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.     

Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.     

Yeast telomerase appears to frequently copy the entire template in vivo

Telomeres derived from the same formation event in wild type strains of Saccharomyces cerevisiae possess the same, precise TG_1–3 sequence for the most internal ~100 bp of the 250–350 bp TG_1–3 repeats. The conservation of this internal domain is thought to reflect the fact that telomere lengthening and shortening, and thus alteration of the precise TG_1–3 sequence, is confined to the terminal region of the telomere. The internal domains of telomeres from yku70Δ and tel1Δ mutants, whose entire telomeres are only ~100 bp, were examined by analyzing 5.1 kb of cloned TG_1–3 sequences from telomeres formed during transformation of wild type, yku70Δ and tel1Δ cells. The internal domains were 97–137 bp in wild type cells, 27–36 bp in yku70Δ cells and 7–9 bp in tel1Δ cells. These data suggest that the majority of the tel1Δ cell TG_1–3 repeats may be resynthesized during shortening and lengthening reactions while a portion of the yku70Δ cell telomeres are protected. TG_1–3 sequences are synthesized by telomerase repeatedly copying an internal RNA template, which introduces a sequence bias into TG_1–3 repeats. Analysis of in vivo-derived telomeres revealed that of the many possible high affinity binding sites for the telomere protein Rap1p in TG_1–3 repeats, only those consistent with telomere hybridization to the ACACAC in the 3′-region of the telomerase RNA template followed by copying of most of the template were present. Copies of the telomerase RNA template made up 40–60% of the TG_1–3 sequences from each strain and could be found in long, tandem repeats. The data suggest that in vivo yeast telomerase frequently allows telomeres to hybridize to the 3′-region of RNA template and copy most of it prior to dissociation, or that in vivo telomere processing events result in the production of TG_1–3 sequences that mimic this process.     

The Pif1 Helicase,a Negative Regulator of Telomerase,Acts Preferentially at Long Telomeres

Telomerase, the enzyme that maintains telomeres, preferentially lengthens short telomeres. The S. cerevisiae Pif1 DNA helicase inhibits both telomerase-mediated telomere lengthening and de novo telomere addition at double strand breaks (DSB). Here, we report that the association of the telomerase subunits Est2 and Est1 at a DSB was increased in the absence of Pif1, as it is at telomeres, suggesting that Pif1 suppresses de novo telomere addition by removing telomerase from the break. To determine how the absence of Pif1 results in telomere lengthening, we used the single telomere extension assay (STEX), which monitors lengthening of individual telomeres in a single cell cycle. In the absence of Pif1, telomerase added significantly more telomeric DNA, an average of 72 nucleotides per telomere compared to the 45 nucleotides in wild type cells, and the fraction of telomeres lengthened increased almost four-fold. Using an inducible short telomere assay, Est2 and Est1 no longer bound preferentially to a short telomere in pif1 mutant cells while binding of Yku80, a telomere structural protein, was unaffected by the status of the PIF1 locus. Two experiments demonstrate that Pif1 binding is affected by telomere length: Pif1 (but not Yku80) -associated telomeres were 70 bps longer than bulk telomeres, and in the inducible short telomere assay, Pif1 bound better to wild type length telomeres than to short telomeres. Thus, preferential lengthening of short yeast telomeres is achieved in part by targeting the negative regulator Pif1 to long telomeres.     

Telomerase-dependent repeat divergence at the 3' ends of yeast telomeres

Yeast telomeres consist of ~300 nt of degenerate repeats with the consensus sequence G_2–3(TG)_1–6. We developed a method for the amplification of a genetically marked telomere by PCR, allowing precise length and sequence determination of the G-rich strand including the 3′ terminus. We examined wild-type cells, telomerase RNA deficient cells and a strain deleted for YKU70, which encodes for a protein involved in telomere maintenance and DNA double strand break repair. The 3′ end of the G-rich strand was found to be at a variable position within the telomeric repeat. No preference for either thymine or guanine as the 3′ base was detected. Comparison of telomere sequences from clonal populations revealed that telomeres consist of a centromere-proximal region of stable sequence and a distal region with differing degenerate repeats. In wild-type as well as yku70-Δ cells, variation in the degenerate telomeric repeats was detected starting 40–100 nt from the 3′ end. Sequence divergence was abolished after deletion of the telomerase RNA gene. Thus, this region defines the domain where telomere shortening and telomerase-mediated extension occurs. Since this domain is much larger than the number of nucleo­tides lost per generation in the absence of telomerase, we propose that telomerase does not extend a given telomere in every cell cycle.     

Modulation of telomere terminal structure by telomerase components in Candida albicans

The telomerase ribonucleoprotein in Candida albicans is presumed to contain at least three Est proteins: CaEst1p, CaEst2p/TERT and CaEst3p. We constructed mutants missing each of the protein subunit of telomerase and analyzed overall telomere dynamics and single-stranded telomere overhangs over the course of many generations. The est1-ΔΔ mutant manifested abrupt telomere loss and recovery, consistent with heightened recombination. Both the est2-ΔΔ and est3-ΔΔ mutant exhibited progressive telomere loss, followed by the gradual emergence of survivors with long telomeres. In no case was telomere loss accompanied by severe growth defects, suggesting that cells with short telomeres can continue to proliferate. Furthermore, the amount of G-strand terminal overhangs was greatly increased in the est2-ΔΔ mutant, but not others. Our results suggest that in addition to their well-characterized function in telomere elongation, both CaEst1p and CaEst2p mediate some aspects of telomere protection in Candida, with the former suppressing excessive recombination, and the latter preventing excessive C-strand degradation.     

Roles of Pif1-like helicases in the maintenance of genomic stability

The Pif1p family of DNA helicases is conserved from yeast to humans. To date, four members of this family have been analyzed in some detail by in vitro and in vivo assays: the two baker's yeast helicases, ScPif1p and Rrm3p, the fission yeast Pfh1p and the human enzyme hPif1p. In vitro, these enzymes are 5′ to 3′ DNA helicase and show little processivity. In vivo, ScPif1p, Rrm3p and probably Pfh1p, function in both the nucleus at specific genomic loci and in mitochondria, where they are needed for the stable maintenance of the genome as accessory helicases to the replication machinery. Interestingly, they act on common DNA substrates but appear to have largely non-overlapping cellular functions, ranging from Okazaki fragment processing, telomerase inhibition, to helping the replication fork progress through non-nucleosomal protein–DNA complexes. For example, both ScPif1p and Rrm3p affect the replication of telomeres, but in a different way: Pif1p inhibits telomerase-mediated telomere elongation by directly removing telomerase from a DNA end, whereas Rrm3p facilitates replication through telomeric DNA. Here we review the current knowledge on the Pif1-like helicases, as a first step towards understanding the basis of their functional specialization and mechanism of action.     

Dynamic regulation of single-stranded telomeres in Saccharomyces cerevisiae

The temperature-sensitive phenotypes of yku70Delta and yku80Delta have provided a useful tool for understanding telomere homeostasis. Mutating the helicase domain of the telomerase inhibitor Pif1 resulted in the inactivation of cell cycle checkpoints and the subsequent rescue of temperature sensitivity of the yku70Delta strain. The inactivation of Pif1 in yku70Delta increased overall telomere length. However, the long G-rich, single-stranded overhangs at the telomeres, which are the major cause of temperature sensitivity, were slightly increased. Interestingly, the rescue of temperature sensitivity in strains having both pif1-m2 and yku70Delta mutations depended on the homologous recombination pathway. Furthermore, the BLM/WRN helicase yeast homolog Sgs1 exacerbated the temperature sensitivity of the yku70Delta strain. Therefore, the yKu70-80 heterodimer and telomerase maintain telomere size, and the helicase activity of Pif1 likely also helps to balance the overall size of telomeres and G-rich, single-stranded overhangs in wild-type cells by regulating telomere protein homeostasis. However, the absence of yKu70 may provide other proteins such as those involved in homologous recombination, Sgs1, or Pif1 additional access to G-rich, single-stranded DNA and may determine telomere size, cell cycle checkpoint activation, and, ultimately, temperature sensitivity.     

At Short Telomeres Tel1 Directs Early Replication and Phosphorylates Rif1

The replication time of Saccharomyces cerevisiae telomeres responds to TG_1–3 repeat length, with telomeres of normal length replicating late during S phase and short telomeres replicating early. Here we show that Tel1 kinase, which is recruited to short telomeres, specifies their early replication, because we find a tel1Δ mutant has short telomeres that nonetheless replicate late. Consistent with a role for Tel1 in driving early telomere replication, initiation at a replication origin close to an induced short telomere was reduced in tel1Δ cells, in an S phase blocked by hydroxyurea. The telomeric chromatin component Rif1 mediates late replication of normal telomeres and is a potential substrate of Tel1 phosphorylation, so we tested whether Tel1 directs early replication of short telomeres by inactivating Rif1. A strain lacking both Rif1 and Tel1 behaves like a rif1Δ mutant by replicating its telomeres early, implying that Tel1 can counteract the delaying effect of Rif1 to control telomere replication time. Proteomic analyses reveals that in yku70Δ cells that have short telomeres, Rif1 is phosphorylated at Tel1 consensus sequences (S/TQ sites), with phosphorylation of Serine-1308 being completely dependent on Tel1. Replication timing analysis of a strain mutated at these phosphorylation sites, however, suggested that Tel1-mediated phosphorylation of Rif1 is not the sole mechanism of replication timing control at telomeres. Overall, our results reveal two new functions of Tel1 at shortened telomeres: phosphorylation of Rif1, and specification of early replication by counteracting the Rif1-mediated delay in initiation at nearby replication origins.     

Recombination-based telomere maintenance is dependent on Tel1-MRN and Rap1 and inhibited by telomerase, Taz1, and Ku in fission yeast

Fission yeast cells survive loss of the telomerase catalytic subunit Trt1 (TERT) through recombination-based telomere maintenance or through chromosome circularization. Although trt1Δ survivors with linear chromosomes can be obtained, they often spontaneously circularize their chromosomes. Therefore, it was difficult to establish genetic requirements for telomerase-independent telomere maintenance. In contrast, when the telomere-binding protein Taz1 is also deleted, taz1Δ trt1Δ cells are able to stably maintain telomeres. Thus, taz1Δ trt1Δ cells can serve as a valuable tool in understanding the regulation of telomerase-independent telomere maintenance. In this study, we show that the checkpoint kinase Tel1 (ATM) and the DNA repair complex Rad32-Rad50-Nbs1 (MRN) are required for telomere maintenance in taz1Δ trt1Δ cells. Surprisingly, Rap1 is also essential for telomere maintenance in taz1Δ trt1Δ cells, even though recruitment of Rap1 to telomeres depends on Taz1. Expression of catalytically inactive Trt1 can efficiently inhibit recombination-based telomere maintenance, but the inhibition requires both Est1 and Ku70. While Est1 is essential for recruitment of Trt1 to telomeres, Ku70 is dispensable. Thus, we conclude that Taz1, TERT-Est1, and Ku70-Ku80 prevent telomere recombination, whereas MRN-Tel1 and Rap1 promote recombination-based telomere maintenance. Evolutionarily conserved proteins in higher eukaryotic cells might similarly contribute to telomere recombination.     

Survival and Growth of Yeast without Telomere Capping by Cdc13 in the Absence of Sgs1, Exo1, and Rad9

Maintenance of telomere capping is absolutely essential to the survival of eukaryotic cells. Telomere capping proteins, such as Cdc13 and POT1, are essential for the viability of budding yeast and mammalian cells, respectively. Here we identify, for the first time, three genetic modifications that allow budding yeast cells to survive without telomere capping by Cdc13. We found that simultaneous inactivation of Sgs1, Exo1, and Rad9, three DNA damage response (DDR) proteins, is sufficient to allow cell division in the absence of Cdc13. Quantitative amplification of ssDNA (QAOS) was used to show that the RecQ helicase Sgs1 plays an important role in the resection of uncapped telomeres, especially in the absence of checkpoint protein Rad9. Strikingly, simultaneous deletion of SGS1 and the nuclease EXO1, further reduces resection at uncapped telomeres and together with deletion of RAD9 permits cell survival without CDC13. Pulsed-field gel electrophoresis studies show that cdc13-1 rad9Δ sgs1Δ exo1Δ strains can maintain linear chromosomes despite the absence of telomere capping by Cdc13. However, with continued passage, the telomeres of such strains eventually become short and are maintained by recombination-based mechanisms. Remarkably, cdc13Δ rad9Δ sgs1Δ exo1Δ strains, lacking any Cdc13 gene product, are viable and can grow indefinitely. Our work has uncovered a critical role for RecQ helicases in limiting the division of cells with uncapped telomeres, and this may provide one explanation for increased tumorigenesis in human diseases associated with mutations of RecQ helicases. Our results reveal the plasticity of the telomere cap and indicate that the essential role of telomere capping is to counteract specific aspects of the DDR.     

Telomere-binding and Stn1p-interacting activities are required for the essential function of Saccharomyces cerevisiae Cdc13p

Yeast Saccharomyces cerevisiae Cdc13p is the telomere-binding protein that protects telomeres and regulates telomere length. It is documented that Cdc13p binds specifically to single-stranded TG_1–3 telomeric DNA sequences and interacts with Stn1p. To localize the region for single-stranded TG_1–3 DNA binding, Cdc13p mutants were constructed by deletion mutagenesis and assayed for their binding activity. Based on in vitro electrophoretic mobility shift assay, a 243-amino-acid fragment of Cdc13p (amino acids 451–693) was sufficient to bind single-stranded TG_1–3 with specificity similar to that of the native protein. Consistent with the in vitro observation, in vivo one-hybrid analysis also indicated that this region of Cdc13p was sufficient to localize itself to telomeres. However, the telomere-binding region of Cdc13p (amino acids 451–693) was not capable of complementing the growth defects of cdc13 mutants. Instead, a region comprising the Stn1p-interacting and telomere-binding region of Cdc13p (amino acids 252–924) complemented the growth defects of cdc13 mutants. These results suggest that binding to telomeres by Cdc13p is not sufficient to account for the cell viability, interaction with Stn1p is also required. Taken together, we have defined the telomere-binding domain of Cdc13p and showed that both binding to telomeres and Stn1p by Cdc13p are required to maintain cell growth.     

S. cerevisiae Tel1p and Mre11p are required for normal levels of Est1p and Est2p telomere association

In diverse organisms, the Mre11 complex and phosphoinositide 3-kinase-related kinases (PIKKs), such as Tel1p and Mec1p from S. cerevisiae, are key mediators of DNA repair and DNA damage checkpoints that also function at telomeres. Here, we use chromatin immunoprecipitation (ChIP) to determine if Mre11p, Tel1p, or Mec1p affects telomere maintenance by promoting recruitment of telomerase subunits to S. cerevisiae telomeres. We find that recruitment of Est2p, the catalytic subunit of telomerase, and Est1p, a telomerase accessory protein, was severely reduced in mre11Delta and tel1Delta cells. In contrast, the levels of Est2p and Est1p binding in late S/G2 phase, the period in the cell cycle when yeast telomerase lengthens telomeres, were indistinguishable in wild-type (WT) and mec1Delta cells. These data argue that Mre11p and Tel1p affect telomere length by promoting telomerase recruitment to telomeres, whereas Mec1p has only a minor role in telomerase recruitment in a TEL1 cell.     

Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae

In diverse organisms, telomerase preferentially elongates short telomeres. We generated a single short telomere in otherwise wild-type (WT) S. cerevisiae cells. The binding of the positive regulators Ku and Cdc13p was similar at short and WT-length telomeres. The negative regulators Rif1p and Rif2p were present at the short telomere, although Rif2p levels were reduced. Two telomerase holoenzyme components, Est1p and Est2p, were preferentially enriched at short telomeres in late S/G2 phase, the time of telomerase action. Tel1p, the yeast ATM-like checkpoint kinase, was highly enriched at short telomeres from early S through G2 phase and even into the next cell cycle. Nonetheless, induction of a single short telomere did not elicit a cell-cycle arrest. Tel1p binding was dependent on Xrs2p and required for preferential binding of telomerase to short telomeres. These data suggest that Tel1p targets telomerase to the DNA ends most in need of extension.     

Kinase-Independent Functions of TEL1 in Telomere Maintenance

TEL1 is important in Saccharomyces cerevisiae telomere maintenance, and its kinase activity is required. Tel1p associates with telomeres in vivo, is enriched at short telomeres, and enhances the binding of telomerase components to short telomeres. However, it is unclear how the kinase activity and telomere association contribute to Tel1p''s overall function in telomere length maintenance. To investigate this question, we generated a set of single point mutants and a double point mutant (tel1^KD) of Tel1p that were kinase deficient and two Xrs2p mutants that failed to bind Tel1p. Using these separation-of-function alleles in a de novo telomere elongation assay, we found, surprisingly, that the tel1^KD allele and xrs2 C-terminal mutants were both partially functional. Combining the tel1^KD and xrs2 C-terminal mutants had an additive effect and resembled the TEL1 null (tel1Δ) phenotype. These data indicate that Tel1p has two separate functions in telomere maintenance and that the Xrs2p-dependent recruitment of Tel1p to telomeres plays an important role even in the absence of its kinase activity.The telomere is a highly ordered complex of proteins and DNA found at the ends of linear chromosomes that functions to protect the ends and prevents them from being recognized as double-strand DNA breaks (51). Telomeres shorten gradually due to incomplete replication (1, 20), and this shortening is counteracted by telomerase, which elongates telomeres (18, 19).Saccharomyces cerevisiae telomeres are composed of 300 ± 50 bp of the sequence TG_1-3/C_1-3A. The yeast telomerase complex consists of Est2p (catalytic subunit), the RNA component TLC1, and two accessory proteins, Est1p and Est3p (50). Cells deficient for any of these telomerase components undergo progressive telomere shortening and a simultaneous decrease in growth rate, described as senescence (24, 27). Typically, a small fraction of cells, termed survivors, escape senescence and maintain telomere length by utilizing RAD52-dependent recombination (24, 26).In addition to the telomerase complex, a number of yeast proteins are important in maintaining telomere length and integrity. These include Tel1p and Mec1p, the yeast homologues of mammalian ATM and ATR, respectively (39). While deletion of TEL1 results in short but stable telomeres, MEC1 deletion has little effect on average telomere length. However, cells lacking TEL1 that have a mutant mec1-21 allele undergo senescence, similar to telomerase null cells (36), suggesting that MEC1 plays a minor but essential role in telomere length maintenance in tel1Δ cells. It has been shown that the protein kinase activities of Tel1p and Mec1p are essential in telomere maintenance, since tel1^KD cells have short telomeres and tel1Δ mec1^KD cells undergo senescence (29).In current models, Tel1p acts to maintain telomere length by regulating the access of telomerase to short telomeres. TEL1 is required for the association of Est1p and Est2p with telomeres in the late S/G₂ phase of the cell cycle (16), the time when telomeres are elongated (9, 31). Additionally, in both yeast and mammalian cells, telomerase preferentially elongates the shortest telomeres (22, 30, 47). Therefore, TEL1 seems to be required mainly for the association of telomerase to short telomeres in yeast. Indeed, Tel1p preferentially binds to short telomeres (4, 21, 38) and is essential for the increased association of Est1p and Est2p to short telomeres during late S/G₂ (38). However, the kinase activity of Tel1p is not required for the telomere association (21). In addition to its role in telomerase recruitment, TEL1 may also regulate telomere length by enhancing the processivity of telomerase at short telomeres (7).The Mre11p, Rad50p, and Xrs2p (MRX) complex also plays important roles in telomere maintenance. Cells lacking any one of these components (mrxΔ) have short and stable telomeres. Since combining mrxΔ with tel1Δ has no synergistic effect on telomere shortening and mrxΔ mec1Δ cells undergo senescence, it was proposed that the MRX complex and Tel1p function in the same telomere maintenance pathway (37). In agreement with this model, the C-terminal region of Xrs2p is essential in recruiting Tel1p both to double-strand breaks (32) and to short telomeres (38). Interestingly, the mammalian functional homologue of Xrs2p, NBS1, interacts with ATM via its extreme C terminus (13), suggesting that the recruitment of Tel1p to telomeres and the recruitment of ATM to DNA damage sites are conserved.It remains a question what exact roles the kinase activity of Tel1p and its telomere binding play in telomere maintenance. Tel1p''s telomere maintenance function seems to be dependent on its kinase activity, since tel1^KD cells have short telomeres (29). It has been proposed that Tel1p may regulate the recruitment of Est1p, and thus the rest of the telomerase complex (12, 23, 54), to telomeres by phosphorylating Cdc13p (3, 48). Other experiments suggest the association of Tel1p to the telomere plays a major role. The preferential binding of Tel1p to short telomeres is lost in xrs2-664 cells (38), which lack the C-terminal 190 amino acids of Xrs2p and have short telomeres, similar to xrs2Δ (41). It has been suggested that the association of Tel1p to telomeres is required for its substrate phosphorylation and, therefore, telomere length maintenance (3, 39).To further analyze the functions of Tel1p in telomere maintenance, we generated a novel kinase-dead allele of TEL1 and new alleles of XRS2 that do not interact with Tel1p. Through these separation-of-function mutants, we show that both sets of alleles are partially active in a de novo telomere elongation assay. However, combining both the tel1^KD and either of the Tel1p interaction-deficient xrs2 alleles resulted in a phenotype resembling the tel1Δ phenotype, suggesting that Tel1p has kinase-dependent and kinase-independent, but telomere binding-dependent, functions in telomere maintenance.     

Mrc1, a non-essential DNA replication protein,is required for telomere end protection following loss of capping by Cdc13, Yku or telomerase

Proteins involved in telomere end protection have previously been identified. In Saccharomyces cerevisiae, Cdc13, Yku and telomerase, mainly, prevent telomere uncapping, thus providing telomere stability and avoiding degradation and death by senescence. Here, we report that in the absence of Mrc1, a component of the replication forks, telomeres of cdc13 or yku70 mutants exhibited increased degradation, while telomerase-negative cells displayed accelerated senescence. Moreover, deletion of MRC1 increased the single-strandedness of the telomeres in cdc13-1 and yku70Δ mutant strains. An mrc1 deletion strain also exhibited slight but stable telomere shortening compared to a wild-type strain. Loss of Mrc1’s checkpoint function alone did not provoke synthetic growth defects in combination with the cdc13-1 mutation. Combinations between the cdc13-1 mutation and deletion of either TOF1 or PSY2, coding for proteins physically interacting with Mrc1, also resulted in a synthetic growth defect. Thus, the present data suggest that non-essential components of the DNA replication machinery, such as Mrc1 and Tof1, may have a role in telomere stability in addition to their role in fork progression.     

“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.