MEC3, MEC1, and DDC2 are essential components of a telomere checkpoint pathway required for cell cycle arrest during senescence in Saccharomyces cerevisiae

When telomerase is absent and/or telomeres become critically short, cells undergo a progressive decline in viability termed senescence. The telomere checkpoint model predicts that cells will respond to a damaged or critically short telomere by transiently arresting and activating repair of the telomere. We examined the senescence of telomerase-deficient Saccharomyces cerevisiae at the cellular level to ask if the loss of telomerase activity triggers a checkpoint response. As telomerase-deficient mutants were serially subcultured, cells exhibited a progressive decline in average growth rate and an increase in the number of cells delayed in the G2/M stage of the cell cycle. MEC3, MEC1, and DDC2, genes important for the DNA damage checkpoint response, were required for the cell cycle delay in telomerase-deficient cells. In contrast, TEL1, RAD9, and RAD53, genes also required for the DNA damage checkpoint response, were not required for the G2/M delay in telomerase-deficient cells. We propose that the telomere checkpoint is distinct from the DNA damage checkpoint and requires a specific set of gene products to delay the cell cycle and presumably to activate telomerase and/or other telomere repair activities.     

Two pathways recruit telomerase to Saccharomyces cerevisiae telomeres

The catalytic subunit of yeast telomerase, Est2p, is a telomere associated throughout most of the cell cycle, while the Est1p subunit binds only in late S/G2 phase, the time of telomerase action. Est2p binding in G1/early S phase requires a specific interaction between telomerase RNA (TLC1) and Ku80p. Here, we show that in four telomerase-deficient strains (cdc13-2, est1Ä, tlc1-SD, and tlc1-BD), Est2p telomere binding was normal in G1/early S phase but reduced to about 40–50% of wild type levels in late S/G2 phase. Est1p telomere association was low in all four strains. Wild type levels of Est2p telomere binding in late S/G2 phase was Est1p-dependent and required that Est1p be both telomere-bound and associated with a stem-bulge region in TLC1 RNA. In three telomerase-deficient strains in which Est1p is not Est2p-associated (tlc1-SD, tlc1-BD, and est2Ä), Est1p was present at normal levels but its telomere binding was very low. When the G1/early S phase and the late S/G2 phase telomerase recruitment pathways were both disrupted, neither Est2p nor Est1p was telomere-associated. We conclude that reduced levels of Est2p and low Est1p telomere binding in late S/G2 phase correlated with an est phenotype, while a WT level of Est2p binding in G1 was not sufficient to maintain telomeres. In addition, even though Cdc13p and Est1p interact by two hybrid, biochemical and genetic criteria, this interaction did not occur unless Est1p was Est2p-associated, suggesting that Est1p comes to the telomere only as part of the holoenzyme. Finally, the G1 and late S/G2 phase pathways for telomerase recruitment are distinct and are likely the only ones that bring telomerase to telomeres in wild-type cells.     

The Saccharomyces cerevisiae WRN homolog Sgs1p participates in telomere maintenance in cells lacking telomerase

Werner syndrome (WS) is marked by early onset of features resembling aging, and is caused by loss of the RecQ family DNA helicase WRN. Precisely how loss of WRN leads to the phenotypes of WS is unknown. Cultured WS fibroblasts shorten their telomeres at an increased rate per population doubling and the premature senescence this loss induces can be bypassed by telomerase. Here we show that WRN co-localizes with telomeric factors in telomerase-independent immortalized human cells, and further that the budding yeast RecQ family helicase Sgs1p influences telomere metabolism in yeast cells lacking telomerase. Telomerase-deficient sgs1 mutants show increased rates of growth arrest in the G2/M phase of the cell cycle as telomeres shorten. In addition, telomerase-deficient sgs1 mutants have a defect in their ability to generate survivors of senescence that amplify telomeric TG1-3 repeats, and SGS1 functions in parallel with the recombination gene RAD51 to generate survivors. Our findings indicate that Sgs1p and WRN function in telomere maintenance, and suggest that telomere defects contribute to the pathogenesis of WS and perhaps other RecQ helicase diseases.     

The Est1 subunit of yeast telomerase binds the Tlc1 telomerase RNA

Est1 is a component of yeast telomerase, and est1 mutants have senescence and telomere loss phenotypes. The exact function of Est1 is not known, and it is not homologous to components of other telomerases. We previously showed that Est1 protein coimmunoprecipitates with Tlc1 (the telomerase RNA) as well as with telomerase activity. Est1 has homology to Ebs1, an uncharacterized yeast open reading frame product, including homology to a putative RNA recognition motif (RRM) of Ebs1. Deletion of EBS1 results in short telomeres. We created point mutations in a putative RRM of Est1. One mutant was unable to complement either the senescence or the telomere loss phenotype of est1 mutants. Furthermore, the mutant protein no longer coprecipitated with the Tlc1 telomerase RNA. Mutants defective in the binding of Tlc1 RNA were nevertheless capable of binding single-stranded TG-rich DNA. Our data suggest that an important role of Est1 in the telomerase complex is to bind to the Tlc1 telomerase RNA via an RRM. Since Est1 can also bind telomeric DNA, Est1 may tether telomerase to the telomere.     

“Poisoning” yeast telomeres distinguishes between redundant telomere capping pathways

In most eukaryotes, telomeres are composed of tandem arrays of species-specific DNA repeats ending with a G-rich 3′ overhang. In budding yeast, Cdc13 binds this overhang and recruits Ten1–Stn1 and the telomerase protein Est1 to protect (cap) and elongate the telomeres, respectively. To dissect and study the various pathways employed to cap and maintain the telomere end, we engineered telomerase to incorporate Tetrahymena telomeric repeats (G₄T₂) onto the telomeres of the budding yeast Kluyveromyces lactis. These heterologous repeats caused telomere–telomere fusions, cell cycle arrest at G2/M, and severely reduced viability—the hallmarks of telomere uncapping. Fusing Cdc13 or Est1 to universal minicircle sequence binding protein (UMSBP), a small protein that binds the single-stranded G₄T₂ repeats, rescued the cell viability and restored telomere capping, but not telomerase-mediated telomere maintenance. Surprisingly, Cdc13–UMSBP-mediated telomere capping was dependent on the homologous recombination factor Rad52, while Est1–UMSBP was not. Thus, our results distinguish between two, redundant, telomere capping pathways.     

Telomere maintenance in fission yeast requires an Est1 ortholog

Telomerase regulation is critical to genome maintenance yet remains poorly understood. Without telomerase's ability to synthesize telomere repeats, chromosome ends shorten progressively, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. In Saccharomyces cerevisiae, telomerase activity in vivo absolutely depends on a set of telomerase accessory proteins that includes Est1p, which appears to recruit or activate telomerase at the site of polymerization. Thus, est1Delta cells have the same cellular senescence phenotype as cells lacking either the catalytic protein subunit of telomerase or its template-containing RNA subunit. While the telomerase protein is highly conserved among eukaryotes, the apparent lack of Est1p homologs has frustrated efforts to describe a common mechanism of telomerase recruitment and activation. Here, we describe SpEst1p, a homolog of Est1p from the evolutionarily distant Schizosaccharomyces pombe. Like ScEst1p, SpEst1p is required for telomerase activity in vivo. Coupled with the identification of an orthologous Est1 protein in humans [10], this suggests a much wider conservation of telomerase regulation than was previously known. Strikingly, in cells with compromised telomere function (taz1Delta), SpEst1p loss confers a lethal germination phenotype, while telomerase loss does not, indicating that SpEst1p plays an unexpected additional role in chromosome end protection.     

The Rap1p-telomere complex does not determine the replicative capacity of telomerase-deficient yeast

Telomeres are nucleoprotein structures that cap the ends of chromosomes and thereby protect their stability and integrity. In the presence of telomerase, the enzyme that synthesizes telomeric repeats, telomere length is controlled primarily by Rap1p, the budding yeast telomeric DNA binding protein which, through its C-terminal domain, nucleates a protein complex that limits telomere lengthening. In the absence of telomerase, telomeres shorten with every cell division, and eventually, cells enter replicative senescence. We have set out to identify the telomeric property that determines the replicative capacity of telomerase-deficient budding yeast. We show that in cells deficient for both telomerase and homologous recombination, replicative capacity is dependent on telomere length but not on the binding of Rap1p to the telomeric repeats. Strikingly, inhibition of Rap1p binding or truncation of the C-terminal tail of Rap1p in Kluyveromyces lactis and deletion of the Rap1p-recruited complex in Saccharomyces cerevisiae lead to a dramatic increase in replicative capacity. The study of the role of telomere binding proteins and telomere length on replicative capacity in yeast may have significant implications for our understanding of cellular senescence in higher organisms.     

Cdc13 prevents telomere uncapping and Rad50-dependent homologous recombination.

Cdc13 performs an essential function in telomere end protection in budding yeast. Here, we analyze the consequences on telomere dynamics of cdc13-induced telomeric DNA damage in proliferating cells. Checkpoint-deficient cdc13-1 cells accumulated DNA damage and eventually senesced. However, these telomerase-proficient cells could survive by using homologous recombination but, contrary to telomerase-deficient cells, did so without prior telomere shortening. Strikingly, homologous recombination in cdc13-1 mec3, as well as in telomerase-deficient cdc13-1 cells, which were Rad52- and Rad50-dependent but Rad51-independent, exclusively amplified the TG(1-3) repeats. This argues that not only short telomeres are substrates for type II recombination. The Cdc13-1 mutant protein harbored a defect in its association with Stn1 and Ten1 but also an additional, unknown, defect that could not be cured by expressing a Cdc13-1- Ten1-Stn1 fusion. We propose that Cdc13 prevents telomere uncapping and inhibits recombination between telomeric sequences through a pathway distinct from and complementary to that used by telomerase.     

Telomerase and ATM/Tel1p protect telomeres from nonhomologous end joining

Telomeres protect chromosome ends from fusing to double-stranded breaks (DSBs). Using a quantitative real-time PCR assay, we show that nonhomologous end joining between a telomere and an inducible DSB was undetectable in wild-type cells, but occurred within a few hours of DSB induction in approximately 1/2000 genomes in telomerase-deficient cells and in >1/1000 genomes in telomerase-deficient cells also lacking the ATM homolog Tel1p. The fused telomeres contained very little telomeric DNA, suggesting that catastrophic telomere shortening preceded fusion. Lengthening of telomeres did not prevent such catastrophic telomere shortening and fusion events. Telomere-DSB fusion also occurred in cells containing a catalytically inactive telomerase and in tel1 mec1 cells where telomerase cannot elongate telomeres. Thus, telomerase and Tel1p function in telomere protection as well as in telomere elongation.     

The role of the Mre11-Rad50-Xrs2 complex in telomerase- mediated lengthening of Saccharomyces cerevisiae telomeres.

BACKGROUND: The Saccharomyces Mre11p, Rad50p, and Xrs2p proteins form a complex, called the MRX complex, that is required to maintain telomere length. Cells lacking any one of the three MRX proteins and Mec1p, an ATM-like protein kinase, undergo telomere shortening and ultimately die, phenotypes characteristic of cells lacking telomerase. The other ATM-like yeast kinase, Tel1p, appears to act in the same pathway as MRX: mec1 tel1 cells have telomere phenotypes similar to those of telomerase-deficient cells, whereas the phenotypes of tel1 cells are not exacerbated by the loss of a MRX protein. RESULTS: The nuclease activity of Mre11p was found to be dispensable for the telomerase-promoting activity of the MRX complex. The association of the single-stranded TG1-3 DNA binding protein Cdc13p with yeast telomeres occurred efficiently in the absence of Tel1p, Mre11p, Rad50p, or Xrs2p. Targeting of catalytically active telomerase to the telomere suppressed the senescence phenotype of mec1 mrx or mec1 tel1 cells. Moreover, when telomerase was targeted to telomeres, telomere lengthening was robust in mec1 mrx and mec1 tel1 cells. CONCLUSIONS: These data rule out models in which the MRX complex is necessary for Cdc13p binding to telomeres or in which the MRX complex is necessary for the catalytic activity of telomerase. Rather, the data suggest that the MRX complex is involved in recruiting telomerase activity to yeast telomeres.     

Ten1 functions in telomere end protection and length regulation in association with Stn1 and Cdc13

In Saccharomyces cerevisiae, Cdc13 has been proposed to mediate telomerase recruitment at telomere ends. Stn1, which associates with Cdc13 by the two-hybrid interaction, has been implicated in telomere maintenance. Ten1, a previously uncharacterized protein, was found to associate physically with both Stn1 and Cdc13. A binding defect between Stn1-13 and Ten1 was responsible for the long telomere phenotype of stn1-13 mutant cells. Moreover, rescue of the cdc13-1 mutation by STN1 was much improved when TEN1 was simultaneously overexpressed. Several ten1 mutations were found to confer telomerase-dependent telomere lengthening. Other, temperature-sensitive, mutants of TEN1 arrested at G(2)/M via activation of the Rad9-dependent DNA damage checkpoint. These ten1 mutant cells were found to accumulate single-stranded DNA in telomeric regions of the chromosomes. We propose that Ten1 is required to regulate telomere length, as well as to prevent lethal damage to telomeric DNA.     

Saccharomyces cerevisiae Est3p dimerizes in vitro and dimerization contributes to efficient telomere replication in vivo

In Saccharomyces cerevisiae at least five genes, EST1, EST2, EST3, TLC1 and CDC13, are required for telomerase activity in vivo. The telomerase catalytic subunit Est2p and telomerase RNA subunit Tlc1 constitute the telomerase core enzyme. Est1p and Est3p are the other subunits of telomerase holoenzyme. In order to dissect the function of Est3p in telomere replication, we over-expressed and purified recombinant wild-type and mutant Est3 proteins. The wild-type protein, as well as the K71A, E104A and T115A mutants were able to dimerize in vitro, while the Est3p-D49A, -K68A or -D166A mutant showed reduced ability to dimerize. Mutations in Est3p that decreased dimerization also appeared to cause telomere shortening in vivo. Double point mutation of Est3p-D49A-K68A and single point mutation of Est3p-K68A showed similar telomere shortening, suggesting that the K68 residue might be more important for telomerase activity. The ectopic co-expression of K71A or T115A mutant with wild-type Est3p using centromere plasmids caused telomere shortening, while co-expression of the D49A, K68A, D86A or F103A mutants with wild-type Est3p had no effect on telomere length regulation. These data suggested that dimerization is important for Est3p function in vivo.     

The Rad51 pathway of telomerase-independent maintenance of telomeres can amplify TG1-3 sequences in yku and cdc13 mutants of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence. We report here that cdc13-1 yku70delta mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events. cdc13-1 yku70delta cells survived telomere deprotection by exclusively amplifying TG(1-3) repeats (type II recombination). In a background lacking telomerase (tlc1delta), this process predominated over type I recombination (amplification of subtelomeric Y' sequences). Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70delta or yku70delta tlc1delta mutants, but also in cdc13-1 YKU70(+) tlc1delta mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway. In addition, delayed senescence was observed in cdc13-1 yku70delta rad51delta and cdc13-1 tlc1delta rad51delta cells. These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG(1-3) sequences. They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70.     

Stn1 is critical for telomere maintenance and long‐term viability of somatic human cells

Disruption of telomere maintenance pathways leads to accelerated entry into cellular senescence, a stable proliferative arrest that promotes aging‐associated disorders in some mammals. The budding yeast CST complex, comprising Cdc13, Stn1, and Ctc1, is critical for telomere replication, length regulation, and end protection. Although mammalian homologues of CST have been identified recently, their role and function for telomere maintenance in normal somatic human cells are still incompletely understood. Here, we characterize the function of human Stn1 in cultured human fibroblasts and demonstrate its critical role in telomere replication, length regulation, and function. In the absence of high telomerase activity, shRNA‐mediated knockdown of hStn1 resulted in aberrant and fragile telomeric structures, stochastic telomere attrition, increased telomere erosion rates, telomere dysfunction, and consequently accelerated entry into cellular senescence. Oxidative stress augmented the defects caused by Stn1 knockdown leading to almost immediate cessation of cell proliferation. In contrast, overexpression of hTERT suppressed some of the defects caused by hStn1 knockdown suggesting that telomerase can partially compensate for hStn1 loss. Our findings reveal a critical role for human Stn1 in telomere length maintenance and function, supporting the model that efficient replication of telomeric repeats is critical for long‐term viability of normal somatic mammalian cells.     

Stimulation of yeast telomerase activity by the ever shorter telomere 3 (Est3) subunit is dependent on direct interaction with the catalytic protein Est2

Telomerase is a multisubunit enzyme that maintains genome stability through its role in telomere replication. Although the Est3 protein is long recognized as an essential telomerase component, how it associates with and functions in the telomerase complex has remained enigmatic. Here we provide the first evidence of a direct interaction between Saccharomyces cerevisiae Est3p and the catalytic protein subunit (Est2p) by demonstrating that recombinant Est3p binds the purified telomerase essential N-terminal (TEN) domain of Est2p in vitro. Mutations in a small cluster of amino acids predicted to lie on the surface of Est3p disrupt this interaction with Est2p, reduce assembly of Est3p with telomerase in vivo, and cause telomere shortening and senescence. We also show that recombinant Est3p stimulates telomerase activity above basal levels in vitro in a manner dependent on the Est2p TEN domain interaction. Together, these results define a direct binding interaction between Est3p and Est2p and reconcile the effect of S. cerevisiae Est3p with previous experiments showing that Est3p homologs in related yeast species influence telomerase activity. Additionally, it contributes functional support to the idea that Est3p is structurally related to the mammalian shelterin protein, TPP1, which also influences telomerase activity through interaction with the Est2p homolog, TERT.     

Cdc13 cooperates with the yeast Ku proteins and Stn1 to regulate telomerase recruitment

The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both on STN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.