G-overhang dynamics at Tetrahymena telomeres

Jacob, Naduparambil K.; Skopp, Rose; Price, Carolyn M.

doi:10.1093/emboj/20.15.4299

Cited by 60 publications

(72 citation statements)

References 45 publications

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“…Tetrahymena cells were grown in 1.5ϫ PPYS at 30°C as previously described (20). CdCl 2 was added to 2 g/ml to maintain the expression of the rescuing POT1a allele.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting ϳ40,000 macronuclear telomeres are composed of 250-to 350-bp G 4 T 2 /C 4 A 2 repeats. These telomeres have a very precise terminal DNA structure, as the C strands end with the sequence 5Ј C 3 A 2 -or 5Ј C 2 A 2 -, while most G strands extend another 14 or 15 nucleotides (nt) or 20 or 21 nt and terminate with the sequence 5Ј-T 2 G 4 T (20). Studies with telomerase-deficient cells have revealed that this precise G-overhang structure is generated by a series of specific G-and C-strand processing reactions (21).…”

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confidence: 99%

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Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Jacob

Lescasse

Linger

et al. 2007

Molecular and Cellular Biology

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The POT1/TEBP telomere proteins are a group of single-stranded DNA (ssDNA)-binding proteins that have long been assumed to protect the G overhang on the telomeric 3 strand. We have found that the Tetrahymena thermophila genome contains two POT1 gene homologs, POT1a and POT1b. The POT1a gene is essential, but POT1b is not. We have generated a conditional POT1a cell line and shown that POT1a depletion results in a monster cell phenotype and growth arrest. However, G-overhang structure is essentially unchanged, indicating that POT1a is not required for overhang protection. In contrast, POT1a is required for telomere length regulation. After POT1a depletion, most telomeres elongate by 400 to 500 bp, but some increase by up to 10 kb. This elongation occurs in the absence of further cell division. The growth arrest caused by POT1a depletion can be reversed by reexpression of POT1a or addition of caffeine. Thus, POT1a is required to prevent a cell cycle checkpoint that is most likely mediated by ATM or ATR (ATM and ATR are protein kinases of the PI-3 protein kinase-like family). Our findings indicate that the essential function of POT1a is to prevent a catastrophic DNA damage response. This response may be activated when nontelomeric ssDNA-binding proteins bind and protect the G overhang.In order to maintain genome integrity, the telomeric DNA from cells with linear chromosomes is packaged into a protective nucleoprotein complex (10). In the absence of this complex, the telomeres are recognized as DNA damage and subject to repair by nonhomologous end joining (33). The resulting chromosome fusions lead to genome instability (36). The protective telomeric complex is composed of a series of unique telomere proteins that bind the double-stranded region of the telomeric DNA and/or the single-strand overhang on the 3Ј G-rich strand (37). Although the exact composition of the complex varies between species, vertebrates, yeasts, plants, and ciliates all use a series of structurally related proteins to protect their telomeres. The G-overhang binding proteins all bind Gstrand DNA through a conserved OB fold motif, while the double-stranded DNA-binding proteins bind via a conserved myb motif (23,37,46).In vertebrate cells, telomeres are packaged by a core complex of six proteins which function both in telomere protection and in telomere length regulation (10). This core complex (which is sometimes called shelterin) contains two doublestranded DNA-binding proteins, TRF1 and TRF2, which anchor the complex along the length of the telomere, and the TRF1/2 interaction partners Rap1, TIN2, and TPP1. The Gstrand binding protein POT1 is also a component of the complex. Although POT1 is secured into the complex through interactions with TPP1 (17, 31, 50), POT1 molecules are also thought to bind the G-strand overhang via their OB foldcontaining DNA-binding domain (45,46). Additional telomere-associated proteins include a number of DNA damage response factors (33). However, these factors appear to bind only transiently during replication of...

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“…Tetrahymena cells were grown in 1.5ϫ PPYS at 30°C as previously described (20). CdCl 2 was added to 2 g/ml to maintain the expression of the rescuing POT1a allele.…”

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Jacob

Lescasse

Linger

et al. 2007

Molecular and Cellular Biology

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“…7). Tetrahymena telomeres are capped by Pot1a in complex with Tpt1, Pat1, and Pat2 (25,26), bound to an overhang of 14 to 15 or 20 to 21 nucleotides with a TGGGGT-3=OH end permutation (45,46). This length of overhang is insufficient for binding both Pot1a and Teb1 (34).…”

Section: Discussionmentioning

confidence: 99%

Direct Single-Stranded DNA Binding by Teb1 Mediates the Recruitment of Tetrahymena thermophila Telomerase to Telomeres

Upton

Hong

Collins

2014

Molecular and Cellular Biology

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The eukaryotic reverse transcriptase telomerase copies its internal RNA template to synthesize telomeric DNA repeats at chromosome ends in balance with sequence loss during cell proliferation. Previous work has established several factors involved in telomerase recruitment to telomeres in yeast and mammalian cells; however, it remains unclear what determines the association of telomerase with telomeres in other organisms. Here we investigate the cell cycle dependence of telomere binding by each of the seven Tetrahymena thermophila telomerase holoenzyme proteins TERT, p65, Teb1, p50, p75, p45, and p19. We observed coordinate cell cycle-regulated recruitment and release of all of the subunits, including the telomeric-repeat DNA-binding subunit Teb1. Using domain truncation and mutagenesis approaches, we investigated which subunits govern the interaction of telomerase holoenzyme with telomeres. Our results show that Teb1 is critical for telomere interaction of other holoenzyme subunits and demonstrate that high-affinity Teb1 DNA-binding activity is necessary and sufficient for cell cycle-regulated telomere association. Overall, these and additional findings indicate that in the ciliate Tetrahymena, telomerase recruitment to telomeres requires direct binding to single-stranded DNA, unlike the indirect DNA recognition through telomere-bound proteins essential in yeast and mammalian cells.

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“…Diverses expériences suggèrent que des extensions G-riches sont présentes sur tous les télomères, aux deux extrémités des chromosomes macronucléaires [9]. Ces extensions sont détectées durant tout le cycle cellulaire [10]. La taille exacte des extensions, ainsi que leur séquence, sont contrôlées très strictement chez les ciliés.…”

Section: Débuts Chez Les Ciliésunclassified

“…La taille exacte des extensions, ainsi que leur séquence, sont contrôlées très strictement chez les ciliés. La majorité des extensions se termine par la séquence 5'-TTGGGGT-3' et plus de 80 % de ces extensions ont 14-15 ou 20-21 nucléotides [10]. Cependant, leur taille peut varier lorsque les cellules sont soumises à certaines restrictions nutritionnelles ou lorsqu'elles sortent du cycle cellulaire.…”

Section: Débuts Chez Les Ciliésunclassified

Structure terminale des chromosomes : le « capuchon télomérique »

LeBel

Wellinger²

2004

Med Sci (Paris)

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maintenant établi dans la communauté scientifique. Ce que l'on connaissait de la machinerie de réplication conventionnelle, incapable de synthétiser l'extrémité de molécules d'ADN linéaires comme les chromosomes eucaryotes, laissait supposer que les télomères devaient subir des pertes de leurs séquences terminales au fil des cycles de réplication. Chez les humains, on observe effectivement un raccourcissement des chromosomes dans la plupart des cellules somatiques au fil des années, et l'hypothèse selon laquelle les télomères joueraient un rôle déterminant dans la sénescence cellulaire a été proposée. Cependant, il existe une enzyme spécifique, la télomérase, d'abord identifiée chez le cilié Tetrahymena en 1985, qui peut prévenir ce raccourcissement naturel des chromosomes en ajoutant des répétitions télomériques à leurs extrémités [3]. Cette enzyme est absente de la plupart des cellules humaines somatiques normales. En revanche, chez l'homme, elle est présente et activée dans les cellules germinales, dans la plupart des lignées de cellules transformées et dans 90% des cellules cancéreuses primaires [4]. Ce lien entre activation de la télomérase et carcinogenèse a suscité un grand enthousiasme dans la communauté scientifique, suggérant que des thérapies anticancé-Les études du généticien H.J. Muller sur les chromosomes de la drosophile ont donné naissance au concept de télomère en 1938. H.J. Muller observa que des délétions ou des inversions induites par irradiation aux rayons X de l'ensemble du génome étaient quasiment indétectables dans les régions terminales des chromosomes [1]. Il en conclut que les extrémités des chromosomes, qu'il nomma télomères, devaient posséder une structure particulière capable de stabiliser les chromosomes. À la même époque, B. McClintock démontra que des chromosomes du maïs ayant subi des cassures double brin étaient capables de fusionner entre eux, contrairement à leurs extrémités qui demeuraient stables [2]. Le modèle le plus récent suggère que les télomères agissent comme des structures qui protègent les chromosomes d'éventuelles fusions de leurs extrémités, de l'intervention de la machinerie de réparation (qui les considère-rait comme des cassures double brin) et de la dégrada-tion par des exonucléases. Ces structures protectrices, que nous proposons d'appeler « capuchons télomé-riques», sont constituées par l'association d'ADN télo-mérique et de protéines spécifiques. Ce nouveau concept de capuchon protecteur des chromosomes est

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G-overhang dynamics at Tetrahymena telomeres

Cited by 60 publications

References 45 publications

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Tetrahymena POT1a Regulates Telomere Length and Prevents Activation of a Cell CycleCheckpoint

Direct Single-Stranded DNA Binding by Teb1 Mediates the Recruitment of Tetrahymena thermophila Telomerase to Telomeres

Structure terminale des chromosomes : le « capuchon télomérique »

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