Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

Some human cancers maintain their telomeres using the alternative lengthening of telomeres (ALT) mechanism; a process thought to involve recombination. Different types of recombinational telomere elongation pathways have been identified in yeasts. In senescing yeast telomerase deletion (ter1-Δ) mutants with very short telomeres, it has been hypothesized that copying a tiny telomeric circle (t-circle) by a rolling circle mechanism is the key event in telomere elongation. In other cases more closely resembling ALT cells, such as the stn1-M1 mutant of Kluyveromyces lactis, the telomeres appear to be continuously unstable and routinely reach very large sizes. By employing two-dimensional gel electrophoresis and electron microscopy, we show that stn1-M1 cells contain abundant double stranded t-circles ranging from ∼100 to 30 000 bp in size. We also observed small single-stranded t-circles, specifically composed of the G-rich telomeric strand and tailed circles resembling rolling circle replication intermediates. The t-circles most likely arose from recombination events that also resulted in telomere truncations. The findings strengthen the possibility that t-circles contribute to telomere maintenance in stn1-M1 and ALT cells.     

Maintenance of very long telomeres by recombination in the Kluyveromyces lactis stn1-M1 mutant involves extreme telomeric turnover, telomeric circles, and concerted telomeric amplification

Some cancers utilize the recombination-dependent process of alternative lengthening of telomeres (ALT) to maintain long heterogeneous telomeres. Here, we studied the recombinational telomere elongation (RTE) of the Kluyveromyces lactis stn1-M1 mutant. We found that the total amount of the abundant telomeric DNA in stn1-M1 cells is subject to rapid variation and that it is likely to be primarily extrachromosomal. Rad50 and Rad51, known to be required for different RTE pathways in Saccharomyces cerevisiae, were not essential for the production of either long telomeres or telomeric circles in stn1-M1 cells. Circles of DNA containing telomeric repeats (t-circles) either present at the point of establishment of long telomeres or introduced later into stn1-M1 cells each led to the formation of long tandem arrays of the t-circle's sequence, which were incorporated at multiple telomeres. These tandem arrays were extraordinarily unstable and showed evidence of repeated rounds of concerted amplification. Our results suggest that the maintenance of telomeres in the stn1-M1 mutant involves extreme turnover of telomeric sequences from processes including both large deletions and the copying of t-circles.     

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.     

Recombination at long mutant telomeres produces tiny single- and double-stranded telomeric circles

Recombinational telomere elongation (RTE) known as alternate lengthening of telomeres is the mechanism of telomere maintenance in up to 5 to 10% of human cancers. The telomeres of yeast mutants lacking telomerase can also be maintained by recombination. Previously, we proposed the roll-and-spread model to explain this elongation in the yeast Kluveromyces lactis. This model suggests that a very small ( approximately 100-bp) circular molecule of telomeric DNA is copied by a rolling circle event to generate a single long telomere. The sequence of this primary elongated telomere is then spread by recombination to all remaining telomeres. Here we show by two-dimensional gel analysis and electron microscopy that small circles of single- and double-stranded telomeric DNA are commonly made by recombination in a K. lactis mutant with long telomeres. These circles were found to be especially abundant between 100 and 400 bp (or nucleotides). Interestingly, the single-stranded circles consist of only the G-rich telomeric strand sequence. To our knowledge this is the first report of single-stranded telomeric circles as a product of telomere dysfunction. We propose that the small telomeric circles form through the resolution of an intratelomeric strand invasion which resembles a t-loop. Our data reported here demonstrate that K. lactis can, in at least some circumstances, make telomeric circles of the very small sizes predicted by the roll-and-spread model. The very small circles seen here are both predicted products of telomere rapid deletion, a process observed in both human and yeast cells, and predicted templates for roll-and-spread RTE.     

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.     

Factors influencing the recombinational expansion and spread of telomeric tandem arrays in Kluyveromyces lactis

We have previously shown that DNA circles containing telomeric repeats and a marker gene can promote the recombinational elongation of telomeres in Kluyveromyces lactis by a mechanism proposed to involve rolling-circle DNA synthesis. Wild-type cells acquire a long tandem array at a single telomere, while telomerase deletion (ter1-Δ) cells, acquire an array and also spread it to multiple telomeres. In this study, we further examine the factors that affect the formation and spread of telomeric tandem arrays. We show that a telomerase⁺ strain with short telomeres and high levels of subtelomeric gene conversion can efficiently form and spread arrays, while a telomere fusion mutant is not efficient at either process. This indicates that an elevated level of gene conversion near telomeres is required for spreading but that growth senescence and a tendency to elongate telomeres in the absence of exogenously added circles are not. Surprisingly, telomeric repeats are frequently deleted from a transforming URA3-telomere circle at or prior to the time of array formation by a mechanism dependent upon the presence of subtelomeric DNA in the circle. We further show that in a ter1-Δ strain, long tandem arrays can arise from telomeres initially containing a single-copy insert of the URA3-telomere sequence. However, the reduced rate of array formation in such strains suggests that single-copy inserts are not typical intermediates in arrays formed from URA3-telomere circles. Using heteroduplex circles, we have demonstrated that either strand of a URA3-telomere circle can be utilized to form telomeric tandem arrays. Consistent with this, we demonstrate that 100-nucleotide single-stranded telomeric circles of either strand can promote recombinational telomere elongation.     

A mutation in the STN1 gene triggers an alternative lengthening of telomere-like runaway recombinational telomere elongation and rapid deletion in yeast

Some human cancer cells achieve immortalization by using a recombinational mechanism termed ALT (alternative lengthening of telomeres). A characteristic feature of ALT cells is the presence of extremely long and heterogeneous telomeres. The molecular mechanism triggering and maintaining this pathway is currently unknown. In Kluyveromyces lactis, we have identified a novel allele of the STN1 gene that produces a runaway ALT-like telomeric phenotype by recombination despite the presence of an active telomerase pathway. Additionally, stn1-M1 cells are synthetically lethal in combination with rad52 and display chronic growth and telomere capping defects including extensive 3' single-stranded telomere DNA and highly elevated subtelomere gene conversion. Strikingly, stn1-M1 cells undergo a very high rate of telomere rapid deletion (TRD) upon reintroduction of STN1. Our results suggest that the protein encoded by STN1, which protects the terminal 3' telomere DNA, can regulate both ALT and TRD.     

Recombinogenic Telomeres in Diploid Sorex granarius (Soricidae,Eulipotyphla) Fibroblast Cells

The telomere structure in the Iberian shrew Sorex granarius is characterized by unique, striking features, with short arms of acrocentric chromosomes carrying extremely long telomeres (up to 300 kb) with interspersed ribosomal DNA (rDNA) repeat blocks. In this work, we investigated the telomere physiology of S. granarius fibroblast cells and found that telomere repeats are transcribed on both strands and that there is no telomere-dependent senescence mechanism. Although telomerase activity is detectable throughout cell culture and appears to act on both short and long telomeres, we also discovered that signatures of a recombinogenic activity are omnipresent, including telomere-sister chromatid exchanges, formation of alternative lengthening of telomeres (ALT)-associated PML-like bodies, production of telomere circles, and a high frequency of telomeres carrying marks of a DNA damage response. Our results suggest that recombination participates in the maintenance of the very long telomeres in normal S. granarius fibroblasts. We discuss the possible interplay between the interspersed telomere and rDNA repeats in the stabilization of the very long telomeres in this organism.     

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.     

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.     

Deletion of the major peroxiredoxin Tsa1 alters telomere length homeostasis

Reactive oxygen species (ROS) are proposed to play a major role in telomere length alterations during aging. The mechanisms by which ROS disrupt telomeres remain unclear. In Saccharomyces cerevisiae, telomere DNA consists of TG_(1–3) repeats, which are maintained primarily by telomerase. Telomere length maintenance can be modulated by the expression level of telomerase subunits and telomerase activity. Additionally, telomerase‐mediated telomere repeat addition is negatively modulated by the levels of telomere‐bound Rap1‐Rif1‐Rif2 protein complex. Using a yeast strain defective in the major peroxiredoxin Tsa1 that is involved in ROS neutralization, we have investigated the effect of defective ROS detoxification on telomere DNA, telomerase, telomere‐binding proteins, and telomere length. Surprisingly, the tsa1 mutant does not show significant increase in steady‐state levels of oxidative DNA lesions at telomeres. The tsa1 mutant displays abnormal telomere lengthening, and reduction in oxidative exposure alleviates this phenotype. The telomere lengthening in the tsa1 cells was abolished by disruption of Est2, subtelomeric DNA, Rap1 C‐terminus, or Rif2, but not by Rif1 deletion. Although telomerase expression and activity are not altered, telomere‐bound Est2 is increased, while telomere‐bound Rap1 is reduced in the tsa1 mutant. We propose that defective ROS scavenging can interfere with pathways that are critical in controlling telomere length homeostasis.     

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.     

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.     

Recombination can either help maintain very short telomeres or generate longer telomeres in yeast cells with weak telomerase activity

Yeast mutants lacking telomerase are able to elongate their telomeres through processes involving homologous recombination. In this study, we investigated telomeric recombination in several mutants that normally maintain very short telomeres due to the presence of a partially functional telomerase. The abnormal colony morphology present in some mutants was correlated with especially short average telomere length and with a requirement for RAD52 for indefinite growth. Better-growing derivatives of some of the mutants were occasionally observed and were found to have substantially elongated telomeres. These telomeres were composed of alternating patterns of mutationally tagged telomeric repeats and wild-type repeats, an outcome consistent with amplification occurring via recombination rather than telomerase. Our results suggest that recombination at telomeres can produce two distinct outcomes in the mutants we studied. In occasional cells, recombination generates substantially longer telomeres, apparently through the roll-and-spread mechanism. However, in most cells, recombination appears limited to helping to maintain very short telomeres. The latter outcome likely represents a simplified form of recombinational telomere maintenance that is independent of the generation and copying of telomeric circles.     

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.     

Fission Yeast Tel1ATM and Rad3ATR Promote Telomere Protection and Telomerase Recruitment

The checkpoint kinases ATM and ATR are redundantly required for maintenance of stable telomeres in diverse organisms, including budding and fission yeasts, Arabidopsis, Drosophila, and mammals. However, the molecular basis for telomere instability in cells lacking ATM and ATR has not yet been elucidated fully in organisms that utilize both the telomere protection complex shelterin and telomerase to maintain telomeres, such as fission yeast and humans. Here, we demonstrate by quantitative chromatin immunoprecipitation (ChIP) assays that simultaneous loss of Tel1^ATM and Rad3^ATR kinases leads to a defect in recruitment of telomerase to telomeres, reduced binding of the shelterin complex subunits Ccq1 and Tpz1, and increased binding of RPA and homologous recombination repair factors to telomeres. Moreover, we show that interaction between Tpz1-Ccq1 and telomerase, thought to be important for telomerase recruitment to telomeres, is disrupted in tel1Δ rad3Δ cells. Thus, Tel1^ATM and Rad3^ATR are redundantly required for both protection of telomeres against recombination and promotion of telomerase recruitment. Based on our current findings, we propose the existence of a regulatory loop between Tel1^ATM/Rad3^ATR kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.     

The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening

Pif1 family helicases are evolutionary conserved 5′–3′ DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.     

Abnormal micronuclear telomeres lead to an unusual cell cycle checkpoint and defects in Tetrahymena oral morphogenesis

Telomere mutants have been well studied with respect to telomerase and the role of telomere binding proteins, but they have not been used to explore how a downstream morphogenic event is related to the mutated telomeric DNA. We report that alterations at the telomeres can have profound consequences on organellar morphogenesis. Specifically, a telomerase RNA mutation termed ter1-43AA results in the loss of germ line micronuclear telomeres in the binucleate protozoan Tetrahymena thermophila. These cells also display a micronuclear mitotic arrest, characterized by an extreme delay in anaphase with an elongated, condensed chromatin and a mitotic spindle apparatus. This anaphase defect suggests telomere fusions and consequently a spindle rather than a DNA damage checkpoint. Most surprisingly, these mutants exhibit unique, dramatic defects in the formation of the cell's oral apparatus. We suggest that micronuclear telomere loss leads to a “dynamic pause” in the program of cortical development, which may reveal an unusual cell cycle checkpoint.     

Specific Localization of the Drosophila Telomere Transposon Proteins and RNAs,Give Insight in Their Behavior,Control and Telomere Biology in This Organism

Drosophila telomeres constitute a remarkable exception to the telomerase mechanism. Although maintaining the same cytological and functional properties as telomerase maintain telomeres, Drosophila telomeres embed the telomere retrotransposons whose specific and highly regulated terminal transposition maintains the appropriate telomere length in this organism. Nevertheless, our current understanding of how the mechanism of the retrotransposon telomere works and which features are shared with the telomerase system is very limited. We report for the first time a detailed study of the localization of the main components that constitute the telomeres in Drosophila, HeT-A and TART RNAs and proteins. Our results in wild type and mutant strains reveal localizations of HeT-A Gag and TART Pol that give insight in the behavior of the telomere retrotransposons and their control. We find that TART Pol and HeT-A Gag only co-localize at the telomeres during the interphase of cells undergoing mitotic cycles. In addition, unexpected protein and RNA localizations with a well-defined pattern in cells such as the ovarian border cells and nurse cells, suggest possible strategies for the telomere transposons to reach the oocyte, and/or additional functions that might be important for the correct development of the organism. Finally, we have been able to visualize the telomere RNAs at different ovarian stages of development in wild type and mutant lines, demonstrating their presence in spite of being tightly regulated by the piRNA mechanism.