Chromosome end protection plasticity revealed by Stn1p and Ten1p bypass of Cdc13p

Genome stability necessitates a mechanism to protect the termini of linear chromosomes from inappropriate degradation or recombination. In many species this protection depends on 'capping' proteins that bind telomeric DNA. The budding yeast Cdc13p binds single-stranded telomeric sequences, prevents lethal degradation of chromosome ends and regulates telomere extension by telomerase. Two Cdc13-interacting proteins, Stn1p and Ten1p, are also required for viability and telomere length regulation. It has been proposed that Cdc13p DNA binding directs a Cdc13p-Stn1p-Ten1p complex to telomeres to mediate end protection. However, the functional significance of these protein interactions, and their respective roles in maintaining telomere integrity, remain undefined. Here, we show that co-overexpressing TEN1 with a truncated form of STN1 efficiently bypasses the essential role of CDC13. We further show that this truncated Stn1p binds directly to Pol12p, a polymerase alpha-primase regulatory subunit, and that Pol12 activity is required for CDC13 bypass. Thus, Stn1p and Ten1p control a Cdc13p-independent telomere capping mechanism that is coupled to the conventional DNA replication machinery.     

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.     

Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition

The budding yeast Cdc13, Stn1 and Ten1 (CST) proteins are proposed to function as an RPA-like complex at telomeres that protects (‘caps'') chromosome ends and regulates their elongation by telomerase. We show that Stn1 has a critical function in both processes through the deployment of two separable domains. The N terminus of Stn1 interacts with Ten1 and carries out its essential capping function. The C terminus of Stn1 binds both Cdc13 and Pol12, and we present genetic data indicating that the Stn1–Cdc13 interaction is required to limit continuous telomerase action. Stn1 telomere association, similar to that of Cdc13, peaks during S phase. Significantly, the magnitude of Stn1 telomere binding is independent of telomere TG tract length, suggesting that the negative effect of Stn1 on telomerase action might be regulated by a modification of CST activity or structure in cis at individual telomeres. Genetic analysis suggests that the Tel1 kinase exerts an effect in parallel with the Stn1 C terminus to counteract its inhibition of telomerase. These data provide new insights into the coordination of telomere capping and telomerase regulation.     

RPA-like proteins mediate yeast telomere function

Cdc13, Stn1 and Ten1 are essential yeast proteins that both protect chromosome termini from unregulated resection and regulate telomere length. Cdc13, which localizes to telomeres through high-affinity binding to telomeric single-stranded DNA, has been extensively characterized, whereas the contribution(s) of the Cdc13-associated Stn1 and Ten1 proteins to telomere function have remained unclear. We show here that Stn1 and Ten1 are DNA-binding proteins with specificity for telomeric DNA substrates. Furthermore, Stn1 and Ten1 show similarities to Rpa2 and Rpa3, subunits of the heterotrimeric replication protein A (RPA) complex, which is the major single-stranded DNA-binding activity in eukaryotic cells. We propose that Cdc13, Stn1 and Ten1 function as a telomere-specific RPA-like complex. Identification of an RPA-like complex that is targeted to a specific region of the genome suggests that multiple RPA-like complexes have evolved, each making individual contributions to genomic stability.     

Cdc13 delivers separate complexes to the telomere for end protection and replication

In Saccharomyces cerevisiae, the telomere binding protein Cdc13 mediates telomere replication by recruiting telomerase, and also performs an essential function in chromosome end protection. We show here that delivery of the Stn1 protein to the telomere, by fusing the DNA binding domain of Cdc13 (DBD(CDC13)) to Stn1, is sufficient to rescue the lethality of a cdc13 null strain and, hence, provide end protection. Telomere replication is still defective in this strain, but can be restored by delivering telomerase to the telomere as a DBD(CDC13)-telomerase fusion. These results establish Stn1 as the primary effector of chromosome end protection, whereas the principal function of Cdc13 is to provide a loading platform to recruit complexes that provide end protection and telomere replication.     

Fission yeast Stn1 maintains stability of repetitive DNA at subtelomere and ribosomal DNA regions

Telomere binding protein Stn1 forms the CST (Cdc13/CTC1-STN1-TEN1) complex in budding yeast and mammals. Likewise, fission yeast Stn1 and Ten1 form a complex indispensable for telomere protection. We have previously reported that stn1-1, a high-temperature sensitive mutant, rapidly loses telomere DNA at the restrictive temperature due to frequent failure of replication fork progression at telomeres and subtelomeres, both containing repetitive sequences. It is unclear, however, whether Stn1 is required for maintaining other repetitive DNAs such as ribosomal DNA. In this study, we have demonstrated that stn1-1 cells, even when grown at the permissive temperature, exhibited dynamic rearrangements in the telomere-proximal regions of subtelomere and ribosomal DNA repeats. Furthermore, Rad52 and γH2A accumulation was observed at ribosomal DNA repeats in the stn1-1 mutant. The phenotypes exhibited by the stn1-1 allele were largely suppressed in the absence of Reb1, a replication fork barrier-forming protein, suggesting that Stn1 is involved in the maintenance of the arrested replication forks. Collectively, we propose that Stn1 maintains the stability of repetitive DNAs at subtelomeres and rDNA regions.     

Tpz1TPP1 SUMOylation reveals evolutionary conservation of SUMO‐dependent Stn1 telomere association

Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang‐binding Cdc13 is increased by Cdc13 SUMOylation. Human and fission yeast CST instead interact with overhang‐binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association, and a SUMO‐Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation.     

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.     

Genetic analysis reveals essential and non-essential amino acids within the telomeric DNA-binding interface of Cdc13p

Cdc13p is a specific single-stranded telomeric DNA-binding protein of Saccharomyces cerevisiae. It is involved in protecting telomeres and regulating telomere length. The telomere-binding domain of Cdc13p is located between residues 497 and 693, and its structure has been resolved by NMR spectroscopy. A series of aromatic, hydrophobic and basic residues located at the DNA-binding surface of Cdc13p are involved in binding to telomeres. Here we applied a genetic approach to analyse the involvements of these residues in telomere binding. A series of mutants within the telomere-binding domain of Cdc13p were identified that failed to complement cdc13 mutants in vivo. Among the amino acids that were isolated, the Tyr522, Arg635, and Ile633 residues were shown to locate at the DNA-binding surface. We further demonstrated that Y522C and R635A mutants failed to bind telomeric DNA in vitro, indicating that these residues are indeed required for telomere binding. We did not, however, isolate other mutant residues located at the DNA-binding surface of Cdc13p beyond these three residues. Instead, a mutant on Lys568 was isolated that did not affect the essential function of Cdc13p. The Lys568 is also located on the DNA-binding surface of Cdc13p. Thus these results suggested that other DNA-binding residues are not essential for telomere binding. In the present study, we have established a genetic test that enabled the identification of telomere-binding residues of Cdc13p in vivo. This type of analysis provides information on those residues that indeed contribute to telomere binding in vivo.     

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.     

Linear chromosome maintenance in the absence of essential telomere-capping proteins

Telomeres were defined by their ability to cap chromosome ends. Proteins with high affinity for the structure at chromosome ends, binding the G-rich, 3' single-stranded overhang at telomeres include Pot1 in humans and fission yeast, TEBP in Oxytricha nova and Cdc13 in budding yeast. Cdc13 is considered essential for telomere capping because budding yeast that lack Cdc13 rapidly accumulate excessive single-stranded DNA (ssDNA) at telomeres, arrest cell division and die. Cdc13 has a separate, critical role in telomerase recruitment to telomeres. Here, we show that neither Cdc13 nor its partner Stn1 are necessary for telomere capping if nuclease activities that are active at uncapped telomeres are attenuated. Recombination-dependent and -independent mechanisms permit maintenance of chromosomes without Cdc13. Our results indicate that the structure of the eukaryotic telomere cap is remarkably flexible and that changes in the DNA damage response allow alternative strategies for telomere capping to evolve.     

The Telomere Capping Complex CST Has an Unusual Stoichiometry,Makes Multipartite Interaction with G-Tails,and Unfolds Higher-Order G-Tail Structures

The telomere-ending binding protein complex CST (Cdc13-Stn1-Ten1) mediates critical functions in both telomere protection and replication. We devised a co-expression and affinity purification strategy for isolating large quantities of the complete Candida glabrata CST complex. The complex was found to exhibit a 2∶4∶2 or 2∶6∶2 stoichiometry as judged by the ratio of the subunits and the native size of the complex. Stn1, but not Ten1 alone, can directly and stably interact with Cdc13. In gel mobility shift assays, both Cdc13 and CST manifested high-affinity and sequence-specific binding to the cognate telomeric repeats. Single molecule FRET-based analysis indicates that Cdc13 and CST can bind and unfold higher order G-tail structures. The protein and the complex can also interact with non-telomeric DNA in the absence of high-affinity target sites. Comparison of the DNA–protein complexes formed by Cdc13 and CST suggests that the latter can occupy a longer DNA target site and that Stn1 and Ten1 may contact DNA directly in the full CST–DNA assembly. Both Stn1 and Ten1 can be cross-linked to photo-reactive telomeric DNA. Mutating residues on the putative DNA–binding surface of Candida albicans Stn1 OB fold domain caused a reduction in its crosslinking efficiency in vitro and engendered long and heterogeneous telomeres in vivo, indicating that the DNA–binding activity of Stn1 is required for telomere protection. Our data provide insights on the assembly and mechanisms of CST, and our robust reconstitution system will facilitate future biochemical analysis of this important complex.     

Cdk1 Regulates the Temporal Recruitment of Telomerase and Cdc13-Stn1-Ten1 Complex for Telomere Replication

In budding yeast (Saccharomyces cerevisiae), the cell cycle-dependent telomere elongation by telomerase is controlled by the cyclin-dependent kinase 1 (Cdk1). The telomere length homeostasis is balanced between telomerase-unextendable and telomerase-extendable states that both require Cdc13. The recruitment of telomerase complex by Cdc13 promotes telomere elongation, while the formation of Cdc13-Stn1-Ten1 (CST) complex at the telomere blocks telomere elongation by telomerase. However, the cellular signaling that regulates the timing of the telomerase-extendable and telomerase-unextendable states is largely unknown. Phosphorylation of Cdc13 by Cdk1 promotes the interaction between Cdc13 and Est1 and hence telomere elongation. Here, we show that Cdk1 also phosphorylates Stn1 at threonine 223 and serine 250 both in vitro and in vivo, and these phosphorylation events are essential for the stability of the CST complexes at the telomeres. By controlling the timing of Cdc13 and Stn1 phosphorylations during cell cycle progression, Cdk1 regulates the temporal recruitment of telomerase complexes and CST complexes to the telomeres to facilitate telomere maintenance.     

Sequential phosphorylation of CST subunits by different cyclin-Cdk1 complexes orchestrate telomere replication

Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. Telomere homeostasis is central to maintaining genomic integrity. In budding yeast, Cdk1 phosphorylates the telomere-specific binding protein, Cdc13, promoting the recruitment of telomerase to telomere and thereby telomere elongation. Cdc13 is also an integral part of the CST (Cdc13-Stn1-Ten1) complex that is essential for telomere capping and counteracting telomerase-dependent telomere elongation. Therefore, telomere length homeostasis is a balance between telomerase-extendable and CST-unextendable states. In our earlier work, we showed that Cdk1 also phosphorylates Stn1 which occurs sequentially following Cdc13 phosphorylation during cell cycle progression. This stabilizes the CST complex at the telomere and results in telomerase inhibition. Hence Cdk1-dependent phosphorylations of Stn1 acts like a molecular switch that drives Cdc13 to complex with Stn1-Ten1 rather than with telomerase. However, the underlying mechanism of how a single cyclin-dependent kinase phosphorylates Cdc13 and Stn1 in temporally distinct windows is largely unclear. Here, we show that S phase cyclins are necessary for telomere maintenance. The S phase and mitotic cyclins facilitate Cdc13 and Stn1 phosphorylation respectively, to exert opposing outcomes at the telomere. Thus, our results highlight a previously unappreciated role for cyclins in telomere replication.     

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.     

Cdc4p, a contractile ring protein essential for cytokinesis in Schizosaccharomyces pombe, interacts with a phosphatidylinositol 4-kinase

The proposed function of Cdc4p, an essential contractile ring protein in Schizosaccharomyces pombe, is that of a myosin essential light chain. However, five conditionally lethal cdc4 alleles exhibit complementation in diploids. Such interallelic complementation is not readily explained if the sole function of Cdc4p is that of a myosin essential light chain. Complementation of cdc4 alleles could occur only if different mutant forms can assemble into an active oligomeric complex or if Cdc4p has more than one essential function. To search for other proteins that may interact with Cdc4p, we performed a two-hybrid screen and identified two such candidates: one similar to Saccharomyces cerevisiae Vps27p and the other a putative phosphatidylinositol (PI) 4-kinase. Binding of Cdc4p to the latter and to myosin heavy chain (Myo2p) was confirmed by immunosorbent assays. Deletion studies demonstrated interaction between the Cdc4p C-terminal domain and the PI 4-kinase C-terminal domain. Furthermore, interaction was abolished by the Cdc4p C-terminal domain point mutation, Gly107 to Ser. This allele also causes failure of cytokinesis. Ectopic expression of the PI 4-kinase C-terminal domain caused cytokinesis defects that were most extreme in cells carrying the G107S allele. We suggest that Cdc4p plays multiple roles in cytokinesis and that interaction with a PI 4-kinase may be important for contractile ring assembly and/or function.     

Telomere cap components influence the rate of senescence in telomerase-deficient yeast cells

Cells lacking telomerase undergo senescence, a progressive reduction in cell division that involves a cell cycle delay and culminates in "crisis," a period when most cells become inviable. In telomerase-deficient Saccharomyces cerevisiae cells lacking components of the nonsense-mediated mRNA decay (NMD) pathway (Upf1,Upf2, or Upf3 proteins), senescence is delayed, with crisis occurring approximately 10 to 25 population doublings later than in Upf+ cells. Delayed senescence is seen in upfDelta cells lacking the telomerase holoenzyme components Est2p and TLC1 RNA, as well as in cells lacking the telomerase regulators Est1p and Est3p. The delay of senescence in upfDelta cells is not due to an increased rate of survivor formation. Rather, it is caused by alterations in the telomere cap, composed of Cdc13p, Stn1p, and Ten1p. In upfDelta mutants, STN1 and TEN1 levels are increased. Increasing the levels of Stn1p and Ten1p in Upf+ cells is sufficient to delay senescence. In addition, cdc13-2 mutants exhibit delayed senescence rates similar to those of upfDelta cells. Thus, changes in the telomere cap structure are sufficient to affect the rate of senescence in the absence of telomerase. Furthermore, the NMD pathway affects the rate of senescence in telomerase-deficient cells by altering the stoichiometry of telomere cap components.     

Rif1 supports the function of the CST complex in yeast telomere capping

Telomere integrity in budding yeast depends on the CST (Cdc13-Stn1-Ten1) and shelterin-like (Rap1-Rif1-Rif2) complexes, which are thought to act independently from each other. Here we show that a specific functional interaction indeed exists among components of the two complexes. In particular, unlike RIF2 deletion, the lack of Rif1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. This synthetic interaction between Rif1 and the CST complex occurs independently of rif1Δ-induced alterations in telomere length. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, both DNA damage checkpoint activation and lethality in cdc13 rif1Δ cells are partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. The functional interaction between Rif1 and the CST complex is specific, because RIF1 deletion does not enhance checkpoint activation in case of CST-independent telomere capping deficiencies, such as those caused by the absence of Yku or telomerase. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex.     

Quantitative fitness analysis shows that NMD proteins and many other protein complexes suppress or enhance distinct telomere cap defects

To better understand telomere biology in budding yeast, we have performed systematic suppressor/enhancer analyses on yeast strains containing a point mutation in the essential telomere capping gene CDC13 (cdc13-1) or containing a null mutation in the DNA damage response and telomere capping gene YKU70 (yku70Δ). We performed Quantitative Fitness Analysis (QFA) on thousands of yeast strains containing mutations affecting telomere-capping proteins in combination with a library of systematic gene deletion mutations. To perform QFA, we typically inoculate 384 separate cultures onto solid agar plates and monitor growth of each culture by photography over time. The data are fitted to a logistic population growth model; and growth parameters, such as maximum growth rate and maximum doubling potential, are deduced. QFA reveals that as many as 5% of systematic gene deletions, affecting numerous functional classes, strongly interact with telomere capping defects. We show that, while Cdc13 and Yku70 perform complementary roles in telomere capping, their genetic interaction profiles differ significantly. At least 19 different classes of functionally or physically related proteins can be identified as interacting with cdc13-1, yku70Δ, or both. Each specific genetic interaction informs the roles of individual gene products in telomere biology. One striking example is with genes of the nonsense-mediated RNA decay (NMD) pathway which, when disabled, suppress the conditional cdc13-1 mutation but enhance the null yku70Δ mutation. We show that the suppressing/enhancing role of the NMD pathway at uncapped telomeres is mediated through the levels of Stn1, an essential telomere capping protein, which interacts with Cdc13 and recruitment of telomerase to telomeres. We show that increased Stn1 levels affect growth of cells with telomere capping defects due to cdc13-1 and yku70Δ. QFA is a sensitive, high-throughput method that will also be useful to understand other aspects of microbial cell biology.     

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.