang. In two successive publications Bianchi and Shore have shown that the level of Cdc13 remains constant whether a telomere has normal length or has been reduced by approximately half upon loxP induced excision of telomeric tract. In contrast Tel1 and Est1 association substantially increases at the shortened telomere. The authors suggest that the phosphorylation state of Cdc13 rather than its abundance controls telomerase recruitment and activation. Because the amount of Tel1 increases, the authors suggest that Tel1 is phosphorylating Cdc13 (Bianchi and Shore, 2007b). Furthermore Bianchi and Shore find that a suddenly shortened telomere replicates early in S phase in contrast to normal length telomeres where replication takes place late in S phase (Bianchi and Shore, 2007a).

The telomere cap

In addition to their roles in telomere elongation, Rap1, telomerase, Cdc13/Stn1/Ten1 and yKu form a protective cap at natural chromosome

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ends. This cap is important for genome stability, because it protects the ends against excessive 5’-strand resection (Bertuch and Lundblad, 2003) and from being recognized as DNA DSBs. The absence of yKu from telomeres leads to their deprotection and excessive 5’-end resection (Bertuch and Lundblad, 2003). As mentioned earlier Rap1 has been shown to suppress non homologous end joining (NHEJ) between telomeres (Pardo and Marcand, 2005) and hence embodies an important protecting function at telomeres.

Cdc13, Stn1 and Ten1 both protect chromosome termini from unregulated resection and regulate telomere length. Stn1 and Ten1 show similarities to Rpa2 and Rpa3, subunits of the heterotrimeric replication protein A (RPA) complex, which is the major single-stranded DNA-binding factor in eukaryotic cells. It has been proposed that Cdc13, Stn1 and Ten1 function as a telomere-specific RPA-like complex (Gao et al., 2007). Not long ago, the so-called KEOPS complex was identified, which promotes both telomere uncapping and elongation (Downey et al., 2006). This evolutionary conserved complex contains among other proteins the protein kinase Bud32.

In cdc13-1 cells, deletion of components of KEOPS diminishes the accumulation of long TG 3’ overhangs that are otherwise present in this telomere capping mutant at non-permissive temperatures, arguing for yet another phosphorylation event that is needed to trigger the 5’ resection at telomeres.

Telomerase components seem themselves to be involved in telomere capping, as suggested by the finding that overexpression of subunits thereof suppress the temperature sensitivity of cdc13-1 and yku mutants (Nugent et al., 1998; Teo and Jackson, 2001).

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Human telomeres are protected by shelterin, six proteins exclusively found at telomeres. Two of these, TRF1 and TRF2, bind double stranded telomeric DNA and a third, POT1, binds to single stranded TTAGGG repeats.

Other components of shelterin are TPP, TIN2 and hRAP1. Mammalian telomeres form a so-called t-loop, a structure that was revealed by electron microscopy (Griffith et al., 1999). T-loops are formed through the strand invasion of the 3’ overhang into the duplex part of the telomere.

Telomere clustering and recruitment to the NE

Telomere organization in the interphase nucleus

The organization of chromosomes in the interphase nucleus into functional subnuclear domains plays an important role in the regulation of a variety of processes including gene activation and silencing and DNA repair (reviewed in Sexton et al., 2007). This includes the clustering of repetitive non-coding heterochromatin, as well as the specific positioning of functional chromosomal elements such as origins of replication, boundary elements, and in lower eukaryotes, centromeres and telomeres (reviewed in Spector, 2003;

Taddei et al., 2004b). Because centromeres in most organisms comprise of thousands of kilobases of heterochromatin, the clustering of centromeres into

“chromocenters” is thought to arise from interactions of heterochromatin proteins that recognize satellite repeats. Budding yeast lacks centromeric heterochromatin, but as discussed in the previous section, each telomere contains ~ 350 bp of an irregular TG1-3 repeat that is able to nucleate a repressive chromatin state (Gottschling et al., 1990). The subtelomeric

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repression that spreads from the TG1-3 repeat resembles heterochromatin in many ways, most notably by being late-replicating, refractile to transcription, and able to transmit its repressed state in a heritable manner (reviewed in Lustig, 1998; Tham and Zakian, 2002). Thus, like centromeric heterochromatin, the 32 telomeres of a haploid budding yeast cell are found to cluster together in 2-8 distinct foci. These foci of budding yeast telomeres are found to localize with high frequency near the nuclear envelope (NE) and it has been shown that deletion of either subunit of yKu leads to delocalization of telomeres (Hediger et al., 2002b; Laroche et al., 1998). So another function of yKu is recruitment of telomeres to the NE.

Esc1 is localized in patches along the nuclear envelope (NE) and serves as an important binding site for telomeric chromatin. Fluorescence imaging can clearly distinguish nuclear pore complexes (NPCs) dispersed between the patches of Esc1 (Taddei et al., 2004a), which have been implicated in the localization-dependent enhancement of the transcriptional efficiency of a subset of genes (reviewed in Akhtar and Gasser, 2007).

Despite the finding that at least some telomeres are affected by deletions of nuclear pore proteins (Therizols et al., 2006), several lines of evidence indicate that pores are not universally required for telomere anchoring. For example, telomeres remain evenly distributed along the NE away from pores in cells where the nuclear pores cluster due to a mutation of NUP133 (Hediger et al., 2002a).

These telomeric foci associated with the nuclear periphery sequester the Silent information regulatory proteins, Sir2, Sir3 and Sir4 from internal sites which are known to nucleate silencing at yeast telomeres (Gotta et al.,

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1996; Palladino et al., 1993). As mentioned in a previous section, the CTD of Rap1 is responsible for the recruitment of the Sir proteins. At this point it is worthwhile to mention that yKu interacts with the Sir complex and deletion of either of the two yKu subunits leads to derepression of subtelomeric genes (Maillet et al., 2001). Thus yKu is needed for telomeric silencing.

Anchoring pathways

There are at least two partially redundant pathways to anchor telomeric chromatin to the NE in budding yeast (Hediger et al., 2002b; Taddei et al., 2004a). One involves the yKu heterodimer, and the other the Sir complex and Esc1, which localizes to the NE and has been shown to interact directly with Sir4 in order to recruit telomeres to the nuclear periphery. When individual chromosomes are monitored there are differences in theier dependence on the yKu or Sir4-Esc1 pathway as well as cell cycle variations in the efficiency of anchoring.

Recently, an integral nuclear membrane protein Mps3 has been shown to also play an important role in anchoring telomeres at the nuclear periphery (Bupp et al., 2007). Mps3 is the single S. cerevisiae member of the conserved family of Sad1/UNC-84 homology (SUN) domain containing proteins. One of the main functions of this family is to form bridges across the inner and outer nuclear membranes of the cells nucleus (Tzur et al., 2006). Of the four human proteins known to possess SUN domains, two localize to the NE. The N- terminal domain (NTD) of human SUN1 itself has been shown to face the nuclear interior and to interact with lamin A, whereas the C terminal domain (CTD) and the SUN domain itself reside in the lumen between inner and outer

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nuclear membrane (Haque et al., 2006). Mps3 shares this topology and mutations in the N-terminus of Mps3 (which faces the nuclear interior) have recently been shown to lead to delocalization of telomeres from the NE (Bupp et al., 2007). This is particularly visible in S phase, and evidence points to Sir4 as the critical mediator for telomere anchoring.

Mps3 has several other functions and is an essential protein. It is a component of the spindle pole body (SPB) (Jaspersen et al., 2002), and like its homologues in fission yeast and worm, has been shown to participate in clustering of telomeres into the meiotic bouquet formation (Conrad et al., 2007). Furthermore Mps3 has been shown to play a role in sister chromatid cohesion, due to an interaction with the ‘establishment of sister chromatid cohesion’ factor Ctf7 (Antoniacci et al., 2004). Interestingly, absence of Ctf18, another factor important for sister chromatid cohesion, has been shown to be required for both yKu and Sir mediated telomere positioning pathways especially during G1. It has been proposed that Ctf18 modifies telomeric chromatin to make it competent for yKu/Sir mediated peripheral localization (Hiraga et al., 2006).

Most interestingly, a link to telomerase was found in a large-scale yeast two-hybrid screen where Mps3 was found to interact with Est1 (Uetz et al., 2000). This finding was confirmed by the Skibbens laboratory using in vitro binding experiments (Antoniacci et al., 2007). In this perspective, it is interesting to note that Est1, an indispensable part of the active holoenzyme, is only recruited to telomeres in S phase (Taggart et al., 2002).

Related to this, it has been shown that the position of telomeres within the nucleus has an impact on telomere length. In human cells, subunits of

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telomerase have been shown to shuttle to and from different subnuclear locations and interference with this movement has an impact on telomere length regulation of the viral activator VP16 to a telomere resulted in its delocalization away from its normal position at the nuclear periphery. This delocalization resulted in a shortening of the terminal TG tract in a tel1 background where telomere length control was partially compromised (Hediger et al., 2006). However, these results did not address where the telomerase subunits are actually localized, and how the position of telomeres affects telomerase activity. It remains to be tested if telomere elongation occurs at the nuclear periphery or whether short telomeres may sojourn there to load components required to switch to the accessible elongation state.

Subtelomeric regions and the formation of telomeric foci

Besides the TG repeats, yeast subtelomeric domains contain middle repetitive elements called X and Y’ (Chan and Tye, 1983; Louis and Haber, 1992), often called Telomere associated sequences (TAS). The extent of spreading of telomere-linked heterochromatin into adjacent nucleosomes is variable among chromosome ends and is regulated at least in part by factors that bind the subtelomeric X element. However, given that these elements are present at all yeast telomeres, it is unlikely that the TG repeat, X element, and/or silent chromatin itself are directly responsible for the selective interactions that produce telomeric foci. Indeed, the principles that guide the formation of telomere clusters are unknown. Because long-range interactions may influence recombination frequencies or coordinate transcriptional programs (Fabre et al., 2005; Louis et al., 1994)F, it is of significant interest to identify the principles that guide the clustering of telomeres in yeast.

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Besides the middle repetitive X element, a longer retrotransposon-like Y’ element is present at about 50% of yeast chromosome ends in a strain- dependent manner. Y’ elements are often flanked by TG1-3repeats suggesting that they may have been inserted by TG-induced recombination (Louis, 1995;

Louis et al., 1994). Also present in subtelomeric domains are coding sequences for related groups of genes, such as the MEL (melibiose) family, which is required for growth on alternative carbon sources (Naumov et al., 1996). This family, like others in subtelomeric regions, encodes metabolic enzymes that are suppressed during growth on glucose, but become up- regulated in response to low glucose and/or oxygen stress. Due to their redundancy and the presence of pseudogenes, only a few of the subtelomeric genes have genetically confirmed phenotypes. Nonetheless, recombination studies have indicated that there is preferred exchange between subtelomeric regions of extensive homology, which may explain the maintenance of partially or fully redundant gene families (Fabre et al., 2005; Louis et al., 1994). It has been argued that gene family amplification may even provide an evolutionary advantage among hemiascomycete yeast species (Fabre et al., 2005; Turakainen et al., 1993a; Turakainen et al., 1993a,b).

Telomerase independent telomere maintenance in yeast

Although normal telomeres are rather refractory to recombination, homologous recombination can provide a mechanism for telomere maintenance when telomerase is absent and telomeres become progressively shorter.

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There are two major pathways of telomerase independent telomere preservation. They are referred to as type I and II in budding yeast, both of which are Rad52-dependent, as all formation of survivors is eliminated if Rad52 is missing. These are distinguished both by their genetic requirements and by the structure of the telomeres themselves. Type I survivors were originally described by Lundblad and Blackburn (Lundblad and Blackburn, 1993) where all telomeric ends are extended by the acquisition or amplification of subtelomeric Y’ elements and neighboring short tracts of telomeric repeat DNA. In the same work, Lundblad and Blackburn described a second pathway, yielding cells referred to as type II survivors. This pathway (studied in more detail by Teng and Zakian, 1999), results in the extensive elongation of telomeric TG repeats, with few alterations to the subtelomeric sequences themselves.

Type II survivor formation depends on many factors. The Mre11- Rad50-Xrs2 (MRX) complex and Rad59 have been shown to be involved in this process (Chen et al., 2001; Le et al., 1999; Teng et al., 2000; Teng and Zakian, 1999; Tsukamoto et al., 2001) as has the helicase Sgs1 (Cohen and Sinclair, 2001; Huang et al., 2001; Johnson et al., 2001; Watt et al., 1996) and Exo1 (Maringele and Lydall, 2002). Furthermore the ATM/ATR kinases Tel1/Mec1 are needed, as absence of each one causes a reduction in formation of type II survivors (Tsai et al., 2002). tel1/mec1 double mutants have been shown to senesce (Ritchie et al., 1999) and the only type of survivors that can be formed is I. Type I survivors depend on the RecA homologue Rad51. Finally, it has been shown that telomerase-negative cells

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require Clb2 in association with Cdc28 (Cdk1), to generate post-senescence survivors of both types at a normal rate

Thesis Research Aims

Since all eukaryotes and few prokaryotes keep their genomes in the form of linear DNA molecules, these organisms depend on an intact telomere structure both to maintain chromosome length due to the semi-conservative nature of DNA replication, as well as to differentiate the chromosome ends from the DSB repair machinery to prevent illicit repair events.

Though it is clear that the 32 telomeres of a haploid S. cerevisiae cell cluster in 2-8 foci at the nuclear periphery, a number of questions remained unanswered. It is unclear “who is where” among telomeric foci; that is whether telomeres always reside in a particular cluster, if some telomeres move freely outside of the clusters. My goal was to shed light on these questions using multicolor fluorescence microscopy to observe to differentially labeled telomeres in living cells. I aimed firstly to investigate the likelihood of a telomere residing within or outside of telomeric clusters, and secondly to see whether the composition of clusters is stable or stochastic. In other words, is telomere position in any given cluster reproducible amongst a population of cells? Alternatively, are telomere-telomere interactions random?

Furthermore, a major question that remained unclear is where the components of telomerase themselves are located within the nucleus, and the relationship between telomere position and telomere length regulation. At the beginning of my thesis it was known that there were at least two different pathways that recruit telomeres to the NE, one relying on the telomere bound

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Sir complex and the peripheral protein Esc1, and the other involving the yKu heterodimer and a yet unknown anchoring partner yKu has been shown to interact with TLC1 (Stellwagen et al., 2003) and my goal was to investigate whether the interaction is involved in the positioning of telomeres at the NE.

Also in this context, I explored the relationship between telomere location and major aspects of telomere biology, namely capping and length regulation.

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