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• W3012500692 abstract "•Telomerase repairs collapsed replication forks at telomeres•Rad51, MRN, and Ctp1 are essential in the absence of telomerase•Ku and Trt1 compete for telomeric free DNA ends Telomeres are difficult-to-replicate sites whereby replication itself may threaten telomere integrity. We investigate, in fission yeast, telomere replication dynamics in telomerase-negative cells to unmask problems associated with telomere replication. Two-dimensional gel analysis reveals that replication of telomeres is severely impaired and correlates with an accumulation of replication intermediates that arises from stalled and collapsed forks. In the absence of telomerase, Rad51, Mre11-Rad50-Nbs1 (MRN) complex, and its co-factor CtIPCtp1 become critical to maintain telomeres, indicating that homologous recombination processes these intermediates to facilitate fork restart. We further show that a catalytically dead mutant of telomerase prevents Ku recruitment to telomeres, suggesting that telomerase and Ku both compete for the binding of telomeric-free DNA ends that are likely to originate from a reversed fork. We infer that Ku removal at collapsed telomeric forks allows telomerase to repair broken telomeres, thereby shielding telomeres from homologous recombination. Telomeres are difficult-to-replicate sites whereby replication itself may threaten telomere integrity. We investigate, in fission yeast, telomere replication dynamics in telomerase-negative cells to unmask problems associated with telomere replication. Two-dimensional gel analysis reveals that replication of telomeres is severely impaired and correlates with an accumulation of replication intermediates that arises from stalled and collapsed forks. In the absence of telomerase, Rad51, Mre11-Rad50-Nbs1 (MRN) complex, and its co-factor CtIPCtp1 become critical to maintain telomeres, indicating that homologous recombination processes these intermediates to facilitate fork restart. We further show that a catalytically dead mutant of telomerase prevents Ku recruitment to telomeres, suggesting that telomerase and Ku both compete for the binding of telomeric-free DNA ends that are likely to originate from a reversed fork. We infer that Ku removal at collapsed telomeric forks allows telomerase to repair broken telomeres, thereby shielding telomeres from homologous recombination. Telomeres are nucleoprotein structures that protect chromosome extremities from degradation and ensure replication of chromosome ends (de Lange, 2018de Lange T. Shelterin-mediated telomere protection.Annu. Rev. Genet. 2018; 52: 223-247Crossref PubMed Scopus (341) Google Scholar, Palm and de Lange, 2008Palm W. de Lange T. How shelterin protects mammalian telomeres.Annu. Rev. Genet. 2008; 42: 301-334Crossref PubMed Scopus (1384) Google Scholar). Telomeric DNA consists of repeated G-rich sequences that are gradually lost with the successive cellular divisions. In germinal and stem cells, telomere length is maintained by the activity of telomerase, a reverse transcriptase that is able to elongate the 3′ overhang by addition of telomeric repeats. Telomeres are difficult regions to replicate and replication forks often stall when they progress through telomeric sequences (Sfeir et al., 2009Sfeir A. Kosiyatrakul S.T. Hockemeyer D. MacRae S.L. Karlseder J. Schildkraut C.L. de Lange T. Mammalian telomeres resemble fragile sites and require TRF1 for efficient replication.Cell. 2009; 138: 90-103Abstract Full Text Full Text PDF PubMed Scopus (706) Google Scholar). Indeed, several sources of endogenous stress impede replication fork progression such as telomeric DNA bound proteins, T-loops, RNA:DNA hybrids, and DNA secondary structures (for review, see Higa et al., 2017Higa M. Fujita M. Yoshida K. DNA replication origins and fork progression at mammalian telomeres.Genes (Basel). 2017; 8: E112Crossref PubMed Scopus (40) Google Scholar, Maestroni et al., 2017bMaestroni L. Matmati S. Coulon S. Solving the telomere replication problem.Genes (Basel). 2017; 8: 55Crossref Scopus (52) Google Scholar). Thus, accurate replication of chromosomal termini is a prerequisite for telomere homeostasis. To limit replication stress at telomeres, shelterin proteins TRF1 and TRF2 recruit and regulate the action of a number of helicases and nucleases (Vannier et al., 2012Vannier J.-B. Pavicic-Kaltenbrunner V. Petalcorin M.I.R. Ding H. Boulton S.J. RTEL1 dismantles T loops and counteracts telomeric G4-DNA to maintain telomere integrity.Cell. 2012; 149: 795-806Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, Ye et al., 2010Ye J. Lenain C. Bauwens S. Rizzo A. Saint-Léger A. Poulet A. Benarroch D. Magdinier F. Morere J. Amiard S. et al.TRF2 and apollo cooperate with topoisomerase 2alpha to protect human telomeres from replicative damage.Cell. 2010; 142: 230-242Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, Zimmermann et al., 2014Zimmermann M. Kibe T. Kabir S. de Lange T. TRF1 negotiates TTAGGG repeat-associated replication problems by recruiting the BLM helicase and the TPP1/POT1 repressor of ATR signaling.Genes Dev. 2014; 28: 2477-2491Crossref PubMed Scopus (120) Google Scholar). This underscores the importance of the telomeric proteins for telomere replication (Gilson and Géli, 2007Gilson E. Géli V. How telomeres are replicated.Nat. Rev. Mol. Cell Biol. 2007; 8: 825-838Crossref PubMed Scopus (342) Google Scholar). The fission yeast Schizosaccharomyces pombe has a shelterin-like complex that includes Taz1TRF1-2, Pot1, Rap1, Poz1, Tpz1TPP1, and Ccq1 proteins (Dehé and Cooper, 2010Dehé P.-M. Cooper J.P. Fission yeast telomeres forecast the end of the crisis.FEBS Lett. 2010; 584: 3725-3733Crossref PubMed Scopus (20) Google Scholar, Moser and Nakamura, 2009Moser B.A. Nakamura T.M. Protection and replication of telomeres in fission yeast.Biochem. Cell Biol. 2009; 87: 747-758Crossref PubMed Scopus (44) Google Scholar). Telomerase that is composed of the catalytic subunit Trt1TERT, the regulatory subunit Est1, and the TER1TERC RNA is constitutively expressed in yeast and guarantees telomere homeostasis (Armstrong and Tomita, 2017Armstrong C.A. Tomita K. Fundamental mechanisms of telomerase action in yeasts and mammals: understanding telomeres and telomerase in cancer cells.Open Biol. 2017; 7: 160338Crossref PubMed Scopus (49) Google Scholar). Rad3ATR- and Tel1ATM-dependent phosphorylation of Ccq1 promotes recruitment of telomerase to telomeres by promoting Ccq1-Est1 interaction (Moser et al., 2011Moser B.A. Chang Y.-T. Kosti J. Nakamura T.M. Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast.Nat. Struct. Mol. Biol. 2011; 18: 1408-1413Crossref PubMed Scopus (62) Google Scholar, Webb and Zakian, 2012Webb C.J. Zakian V.A. Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase-telomere association.Genes Dev. 2012; 26: 82-91Crossref PubMed Scopus (30) Google Scholar, Yamazaki et al., 2012Yamazaki H. Tarumoto Y. Ishikawa F. Tel1(ATM) and Rad3(ATR) phosphorylate the telomere protein Ccq1 to recruit telomerase and elongate telomeres in fission yeast.Genes Dev. 2012; 26: 241-246Crossref PubMed Scopus (52) Google Scholar). Telomerase recruitment occurs in S/G2 phase transition concomitantly with telomere replication (Moser et al., 2009aMoser B.A. Subramanian L. Chang Y.-T. Noguchi C. Noguchi E. Nakamura T.M. Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres.EMBO J. 2009; 28: 810-820Crossref PubMed Scopus (65) Google Scholar). As in mammalian cells, the replication forks slow down in the proximity of telomeric repeats in fission yeast (Miller et al., 2006Miller K.M. Rog O. Cooper J.P. Semi-conservative DNA replication through telomeres requires Taz1.Nature. 2006; 440: 824-828Crossref PubMed Scopus (200) Google Scholar), and efficient replication is required for telomere maintenance (Chang et al., 2013Chang Y.-T. Moser B.A. Nakamura T.M. Fission yeast shelterin regulates DNA polymerases and Rad3(ATR) kinase to limit telomere extension.PLoS Genet. 2013; 9: e1003936Crossref PubMed Scopus (26) Google Scholar, Moser et al., 2009bMoser B.A. Subramanian L. Khair L. Chang Y.-T. Nakamura T.M. Fission yeast Tel1(ATM) and Rad3(ATR) promote telomere protection and telomerase recruitment.PLoS Genet. 2009; 5: e1000622Crossref PubMed Scopus (48) Google Scholar). Indeed, telomeric proteins such as Taz1 and the Stn1-Ten1 complex are known to promote efficient replication of telomeres (Matmati et al., 2018Matmati S. Vaurs M. Escandell J.M. Maestroni L. Nakamura T.M. Ferreira M.G. Géli V. Coulon S. The fission yeast Stn1-Ten1 complex limits telomerase activity via its SUMO-interacting motif and promotes telomeres replication.Sci. Adv. 2018; 4: eaar2740Crossref PubMed Scopus (10) Google Scholar, Miller et al., 2006Miller K.M. Rog O. Cooper J.P. Semi-conservative DNA replication through telomeres requires Taz1.Nature. 2006; 440: 824-828Crossref PubMed Scopus (200) Google Scholar, Takikawa et al., 2017Takikawa M. Tarumoto Y. Ishikawa F. Fission yeast Stn1 is crucial for semi-conservative replication at telomeres and subtelomeres.Nucleic Acids Res. 2017; 45: 1255-1269Crossref PubMed Scopus (14) Google Scholar) as well as the RPA heterotrimer and Pfh1 helicase (Audry et al., 2015Audry J. Maestroni L. Delagoutte E. Gauthier T. Nakamura T.M. Gachet Y. Saintomé C. Géli V. Coulon S. RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.EMBO J. 2015; 34: 1942-1958Crossref PubMed Scopus (62) Google Scholar, Luciano et al., 2012Luciano P. Coulon S. Faure V. Corda Y. Bos J. Brill S.J. Gilson E. Simon M.N. Géli V. RPA facilitates telomerase activity at chromosome ends in budding and fission yeasts.EMBO J. 2012; 31: 2034-2046Crossref PubMed Scopus (35) Google Scholar, McDonald et al., 2014McDonald K.R. Sabouri N. Webb C.J. Zakian V.A. The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening.DNA Repair (Amst.). 2014; 24: 80-86Crossref PubMed Scopus (24) Google Scholar). In fission yeast, deleting either of the protein subunits of telomerase or its RNA template leads to replicative senescence (Nakamura et al., 1998Nakamura T.M. Cooper J.P. Cech T.R. Two modes of survival of fission yeast without telomerase.Science. 1998; 282: 493-496Crossref PubMed Scopus (228) Google Scholar, Webb and Zakian, 2008Webb C.J. Zakian V.A. Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA.Nat. Struct. Mol. Biol. 2008; 15: 34-42Crossref PubMed Scopus (88) Google Scholar). In the absence of telomerase activity, telomeres gradually shorten until the cells either cease dividing or die (crisis). This definitive arrest is caused by a DNA damage checkpoint that is activated as a result of unprotected short telomeres being recognized as irreparable double-strand breaks (DSBs). In S. cerevisiae, cell division capacity declines with time in the absence of telomerase activity due to the occurrence of stochastic events during replicative senescence that slow down or stop cell cycle (Churikov et al., 2016Churikov D. Charifi F. Eckert-Boulet N. Silva S. Simon M.N. Lisby M. Géli V. SUMO-dependent relocalization of eroded telomeres to nuclear pore complexes controls telomere recombination.Cell Rep. 2016; 15: 1242-1253Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, Churikov et al., 2014Churikov D. Charifi F. Simon M.N. Géli V. Rad59-facilitated acquisition of Y′ elements by short telomeres delays the onset of senescence.PLoS Genet. 2014; 10: e1004736Crossref PubMed Scopus (25) Google Scholar, Xu et al., 2015Xu Z. Fallet E. Paoletti C. Fehrmann S. Charvin G. Teixeira M.T. Two routes to senescence revealed by real-time analysis of telomerase-negative single lineages.Nat. Commun. 2015; 6: 7680Crossref PubMed Scopus (34) Google Scholar). This led us to investigate the replication dynamic in S. pombe cells lacking telomerase to unmask the replication stress occurring at telomeres (Simon et al., 2016Simon M.N. Churikov D. Géli V. Replication stress as a source of telomere recombination during replicative senescence in Saccharomyces cerevisiae.FEMS Yeast Res. 2016; 16 (fow085)Crossref PubMed Scopus (18) Google Scholar). Our results indicate that telomerase repairs collapsed telomeric replication forks likely by binding to reversed fork. To investigate the replication dynamic at telomeres in cells lacking telomerase, we performed two-dimensional (2D) gel electrophoresis analysis as previously described (Audry et al., 2015Audry J. Maestroni L. Delagoutte E. Gauthier T. Nakamura T.M. Gachet Y. Saintomé C. Géli V. Coulon S. RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.EMBO J. 2015; 34: 1942-1958Crossref PubMed Scopus (62) Google Scholar, Miller et al., 2006Miller K.M. Rog O. Cooper J.P. Semi-conservative DNA replication through telomeres requires Taz1.Nature. 2006; 440: 824-828Crossref PubMed Scopus (200) Google Scholar). We performed replicative senescence kinetics (Webb and Zakian, 2008Webb C.J. Zakian V.A. Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA.Nat. Struct. Mol. Biol. 2008; 15: 34-42Crossref PubMed Scopus (88) Google Scholar) by sampling daily dilutions of the liquid cultures established from the ter1Δ colonies. To ensure unique arrangement of the subtelomeric region, the ter1Δ colonies were obtained by transformation of a single parental strain (Figure 1A). In this representative experiment, crisis was reached after 9 days of growth corresponding to approximately 100–110 population doublings. Southern blot performed with a telomeric probe showed that crisis was concomitant with the disappearance of telomeric signal (Figure 1B). We next performed the first dimension of the 2D gel analysis of the ter1+ and ter1Δ cells (Figure 1C). This Southern blot was revealed with a subtelomeric probe (STE1) to identify subtelomeric regions (NsiI pattern) of S. pombe strains allowing to predict 2D gel profile. In the ter1+ parental strain NsiI digestion of genomic DNA released four telomere-containing restriction fragments from the six chromosome ends (Figure 1C, left panel), indicating that up to four Y-arcs might be detected after the second dimension. In telomerase-negative cells (ter1Δ), migration in the first dimension at different time points of the senescence showed that NsiI fragments faded with time and that additional high-molecular-weight bands and a smeared signal appeared (Figure 1C, right panel). This indicates that several additional replication intermediates (RIs) might be detected by the second dimension of ter1Δ samples. Migration in the second dimension of the ter1+ sample revealed three Y-arcs containing strong pausing sites (Figure 1D, left panel). In agreement with the pattern observed in the first dimension, migration in the second dimension of the telomerase-negative samples revealed two Y-arcs at days 2 and 4 (Figure 1D). At day 4, the Y-arc signal slowly faded and spread into RIs of higher molecular complexity fragments that include Y-arcs like structures and likely X-shaped structures. At day 6, Y-arcs were barely visible, signal became fuzzy, and high-molecular-weight structures (HMWs) were detected, mimicking to some extent taz1Δ phenotype (Miller et al., 2006Miller K.M. Rog O. Cooper J.P. Semi-conservative DNA replication through telomeres requires Taz1.Nature. 2006; 440: 824-828Crossref PubMed Scopus (200) Google Scholar) (Figure 1D). At day 8, RIs were undistinguishable while a single telomeric arc (T-arc) was detectable as previously observed in rpa1D223Y mutant reflecting severe replication defects (Audry et al., 2015Audry J. Maestroni L. Delagoutte E. Gauthier T. Nakamura T.M. Gachet Y. Saintomé C. Géli V. Coulon S. RPA prevents G-rich structure formation at lagging-strand telomeres to allow maintenance of chromosome ends.EMBO J. 2015; 34: 1942-1958Crossref PubMed Scopus (62) Google Scholar). Another example of a ter1Δ clone for which we analyzed in parallel the senescence profile and RIs by 2D gel is shown in Figure S1. Importantly, the appearance of these abnormal RIs correlated with the senescence profile, suggesting that the loss of growth capacity is related to replication defects that would be normally processed when telomerase is present. To discriminate whether RIs accumulation resulted from a telomerase recruitment defect or telomere shortening, we monitored replication dynamics of the tpz1K75A mutant that has short telomeres. Although the tpz1K75A mutation lies in the TEL patch-like domain of Tpz1, it affects telomere elongation by telomerase rather than telomerase recruitment to telomeres (Armstrong et al., 2014Armstrong C.A. Pearson S.R. Amelina H. Moiseeva V. Tomita K. Telomerase activation after recruitment in fission yeast.Curr. Biol. 2014; 24: 2006-2011Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar). In the tpz1+ parental strain (tpz1-HA), analysis of the NsiI pattern revealed multiple subtelomeric fragments that appeared as Y-arcs in the second dimension (Figures 1E and 1F). In the tpz1K75A mutant, we observed a strong accumulation of additional RIs including HMWs and a T-arc that was barely visible (Figure 1F). This result suggests that telomere shortening per se induces a replication stress; however, this stress may also originate from the low processivity of telomerase in the tpz1K75A mutant. Finally, we wanted to determine whether the lack of the main DNA damage response kinase Rad3, which is also involved in telomerase recruitment, affects senescence profiles in telomerase-negative cells. The rad3Δ trt1Δ cells were obtained from tetrad dissection of the rad3+/rad3Δ trt1+/trt1Δ diploid strain that displayed wild-type telomere length (Figure S1D). As previously described (Nakamura et al., 2002Nakamura T.M. Moser B.A. Russell P. Telomere binding of checkpoint sensor and DNA repair proteins contributes to maintenance of functional fission yeast telomeres.Genetics. 2002; 161: 1437-1452Crossref PubMed Google Scholar), rad3Δ trt1Δ clones had a slow growth phenotype, and their senescence profiles were more heterogeneous than those of the trt1Δ clones (Figure S1E). Because of the slow growth phenotype of rad3Δ trt1Δ mutant, it was difficult to obtain a sufficient amount of cells to perform 2D gel analysis. To assess whether the absence of Trt1 activates a DNA damage response (DDR), we monitored checkpoint activation by following Chk1-myc and Cds1-myc phosphorylation in trt1Δ cells at different time points of the senescence (Figures S1F–S1H). Phosphorylation of Chk1 was detected only at time points of the senescence preceding the crisis, while phosphorylation of Cds1 was not observed. Taken together, these data indicate that telomere replication in the absence of telomerase does not activate the phosphorylation of these two kinases that act downstream of the Rad3 during canonical DDR. Homologous recombination (HR) requires Exo1-dependent DNA resection to promote homology search and strand invasion at DSBs (Langerak et al., 2011Langerak P. Mejia-Ramirez E. Limbo O. Russell P. Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks.PLoS Genet. 2011; 7: e1002271Crossref PubMed Scopus (167) Google Scholar, Mimitou and Symington, 2008Mimitou E.P. Symington L.S. Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.Nature. 2008; 455: 770-774Crossref PubMed Scopus (767) Google Scholar, Zhu et al., 2008Zhu Z. Chung W.-H. Shim E.Y. Lee S.E. Ira G. Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.Cell. 2008; 134: 981-994Abstract Full Text Full Text PDF PubMed Scopus (790) Google Scholar). Recently, Exo1 was reported to also process terminally arrested forks in S. pombe (Ait Saada et al., 2017Ait Saada A. Teixeira-Silva A. Iraqui I. Costes A. Hardy J. Paoletti G. Fréon K. Lambert S.A.E. Unprotected replication forks are converted into mitotic sister chromatid bridges.Mol. Cell. 2017; 66: 398-410.e4Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). HR, as well as Ku, were both reported to restrain the Exo1-nucleolytic activity at arrested forks (Ait Saada et al., 2017Ait Saada A. Teixeira-Silva A. Iraqui I. Costes A. Hardy J. Paoletti G. Fréon K. Lambert S.A.E. Unprotected replication forks are converted into mitotic sister chromatid bridges.Mol. Cell. 2017; 66: 398-410.e4Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, Teixeira-Silva et al., 2017Teixeira-Silva A. Ait Saada A. Hardy J. Iraqui I. Nocente M.C. Fréon K. Lambert S.A.E. The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks.Nat. Commun. 2017; 8: 1982Crossref PubMed Scopus (60) Google Scholar). We thus examined the senescence profile and telomere RIs by 2D gel in the exo1Δ trt1Δ cells obtained from tetrad dissection of the exo1+/exo1Δ trt1+/trt1Δ diploid strain. In contrast to budding yeast in which deletion of exo1 does not impair growth of telomerase minus cells (est2Δ) (Bertuch and Lundblad, 2004Bertuch A.A. Lundblad V. EXO1 contributes to telomere maintenance in both telomerase-proficient and telomerase-deficient Saccharomyces cerevisiae.Genetics. 2004; 166: 1651-1659Crossref PubMed Scopus (80) Google Scholar), we found that the absence of Exo1 accelerated senescence in S. pombe (crisis at day 7; Figure 2A) and resulted in the rapid loss of telomeres (Figure 2B), suggesting that Exo1 is required to support efficient telomere maintenance in the absence of telomerase. In exo1Δ trt1+ cells, analysis of NsiI pattern revealed three subtelomeric fragments of a similar size (Figure 2C, left panel) that appeared in the second dimension as a thick Y-arc with marked pausing sites (Figure 2D, left panel). X-shaped molecules emanated from the main pausing site and some RIs were also detected (red arrow). Although telomeres are mainly replicated as simple Y-arcs, these results indicate that Exo1 participates in telomere replication in telomerase-positive cells. In exo1Δ trt1Δ cells, telomere shortening occurred prematurely (Figure 2B). The NsiI pattern observed with exo1Δ trt1Δ clone in the first dimension is different from exo1Δ trt1+ because exo1Δ trt1Δ clones were obtained from tetrad dissection of the exo1+/exo1Δ trt1+/trt1Δ diploid strain (Figure 2C). 2D gel analysis revealed numerous RIs with atypical appearance (Figure 2D). In the exo1Δ trt1Δ mutant, RIs were detected at earlier time points compared with the single trt1Δ mutant (Figure 1D). Remarkably, Y-arc like structures, X-shaped structures, HMWs, and T-arcs progressively accumulated with ongoing replicative senescence. We inferred from these results that Exo1 processes RIs, especially in the absence of telomerase. In exo1Δ trt1Δ cells, accumulation of RIs correlated with accelerated telomere shortening and senescence. These results support the hypothesis that telomerase promotes efficient replication of telomeres by preventing engagement of the telomeric RIs in HR. To investigate the roles of HR during telomeres replication in telomerase-negative cells, we constructed trt1Δ/trt1+ diploid strain combined with either rad51, mre11, or ctp1 deletions. Tetrad analysis revealed that the spores bearing trt1Δ and rad51Δ deletions were either synthetically lethal or extremely sick, preventing their propagation (Figure 3A). The same phenotype was observed when trt1Δ was associated with rad51-II3A (Figure 3B), a mutant of Rad51 that is able to form a stable nucleoprotein filament on single-stranded DNA (ssDNA) but unable to perform the strand-exchange reaction (Ait Saada et al., 2017Ait Saada A. Teixeira-Silva A. Iraqui I. Costes A. Hardy J. Paoletti G. Fréon K. Lambert S.A.E. Unprotected replication forks are converted into mitotic sister chromatid bridges.Mol. Cell. 2017; 66: 398-410.e4Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Chromatin immunoprecipitation (ChIP) experiments indicated that Rad51 was recruited to telomeres during replicative senescence (Figures 3C and S2), its binding being maximum at day 4 concomitantly with the visualization of Y-arcs-like structures, X-shaped molecules, and HMWs by 2D gel (Figure 1D). We concluded that Rad51 fulfills essential functions in the absence of telomerase, likely by processing RIs through strand-exchange reaction. Rad55 is known to assist Rad51 in the formation of the nucleoprotein filament. Although trt1Δ rad55Δ spore was viable, we were unable to further grow double mutants in liquid culture, thus confirming the essential role of HR in the absence of telomerase (Figure 3D). MRN (Mre11-Rad50-Nbs1) complex and its co-factor Ctp1 are required for DSB repair by HR. MRN-Ctp1 have been proposed to release Ku from DNA ends and to initiate resection (Jensen and Russell, 2016Jensen K.L. Russell P. Ctp1-dependent clipping and resection of DNA double-strand breaks by Mre11 endonuclease complex are not genetically separable.Nucleic Acids Res. 2016; 44: 8241-8249Crossref PubMed Scopus (12) Google Scholar, Langerak et al., 2011Langerak P. Mejia-Ramirez E. Limbo O. Russell P. Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks.PLoS Genet. 2011; 7: e1002271Crossref PubMed Scopus (167) Google Scholar, Limbo et al., 2007Limbo O. Chahwan C. Yamada Y. de Bruin R.A.M. Wittenberg C. Russell P. Ctp1 is a cell-cycle-regulated protein that functions with Mre11 complex to control double-strand break repair by homologous recombination.Mol. Cell. 2007; 28: 134-146Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Tetrad analysis revealed that mre11Δ trt1Δ and ctp1Δ trt1Δ were synthetically lethal or sick (Figures 3E and 3F), showing that both are required in the absence of telomerase. When trt1Δ was combined with the mre11-D65N mutant that is deficient in nuclease activity but proficient in MRN complex formation (Hartsuiker et al., 2009Hartsuiker E. Neale M.J. Carr A.M. Distinct requirements for the Rad32(Mre11) nuclease and Ctp1(CtIP) in the removal of covalently bound topoisomerase I and II from DNA.Mol. Cell. 2009; 33: 117-123Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), the double mutant was viable in contrast to the mre11Δ trt1Δ mutant (Figure S3A). Nevertheless, this mutation accelerated senescence since the crisis was reached at day 5 (Figure S3B), suggesting that both the nuclease activity of Mre11 and the structural function of MRN were required to maintain telomeres in the absence of telomerase. Taken together, these results show that Rad51, MRN, and Ctp1 are essential factors in telomerase minus cells that ensure telomere maintenance and sustain cell viability. Noteworthy, we observed that the Rad8 ubiquitin ligase/DNA helicase was dispensable for viability of telomerase-negative cells during senescence (Figures S3C and S3D), although its S. cerevisiae counterpart Rad5 has been shown to promote the viability of cells in the absence of telomerase (Fallet et al., 2014Fallet E. Jolivet P. Soudet J. Lisby M. Gilson E. Teixeira M.T. Length-dependent processing of telomeres in the absence of telomerase.Nucleic Acids Res. 2014; 42: 3648-3665Crossref PubMed Scopus (52) Google Scholar). Taz1 is known to promote efficient replication of telomeric DNA (Miller et al., 2006Miller K.M. Rog O. Cooper J.P. Semi-conservative DNA replication through telomeres requires Taz1.Nature. 2006; 440: 824-828Crossref PubMed Scopus (200) Google Scholar). Indeed, Taz1 loss leads to stalled replication forks, and telomere maintenance in taz1Δ cells relies on the telomerase, which is recruited throughout the cell cycle (Dehé et al., 2012Dehé P.-M. Rog O. Ferreira M.G. Greenwood J. Cooper J.P. Taz1 enforces cell-cycle regulation of telomere synthesis.Mol. Cell. 2012; 46: 797-808Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Because taz1Δ cells accumulate RIs, we wondered how deletion of rad51 and mre11 would impact the viability of the double mutants. Like rad51Δ trt1Δ, the synthetic lethality (or sickness) between rad51Δ and taz1Δ did not allow us to propagate rad51Δ taz1Δ cells (Figure 3G). In contrast, the absence of Mre11 neither impaired the growth of taz1Δ mutant (Figure 3H) nor its telomere maintenance (Figure 3J). Thus, while Rad51 is required to sustain viability in the absence of either Trt1 or Taz1, Mre11 is only required in the absence of Trt1, while it is dispensable in taz1Δ cells pointing out the specific role of Mre11 in telomerase-negative cells. Along the same lines, we observed that the deletion of pku70 in the absence of telomerase was deleterious for cell viability, although pku70Δ trt1Δ spores were able to form colonies (Figures 4A and 4B ), consistent with previous observations (Baumann and Cech, 2000Baumann P. Cech T.R. Protection of telomeres by the Ku protein in fission yeast.Mol. Biol. Cell. 2000; 11: 3265-3275Crossref PubMed Scopus (127) Google Scholar). Of note, this contrasts with the absence of genetic interaction between taz1Δ and pku70Δ (Figures 3I and 3J). ChIP experiments revealed that Ku70 was recruited at early time points of the replicative senescence (Figure 4C). Because it has been shown recently that Ku complex can load onto the terminally arrested forks that undergo reversal (Teixeira-Silva et al., 2017Teixeira-Silva A. Ait Saada A. Hardy J. Iraqui I. Nocente M.C. Fréon K. Lambert S.A.E. The end-joining factor Ku acts in the end-resection of double strand break-free arrested replication forks.Nat. Commun. 2017; 8: 1982Crossref PubMed Scopus (60) Google Scholar), we hypothesiz" @default.
• W3012500692 created "2020-03-23" @default.
• W3012500692 creator A5005900340 @default.
• W3012500692 creator A5007222749 @default.
• W3012500692 creator A5034247890 @default.
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• W3012500692 date "2020-03-01" @default.
• W3012500692 modified "2023-10-16" @default.
• W3012500692 title "Telomerase Repairs Collapsed Replication Forks at Telomeres" @default.
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