8 research outputs found
Le rÎle de la protéine RAP1 dans la protection des télomÚres humains
In mammals, the shelterin complex is the guardian of telomere stability. It operates through a set of six proteins (TRF1, TRF2, POT1, RAP1, TPP1 and TIN2) that binds telomeric DNA and protects it from being recognized as DNA double-strand breaks and therefore control DNA repair and DNA damage response pathways. Among them, RAP1 and TRF2 cooperate and together protect chromosome extremities from end-to-end fusions. TRF2 is seen as a major factor to control telomere DNA topology by wrapping DNA around itself in a right handed manner. This property of TRF2 is required to promote the formation of t-loops, special DNA structures at telomeres that are considered as protective barriers to DNA damage response and fusion. Here we demonstrate two independent situations where RAP1 dysfunction is critical for telomere protection. First, in cells expressing a wrapping-deficient TRF2 allele that cannot form t-loops, RAP1 appears as a backup anti-fusion mechanism. Second, RAP1 downregulation in replicative senescent cells leads to telomere fusions and DNA damage response activation. This is consistent with similar observations in HeLa cells treated with the telomerase inhibitor BIBR1532, and in which RAP1 expression was abolished by an inducible shRNA system. In addition, we show that fusions triggered by RAP1 loss are dependent upon ligase IV, which is a key player of the classical non-homologous end-joining (c-NHEJ) repair pathway. Altogether, these results indicate that RAP1 takes over telomere protection when TRF2 cannot properly function or in the normal physiological situation, such as replicative senescence.Les tĂ©lomĂšres sont des sĂ©quences dâADN, gĂ©nĂ©ralement rĂ©pĂ©tĂ©es en tandem, localisĂ©es Ă lâextrĂ©mitĂ© des chromosomes linĂ©aires. Une des fonctions principales des tĂ©lomĂšres est de diffĂ©rencier lâextrĂ©mitĂ© des chromosomes des cassures double-brin, et ainsi de prĂ©venir lâactivation des voies de rĂ©paration de lâADN. Chez les mammifĂšres, cette fonction est plus spĂ©cifiquement assurĂ©e par le complexe shelterin. Il sâagit dâun complexe hĂ©tĂ©rogĂšne composĂ© de six protĂ©ines distinctes : TRF1, TRF2, POT1, RAP1, TPP1 et TIN2, qui interagit spĂ©cifiquement avec lâADN tĂ©lomĂ©rique. Au sein de ce complexe, les protĂ©ines RAP1 et TRF2 coopĂšrent afin dâempĂȘcher lâextrĂ©mitĂ© des chromosomes dâĂȘtre perçue comme un dommage de lâADN, ce qui autrement aboutirait Ă des fusions inter-chromosomiques suite au processus de rĂ©paration. La protĂ©ine TRF2 se lie directement Ă la molĂ©cule dâADN dans laquelle elle sâenroule de façon spĂ©cifique. Cette propriĂ©tĂ© est primordiale pour gĂ©nĂ©rer une structure dâADN en forme de boucle, appelĂ©e t-loop, et dont le bon fonctionnement des tĂ©lomĂšres dĂ©pend. Les travaux effectuĂ©s au cours de cette thĂšse ont mis en Ă©vidence deux scenarii indĂ©pendants dans lesquels la protĂ©ine RAP1 assure un rĂŽle critique dans la stabilitĂ© des tĂ©lomĂšres. PremiĂšrement, RAP1 peut prĂ©venir les fusions inter-chromosomiques dans des cellules exprimant une forme altĂ©rĂ©e de TRF2 incapable de former des t-loops. DeuxiĂšmement, lâinhibition de RAP1 dans des cellules en sĂ©nescence rĂ©plicative conduit Ă lâactivation des voies de rĂ©paration de lâADN et Ă la formation de fusions inter-chromosomiques. Ces observations font Ă©cho Ă des rĂ©sultats prĂ©cĂ©dents obtenus dans des cellules HeLa traitĂ©es avec lâinhibiteur de la tĂ©lomĂ©rase BIBR1532, et dont lâexpression de la protĂ©ine RAP1 Ă©tait abolie par shRNA. De plus, jâai montrĂ© que les fusions interchromosomiques engendrĂ©es par la perte de RAP1 sont dĂ©pendantes de la ligase IV, qui est un acteur principal de la voie de rĂ©paration de lâADN par recombinaison non-homologue (NHEJ). Dans lâensemble, ces travaux dĂ©montrent lâimportance de la protĂ©ine RAP1 dans la stabilitĂ© des tĂ©lomĂšres lorsque la protĂ©ine TRF2 est non fonctionnelle, mais aussi dans des situations physiologiques telles que la sĂ©nescence rĂ©plicative
Investigation into the role of human RAP1 in telomere protection
Les tĂ©lomĂšres sont des sĂ©quences dâADN, gĂ©nĂ©ralement rĂ©pĂ©tĂ©es en tandem, localisĂ©es Ă lâextrĂ©mitĂ© des chromosomes linĂ©aires. Une des fonctions principales des tĂ©lomĂšres est de diffĂ©rencier lâextrĂ©mitĂ© des chromosomes des cassures double-brin, et ainsi de prĂ©venir lâactivation des voies de rĂ©paration de lâADN. Chez les mammifĂšres, cette fonction est plus spĂ©cifiquement assurĂ©e par le complexe shelterin. Il sâagit dâun complexe hĂ©tĂ©rogĂšne composĂ© de six protĂ©ines distinctes : TRF1, TRF2, POT1, RAP1, TPP1 et TIN2, qui interagit spĂ©cifiquement avec lâADN tĂ©lomĂ©rique. Au sein de ce complexe, les protĂ©ines RAP1 et TRF2 coopĂšrent afin dâempĂȘcher lâextrĂ©mitĂ© des chromosomes dâĂȘtre perçue comme un dommage de lâADN, ce qui autrement aboutirait Ă des fusions inter-chromosomiques suite au processus de rĂ©paration. La protĂ©ine TRF2 se lie directement Ă la molĂ©cule dâADN dans laquelle elle sâenroule de façon spĂ©cifique. Cette propriĂ©tĂ© est primordiale pour gĂ©nĂ©rer une structure dâADN en forme de boucle, appelĂ©e t-loop, et dont le bon fonctionnement des tĂ©lomĂšres dĂ©pend. Les travaux effectuĂ©s au cours de cette thĂšse ont mis en Ă©vidence deux scenarii indĂ©pendants dans lesquels la protĂ©ine RAP1 assure un rĂŽle critique dans la stabilitĂ© des tĂ©lomĂšres. PremiĂšrement, RAP1 peut prĂ©venir les fusions inter-chromosomiques dans des cellules exprimant une forme altĂ©rĂ©e de TRF2 incapable de former des t-loops. DeuxiĂšmement, lâinhibition de RAP1 dans des cellules en sĂ©nescence rĂ©plicative conduit Ă lâactivation des voies de rĂ©paration de lâADN et Ă la formation de fusions inter-chromosomiques. Ces observations font Ă©cho Ă des rĂ©sultats prĂ©cĂ©dents obtenus dans des cellules HeLa traitĂ©es avec lâinhibiteur de la tĂ©lomĂ©rase BIBR1532, et dont lâexpression de la protĂ©ine RAP1 Ă©tait abolie par shRNA. De plus, jâai montrĂ© que les fusions interchromosomiques engendrĂ©es par la perte de RAP1 sont dĂ©pendantes de la ligase IV, qui est un acteur principal de la voie de rĂ©paration de lâADN par recombinaison non-homologue (NHEJ). Dans lâensemble, ces travaux dĂ©montrent lâimportance de la protĂ©ine RAP1 dans la stabilitĂ© des tĂ©lomĂšres lorsque la protĂ©ine TRF2 est non fonctionnelle, mais aussi dans des situations physiologiques telles que la sĂ©nescence rĂ©plicative.In mammals, the shelterin complex is the guardian of telomere stability. It operates through a set of six proteins (TRF1, TRF2, POT1, RAP1, TPP1 and TIN2) that binds telomeric DNA and protects it from being recognized as DNA double-strand breaks and therefore control DNA repair and DNA damage response pathways. Among them, RAP1 and TRF2 cooperate and together protect chromosome extremities from end-to-end fusions. TRF2 is seen as a major factor to control telomere DNA topology by wrapping DNA around itself in a right handed manner. This property of TRF2 is required to promote the formation of t-loops, special DNA structures at telomeres that are considered as protective barriers to DNA damage response and fusion. Here we demonstrate two independent situations where RAP1 dysfunction is critical for telomere protection. First, in cells expressing a wrapping-deficient TRF2 allele that cannot form t-loops, RAP1 appears as a backup anti-fusion mechanism. Second, RAP1 downregulation in replicative senescent cells leads to telomere fusions and DNA damage response activation. This is consistent with similar observations in HeLa cells treated with the telomerase inhibitor BIBR1532, and in which RAP1 expression was abolished by an inducible shRNA system. In addition, we show that fusions triggered by RAP1 loss are dependent upon ligase IV, which is a key player of the classical non-homologous end-joining (c-NHEJ) repair pathway. Altogether, these results indicate that RAP1 takes over telomere protection when TRF2 cannot properly function or in the normal physiological situation, such as replicative senescence
The Telomeric Protein TRF2 Regulates Replication Origin Activity within Pericentromeric Heterochromatin
Heterochromatic regions render the replication process particularly difficult due to the high level of chromatin compaction and the presence of repeated DNA sequences. In humans, replication through pericentromeric heterochromatin requires the binding of a complex formed by the telomeric factor TRF2 and the helicase RTEL1 in order to relieve topological barriers blocking fork progression. Since TRF2 is known to bind the Origin Replication Complex (ORC), we hypothesized that this factor could also play a role at the replication origins (ORI) of these heterochromatin regions. By performing DNA combing analysis, we found that the ORI density is higher within pericentromeric satellite DNA repeats than within bulk genomic DNA and decreased upon TRF2 downregulation. Moreover, we showed that TRF2 and ORC2 interact in pericentromeric DNA, providing a mechanism by which TRF2 is involved in ORI activity. Altogether, our findings reveal an essential role for TRF2 in pericentromeric heterochromatin replication by regulating both replication initiation and elongation
Human RAP1 specifically protects telomeres of senescent cells from DNA damage
Repressor/activator protein 1 (RAP1) is a highly evolutionarily conserved protein found at telomeres. Although yeast Rap1 is a key telomere capping protein preventing nonâhomologous end joining (NHEJ) and consequently telomere fusions, its role at mammalian telomeres in vivo is still controversial. Here, we demonstrate that RAP1 is required to protect telomeres in replicative senescent human cells. Downregulation of RAP1 in these cells, but not in young or dividing preâsenescent cells, leads to telomere uncapping and fusions. The antiâfusion effect of RAP1 was further explored in a HeLa cell line where RAP1 expression was depleted through an inducible CRISPR/Cas9 strategy. Depletion of RAP1 in these cells gives rise to telomere fusions only when telomerase is inhibited. We further show that the fusions triggered by RAP1 loss are dependent upon DNA ligase IV. We conclude that human RAP1 is specifically involved in protecting critically short telomeres. This has important implications for the functions of telomeres in senescent cells
Mitochondrial function in skeletal myofibers is controlled by a TRF2âSIRT3 axis over lifetime
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Genome-wide Control of Heterochromatin Replication by the Telomere Capping Protein TRF2
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TRF2-Mediated Control of Telomere DNA Topology as a Mechanism for Chromosome-End Protection.
International audienceThe shelterin proteins protect telomeres against activation of the DNA damage checkpoints and recombinational repair. We show here that a dimer of the shelterin subunit TRF2 wraps ⌠90 bp of DNA through several lysine and arginine residues localized around its homodimerization domain. The expression of a wrapping-deficient TRF2 mutant, named Top-less, alters telomeric DNA topology, decreases the number of terminal loops (t-loops), and triggers the ATM checkpoint, while still protecting telomeres against non-homologous end joining (NHEJ). In Top-less cells, the protection against NHEJ is alleviated if the expression of the TRF2-interacting protein RAP1 is reduced. We conclude that a distinctive topological state of telomeric DNA, controlled by the TRF2-dependent DNA wrapping and linked to t-loop formation, inhibits both ATM activation and NHEJ. The presence of RAP1 at telomeres appears as a backup mechanism to prevent NHEJ when topology-mediated telomere protection is impaired