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.     

Deletion of Ogg1 DNA glycosylase results in telomere base damage and length alteration in yeast

Telomeres consist of short guanine‐rich repeats. Guanine can be oxidized to 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) and 2,6‐diamino‐4‐hydroxy‐5‐formamidopyrimidine (FapyG). 8‐oxoguanine DNA glycosylase (Ogg1) repairs these oxidative guanine lesions through the base excision repair (BER) pathway. Here we show that in Saccharomyces cerevisiae ablation of Ogg1p leads to an increase in oxidized guanine level in telomeric DNA. The ogg1 deletion (ogg1Δ) strain shows telomere lengthening that is dependent on telomerase and/or Rad52p‐mediated homologous recombination. 8‐oxoG in telomeric repeats attenuates the binding of the telomere binding protein, Rap1p, to telomeric DNA in vitro. Moreover, the amount of telomere‐bound Rap1p and Rif2p is reduced in ogg1Δ strain. These results suggest that oxidized guanines may perturb telomere length equilibrium by attenuating telomere protein complex to function in telomeres, which in turn impedes their regulation of pathways engaged in telomere length maintenance. We propose that Ogg1p is critical in maintaining telomere length homoeostasis through telomere guanine damage repair, and that interfering with telomere length homoeostasis may be one of the mechanism(s) by which oxidative DNA damage inflicts the genome.     

Yeast Est2p affects telomere length by influencing association of Rap1p with telomeric chromatin

In Saccharomyces cerevisiae, the sequence-specific binding of the negative regulator Rap1p provides a mechanism to measure telomere length: as the telomere length increases, the binding of additional Rap1p inhibits telomerase activity in cis. We provide evidence that the association of Rap1p with telomeric DNA in vivo occurs in part by sequence-independent mechanisms. Specific mutations in EST2 (est2-LT) reduce the association of Rap1p with telomeric DNA in vivo. As a result, telomeres are abnormally long yet bind an amount of Rap1p equivalent to that observed at wild-type telomeres. This behavior contrasts with that of a second mutation in EST2 (est2-up34) that increases bound Rap1p as expected for a strain with long telomeres. Telomere sequences are subtly altered in est2-LT strains, but similar changes in est2-up34 telomeres suggest that sequence abnormalities are a consequence, not a cause, of overelongation. Indeed, est2-LT telomeres bind Rap1p indistinguishably from the wild type in vitro. Taken together, these results suggest that Est2p can directly or indirectly influence the binding of Rap1p to telomeric DNA, implicating telomerase in roles both upstream and downstream of Rap1p in telomere length homeostasis.     

Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype

Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation.     

Binding of Multiple Rap1 Proteins Stimulates Chromosome Breakage Induction during DNA Replication

Telomeres, the ends of linear eukaryotic chromosomes, have a specialized chromatin structure that provides a stable chromosomal terminus. In budding yeast Rap1 protein binds to telomeric TG repeat and negatively regulates telomere length. Here we show that binding of multiple Rap1 proteins stimulates DNA double-stranded break (DSB) induction at both telomeric and non-telomeric regions. Consistent with the role of DSB induction, Rap1 stimulates nearby recombination events in a dosage-dependent manner. Rap1 recruits Rif1 and Rif2 to telomeres, but neither Rif1 nor Rif2 is required for DSB induction. Rap1-mediated DSB induction involves replication fork progression but inactivation of checkpoint kinase Mec1 does not affect DSB induction. Rap1 tethering shortens artificially elongated telomeres in parallel with telomerase inhibition, and this telomere shortening does not require homologous recombination. These results suggest that Rap1 contributes to telomere homeostasis by promoting chromosome breakage.     

spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast.

Telomeres are essential for genome integrity. scRap1 (S. cerevisiae Rap1) directly binds to telomeric DNA and regulates telomere length and telomere position effect (TPE) by recruiting two different groups of proteins to its RCT (Rap1 C-terminal) domain. The first group, Rif1 and Rif2, regulates telomere length. The second group, Sir3 and Sir4, is involved in heterochromatin formation. On the other hand, human TRF1 and TRF2, as well as their fission yeast homolog, Taz1, directly bind to telomeric DNA and negatively regulate telomere length. Taz1 also plays important roles in TPE and meiosis. Human Rap1, the ortholog of scRap1, negatively regulates telomere length and appears to be recruited to telomeres by interacting with TRF2. Here, we describe two novel fission yeast proteins, spRap1 (S. pombe Rap1) and spRif1 (S. pombe Rif1), which are orthologous to scRap1 and scRif1, respectively. spRap1 and spRif1 are independently recruited to telomeres by interacting with Taz1. The rap1 mutant is severely defective in telomere length control, TPE, and telomere clustering toward the spindle pole body (SPB) at the premeiotic horsetail stage, indicating that spRap1 has critical roles in these telomere functions. The rif1 mutant also shows some defects in telomere length control and meiosis. Our results indicate that Taz1 provides binding sites for telomere regulators, spRap1 and spRif1, which perform the essential telomere functions. This study establishes the similarity of telomere organization in fission yeast and humans.     

Rap1 affects the length and heterogeneity of human telomeres

Telomere length is controlled in part by cis-acting negative regulators that limit telomere extension by telomerase. In budding yeast, the major telomere length regulator scRap1 binds to telomeric DNA and acts to inhibit telomere elongation in cis. Because the human Rap1 ortholog hRap1 does not bind to telomeric DNA directly but is recruited to telomeres by TRF2, we examined its role in telomere length control. The data are consistent with hRap1 being a negative regulator of telomere length, indicating functional conservation. Deletion mapping confirmed that hRap1 is tethered to telomeres through interaction of its C terminus with TRF2. The telomere length phenotypes of hRap1 deletion mutants implicated both the BRCT and Myb domain as protein interaction domains involved in telomere length regulation. By contrast, scRap1 binds to telomeres with its Myb domains and uses its C terminus to recruit the telomere length regulators Rif1 and Rif2. Together, our data show that although the role of Rap1 at telomeres has been largely conserved, the domains of Rap1 have undergone extensive functional changes during eukaryotic evolution. Surprisingly, hRap1 alleles lacking the BRCT domain diminished the heterogeneity of human telomeres, indicating that hRap1 also plays a role in the regulation of telomere length distribution.     

Telomerase RNA template mutations reveal sequence-specific requirements for the activation and repression of telomerase action at telomeres

Telomeric DNA is maintained within a length range characteristic of an organism or cell type. Significant deviations outside this range are associated with altered telomere function. The yeast telomere-binding protein Rap1p negatively regulates telomere length. Telomere elongation is responsive to both the number of Rap1p molecules bound to a telomere and the Rap1p-centered DNA-protein complex at the extreme telomeric end. Previously, we showed that a specific trinucleotide substitution in the Saccharomyces cerevisiae telomerase gene (TLC1) RNA template abolished the enzymatic activity of telomerase, causing the same cell senescence and telomere shortening phenotypes as a complete tlc1 deletion. Here we analyze effects of six single- and double-base changes within these same three positions. All six mutant telomerases had in vitro enzymatic activity levels similar to the wild-type levels. The base changes predicted from the mutations all disrupted Rap1p binding in vitro to the corresponding duplex DNAs. However, they caused two classes of effects on telomere homeostasis: (i) rapid, RAD52-independent telomere lengthening and poor length regulation, whose severity correlated with the decrease in in vitro Rap1p binding affinity (this is consistent with loss of negative regulation of telomerase action at these telomeres; and (ii) telomere shortening that, depending on the template mutation, either established a new short telomere set length with normal cell growth or was progressive and led to cellular senescence. Hence, disrupting Rap1p binding at the telomeric terminus is not sufficient to deregulate telomere elongation. This provides further evidence that both positive and negative cis-acting regulators of telomerase act at telomeres.     

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.     

Genetic dissection of the Kluyveromyces lactis telomere and evidence for telomere capping defects in TER1 mutants with long telomeres

In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3' of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3' terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.     

A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance

RAP1 is one of the components of shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere length maintenance and protection has remained elusive. RAP1 also binds subtelomeric repeats and along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres, and its deficiency causes a premature aging in humans and mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact on mouse survival, mice lacking both RAP1 and telomerase show a progressively decreased survival with increasing mouse generations compared to telomerase single mutants. Telomere shortening is more pronounced in Rap1^?/? Terc^?/? doubly deficient mice than in the single‐mutant Terc^?/? counterparts, leading to an earlier onset of telomere‐induced DNA damage and degenerative pathologies. Telomerase deficiency abolishes obesity and liver steatohepatitis provoked by RAP1 deficiency. Using genomewide ChIP sequencing, we find that progressive telomere shortening owing to telomerase deficiency leads to re‐localization of RAP1 from telomeres and subtelomeric regions to extratelomeric sites in a genomewide manner. These findings suggest that although in the presence of sufficient telomere reserve RAP1 is not a key factor for telomere maintenance and protection, it plays a crucial role in the context of telomerase deficiency, thus in agreement with its evolutionary conservation as a telomere component from yeast to humans.     

Telomeric protein distributions and remodeling through the cell cycle in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, telomeric DNA is protected by a nonnucleosomal protein complex, tethered by the protein Rap1. Rif and Sir proteins, which interact with Rap1p, are thought to have further interactions with conventional nucleosomic chromatin to create a repressive structure that protects the chromosome end. We showed by microarray analysis that Rif1p association with the chromosome ends extends to subtelomeric regions many kilobases internal to the terminal telomeric repeats and correlates strongly with the previously determined genomic footprints of Rap1p and the Sir2-4 proteins in these regions. Although the end-protection function of telomeres is essential for genomic stability, telomeric DNA must also be copied by the conventional DNA replication machinery and replenished by telomerase, suggesting that transient remodeling of the telomeric chromatin might result in distinct protein complexes at different stages of the cell cycle. Using chromatin immunoprecipitation, we monitored the association of Rap1p, Rif1p, Rif2p, and the protein component of telomerase, Est2p, with telomeric DNA through the cell cycle. We provide evidence for dynamic remodeling of these components at telomeres.     

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.     

Telomerase-independent lengthening of yeast telomeres occurs by an abrupt Rad50p-dependent, Rif-inhibited recombinational process

Type II survivors arise in Saccharomyces cells lacking telomerase by a recombinational pathway that results in very long and heterogeneous length telomeres. Here we show that type II telomeres appeared abruptly in a population of cells with very short telomeres. Once established, these long telomeres progressively shortened. Short telomeres were substrates for rare, one-step lengthening events. The generation of type II survivors was absolutely Rad50p dependent. In a telomerase-proficient cell, the telomere-binding Rif proteins inhibited telomerase lengthening of telomeres. In a telomerase-deficient strain, Rif proteins, especially Rif2p, inhibited type II recombination. These data argue that only short telomeres are substrates for type II recombination and suggest that the donor for this recombination is not a chromosomal telomere.     

Sudden telomere lengthening triggers a Rad53-dependent checkpoint in Saccharomyces cerevisiae

Telomeres are specialized functional complexes that ensure chromosome stability by protecting chromosome ends from fusions and degradation and avoiding chromosomal termini from being sensed as DNA breaks. Budding yeast Tel1 is required both for telomere metabolism and for a Rad53-dependent checkpoint responding to unprocessed double-strand breaks. We show that overexpression of a GAL1-TEL1 fusion causes transient telomere lengthening and activation of a Rad53-dependent G2/M checkpoint in cells whose telomeres are short due to the lack of either Tel1 or Yku70. Sudden telomere elongation and checkpoint-mediated cell cycle arrest are also triggered in wild-type cells by overproducing a protein fusion between the telomeric binding protein Cdc13 and the telomerase-associated protein Est1. Checkpoint activation by GAL1-TEL1 requires ongoing telomere elongation. In fact, it is turned off concomitantly with telomeres reaching a new stable length and is partially suppressed by deletion of the telomerase EST2 gene. Moreover, both telomere length rebalancing and checkpoint inactivation under galactose-induced conditions are accelerated by high levels of either the Sae2 protein, involved in double-strand breaks processing, or the negative telomere length regulator Rif2. These data suggest that sudden telomere lengthening elicits a checkpoint response that inhibits the G2/M transition.     

Counting of Rif1p and Rif2p on Saccharomyces cerevisiae telomeres regulates telomere length

Telomere length is negatively regulated by proteins of the telomeric DNA-protein complex. Rap1p in Saccharomyces cerevisiae binds the telomeric TG(1-3) repeat DNA, and the Rap1p C terminus interacts with Rif1p and Rif2p. We investigated how these three proteins negatively regulate telomere length. We show that direct tethering of each Rif protein to a telomere shortens that telomere proportionally to the number of tethered molecules, similar to previously reported counting of Rap1p. Surprisingly, Rif proteins could also regulate telomere length even when the Rap1p C terminus was absent, and tethered Rap1p counting was completely dependent on the Rif proteins. Thus, Rap1p counting is in fact Rif protein counting. In genetic settings that cause telomeres to be abnormally long, tethering even a single Rif2p molecule was sufficient for maximal effectiveness in preventing the telomere overelongation. We show that a heterologous protein oligomerization domain, the mammalian PDZ domain, when fused to Rap1p can confer telomere length control. We propose that a nucleation and spreading mechanism is involved in forming the higher-order telomere structure that regulates telomere length.