Stn1-Ten1 complex and telomerase limit replication stress at telomeres in Schizosaccharomyces pombe

Les télomères sont des structures nucléoprotéiques qui protègent l’extrémité des chromosomes et assurent leur réplication. Ils sont constitués de séquences répétées, qui raccourcissent progressivement à chaque phase de réplication de l’ADN. La télomérase permet de rallonger ces séquences télomériques. Les télomères sont des régions difficiles à répliquer, pouvant générer un stress pour les cellules. Le but de ma thèse est de comprendre les mécanismes moléculaires mis en œuvre par la cellule pour réduire le stress réplicatif aux télomères et maintenir ainsi la stabilité du génome. Dans un premier temps, je me suis intéressé à l’étude du complexe Stn1-Ten1 qui contrôle négativement la télomérase. Nous avons pu identifier un domaine SIM dans Stn1, essentiel à l’interaction avec Tpz1-SUMO. J’ai montré que ce domaine joue un rôle important dans le recrutement du complexe aux télomères. Mes résultats suggèrent aussi que Stn1-Ten1 participerait activement à la réplication des télomères. Dans un second temps, j’ai étudié la dynamique de la réplication aux télomères en absence de la télomérase. Dans ce contexte, les cellules ne sont plus capables de répliquer de manière efficace leurs télomères. La réplication des télomères repose alors sur la voie de recombinaison homologue et des acteurs impliqués dans la réparation des cassures double-brin. Mes résultats suggèrent que la télomérase fonctionnerait comme une enzyme de réparation des fourches de réplication effondrées aux télomères. J’ai donc identifié deux nouveaux acteurs, le complexe Stn1-Ten1 et la télomérase, impliqués dans la réplication et la maintenance des télomères chez S. pombe.Telomeres are nucleoprotein structures that protect the end of chromosomes and allow efficient replication. They consist of repeated sequences, which progressively shorten after each cell division. Telomerase allows to extend these telomeric sequences. Telomeres are regions that are difficult to replicate. Which is a main source of replication stress. The goal of my thesis is to understand the molecular mechanisms involved to reduce telomere replicative stress and thus maintain the stability of the genome. At first, I was interested in the study of Stn1-Ten1 complex that negatively controls telomerase. We were able to identify a SIM domain in Stn1, which is essential for interaction with Tpz1-SUMO. I have shown that this domain plays an important role in the recruitment of the telomere complex. My results also suggest that Stn1-Ten1 would actively participate in telomere replication. Second, I studied the dynamics of telomere replication in the absence of telomerase. In this context, cells are no able to efficiently replicate their telomeres. The telomere replication is then based on the homologous recombination pathway and the actors involved in the repair of double-strand breaks. My results suggest that telomerase would function as a repair enzyme for replication forks collapsed to telomeres. To conclude, I identified two new actors, the Stn1-Ten1 complex and telomerase, involved in telomere replication and maintenance in S. pombe

Solving the Telomere Replication Problem

Telomeres are complex nucleoprotein structures that protect the extremities of linear chromosomes. Telomere replication is a major challenge because many obstacles to the progression of the replication fork are concentrated at the ends of the chromosomes. This is known as the telomere replication problem. In this article, different and new aspects of telomere replication, that can threaten the integrity of telomeres, will be reviewed. In particular, we will focus on the functions of shelterin and the replisome for the preservation of telomere integrit

Telomerase Repairs Collapsed Replication Forks at Telomeres

International audienceGraphical Abstract Highlights d Telomerase repairs collapsed replication forks at telomeres d Rad51, MRN, and Ctp1 are essential in the absence of telomerase d Ku and Trt1 compete for telomeric free DNA ends In Brief Matmati et al. investigate replication dynamics in telomerase-negative fission yeast cells. Their results reveal that telomerase repairs collapsed replication forks at telomeres and shields telomeres from homologous recombination

Eroded telomeres are rearranged in quiescent fission yeast cells through duplications of subtelomeric sequences

How both telomere stability is regulated and dysfunctional telomeres processed in quiescent cells is poorly understood. Here, the authors provide evidence that eroded telomeres in quiescent fission yeast are rearranged by homologous recombination through duplications of subtelomeric sequences

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast

Abstract Telomeres are nucleoprotein complexes that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich overhang and inhibits the activation of DNA damage checkpoints. In this study, we explore new functions of fission yeast Pot1 by using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and overexpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 plays a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Polα-primase complex to telomeres, thus promoting lagging-strand DNA synthesis at stalled replication forks

Pot1 promotes telomere DNA replication via the Stn1-Ten1 complex in fission yeast

International audienceTelomeres are nucleoprotein comple x es that protect the chromosome-ends from eliciting DNA repair while ensuring their complete duplication. Pot1 is a subunit of telomere capping complex that binds to the G-rich o v erhang and inhibits the activation of DNA damage c hec kpoints. In this study, w e e xplore ne w functions of fission y east Pot1 b y using a pot1-1 temperature sensitive mutant. We show that pot1 inactivation impairs telomere DNA replication resulting in the accumulation of ssDNA leading to the complete loss of telomeric DNA. Recruitment of Stn1 to telomeres, an auxiliary factor of DNA lagging strand synthesis, is reduced in pot1-1 mutants and o v ere xpression of Stn1 rescues loss of telomeres and cell viability at restrictive temperature. We propose that Pot1 pla y s a crucial function in telomere DNA replication by recruiting Stn1-Ten1 and Pol α-primase complex to telomeres via Tpz1, thus promoting lagging-strand DNA synthesis at stalled replication forks

The fission yeast Stn1-Ten1 complex limits telomerase activity via its SUMO-interacting motif and promotes telomeres replication

International audienceMammalian CST (CTC1-STN1-TEN1) complex fulfills numerous functions including rescue of the stalled replication forks and termination of telomerase action. In fission yeast lacking the CTC1 ortholog, the Stn1-Ten1 complex restricts telomerase action via its sumoylation-mediated interaction with Tpz(1TPPl). We identify a small ubiquitin-like modifier (SUMO)-interacting motif (SIM) in the carboxyl-terminal part of Stn1 and show that this domain is crucial for SUMO and Tpz1-SUMO interactions. Point mutations in the SIM (Stn1-226) lead to telomere elongation, impair Stn1-Ten1 recruitment to telomeres, and enhance telomerase binding, revealing that Stn1 SIM domain contributes to the inhibition of telomerase activity at chromosome ends. Our results suggest that Stn1-Ten1 promotes DNA synthesis at telomeres to limit single-strand DNA accumulation. We further demonstrate that Stn1 functions in the replication of telomeric and subtelomeric regions in a Taz1-independent manner. Genetic analysis reveals that misregulation of origin firing and/or telomerase inhibition circumvents the replication defects of the stnl-226 mutant. Together, our results show that the Stn1-Ten1 complex has a dual function at telomeres by limiting telomerase action and promoting chromosome end replication

Ssu72 phosphatase is a conserved telomere replication terminator

International audienceTelomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA poly-merases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/ Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA. Here, we show that the evolutionarily conserved phos-phatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72Δ mutants are defective in telomere replication and exhibit long 3 0-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication

Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast

Summary: Efficient replication of terminal DNA is crucial to maintain telomere stability. In fission yeast, Taz1 and the Stn1-Ten1 (ST) complex play prominent roles in DNA-ends replication. However, their function remains elusive. Here, we have analyzed genome-wide replication and show that ST does not affect genome-wide replication but is crucial for the efficient replication of a subtelomeric region called STE3-2. We further show that, when ST function is compromised, a homologous recombination (HR)-based fork restart mechanism becomes necessary for STE3-2 stability. While both Taz1 and Stn1 bind to STE3-2, we find that the STE3-2 replication function of ST is independent of Taz1 but relies on its association with the shelterin proteins Pot1-Tpz1-Poz1. Finally, we demonstrate that the firing of an origin normally inhibited by Rif1 can circumvent the replication defect of subtelomeres when ST function is compromised. Our results help illuminate why fission yeast telomeres are terminal fragile sites