Fission Yeast Telomeres Research Articles

Fate of telomere entanglements is dictated by the timing of anaphase midregion nuclear envelope breakdown

Persisting replication intermediates can confer mitotic catastrophe. Loss of the fission yeast telomere protein Taz1 (ortholog of mammalian TRF1/TRF2) causes telomeric replication fork (RF) stalling and consequently, telomere entanglements that stretch between segregating mitotic chromosomes. At ≤20 °C, these entanglements fail to resolve, resulting in lethality. Rif1, a conserved DNA replication/repair protein, hinders the resolution of telomere entanglements without affecting their formation. At mitosis, local nuclear envelope (NE) breakdown occurs in the cell’s midregion. Here we demonstrate that entanglement resolution occurs in the cytoplasm following this NE breakdown. However, in response to taz1Δ telomeric entanglements, Rif1 delays midregion NE breakdown at ≤20 °C, in turn disfavoring entanglement resolution. Moreover, Rif1 overexpression in an otherwise wild-type setting causes cold-specific NE defects and lethality, which are rescued by membrane fluidization. Hence, NE properties confer the cold-specificity of taz1Δ lethality, which stems from postponement of NE breakdown. We propose that such postponement promotes clearance of simple stalled RFs, but resolution of complex entanglements (involving strand invasion between nonsister telomeres) requires rapid exposure to the cytoplasm.

Stn1-Ten1 and Taz1 independently promote replication of subtelomeric fragile sequences in fission yeast

Efficient replication of terminal DNA is crucial to maintain telomere stability. In fission yeast, Taz1 and the Stn1-Ten1 (ST) complex play prominent roles in DNA-ends replication. However, their function remains elusive. Here, we have analyzed genome-wide replication and show that ST does not affect genome-wide replication but is crucial for the efficient replication of a subtelomeric region called STE3-2. We further show that, when ST function is compromised, a homologous recombination (HR)-based fork restart mechanism becomes necessary for STE3-2 stability. While both Taz1 and Stn1 bind to STE3-2, we find that the STE3-2 replication function of ST is independent of Taz1 but relies on its association with the shelterin proteins Pot1-Tpz1-Poz1. Finally, we demonstrate that the firing of an origin normally inhibited by Rif1 can circumvent the replication defect of subtelomeres when ST function is compromised. Our results help illuminate why fission yeast telomeres are terminal fragile sites.

Rap1 prevents fusions between long telomeres in fission yeast

The conserved Rap1 protein is part of the shelterin complex that plays critical roles in chromosome end protection and telomere length regulation. Previous studies have addressed how fission yeast Rap1 contributes to telomere length maintenance, but the mechanism by which the protein inhibits end fusions has remained elusive. Here, we use a mutagenesis screen in combination with high‐throughput sequencing to identify several amino acid positions in Rap1 that have key roles in end protection. Interestingly, mutations at these sites render cells susceptible to genome instability in a conditional manner, whereby longer telomeres are prone to undergoing end fusions, while telomeres within the normal length range are sufficiently protected. The protection of long telomeres is in part dependent on their nuclear envelope attachment mediated by the Rap1–Bqt4 interaction. Our data demonstrate that long telomeres represent a challenge for the maintenance of genome integrity, thereby providing an explanation for species‐specific upper limits on telomere length.

Shelterin

Schmutz and de Lange discuss the shelterin complex, which binds to mammalian telomeres and allows for the natural ends of chromosomes to be distinguished from sites of DNA damage.

Evolution of Telomeres in Schizosaccharomyces pombe and Its Possible Relationship to the Diversification of Telomere Binding Proteins.

Telomeres of nuclear chromosomes are usually composed of an array of tandemly repeated sequences that are recognized by specific Myb domain containing DNA-binding proteins (telomere-binding proteins, TBPs). Whereas in many eukaryotes the length and sequence of the telomeric repeat is relatively conserved, telomeric sequences in various yeasts are highly variable. Schizosaccharomyces pombe provides an excellent model for investigation of co-evolution of telomeres and TBPs. First, telomeric repeats of S. pombe differ from the canonical mammalian type TTAGGG sequence. Second, S. pombe telomeres exhibit a high degree of intratelomeric heterogeneity. Third, S. pombe contains all types of known TBPs (Rap1p [a version unable to bind DNA], Tay1p/Teb1p, and Taz1p) that are employed by various yeast species to protect their telomeres. With the aim of reconstructing evolutionary paths leading to a separation of roles between Teb1p and Taz1p, we performed a comparative analysis of the DNA-binding properties of both proteins using combined qualitative and quantitative biochemical approaches. Visualization of DNA-protein complexes by electron microscopy revealed qualitative differences of binding of Teb1p and Taz1p to mammalian type and fission yeast telomeres. Fluorescence anisotropy analysis quantified the binding affinity of Teb1p and Taz1p to three different DNA substrates. Additionally, we carried out electrophoretic mobility shift assays using mammalian type telomeres and native substrates (telomeric repeats, histone-box sequences) as well as their mutated versions. We observed relative DNA sequence binding flexibility of Taz1p and higher binding stringency of Teb1p when both proteins were compared directly to each other. These properties may have driven replacement of Teb1p by Taz1p as the TBP in fission yeast.

Swi1Timeless Prevents Repeat Instability at Fission Yeast Telomeres.

Genomic instability associated with DNA replication stress is linked to cancer and genetic pathologies in humans. If not properly regulated, replication stress, such as fork stalling and collapse, can be induced at natural replication impediments present throughout the genome. The fork protection complex (FPC) is thought to play a critical role in stabilizing stalled replication forks at several known replication barriers including eukaryotic rDNA genes and the fission yeast mating-type locus. However, little is known about the role of the FPC at other natural impediments including telomeres. Telomeres are considered to be difficult to replicate due to the presence of repetitive GT-rich sequences and telomere-binding proteins. However, the regulatory mechanism that ensures telomere replication is not fully understood. Here, we report the role of the fission yeast Swi1Timeless, a subunit of the FPC, in telomere replication. Loss of Swi1 causes telomere shortening in a telomerase-independent manner. Our epistasis analyses suggest that heterochromatin and telomere-binding proteins are not major impediments for telomere replication in the absence of Swi1. Instead, repetitive DNA sequences impair telomere integrity in swi1Δ mutant cells, leading to the loss of repeat DNA. In the absence of Swi1, telomere shortening is accompanied with an increased recruitment of Rad52 recombinase and more frequent amplification of telomere/subtelomeres, reminiscent of tumor cells that utilize the alternative lengthening of telomeres pathway (ALT) to maintain telomeres. These results suggest that Swi1 ensures telomere replication by suppressing recombination and repeat instability at telomeres. Our studies may also be relevant in understanding the potential role of Swi1Timeless in regulation of telomere stability in cancer cells.

Ccq1-Tpz1TPP1 interaction facilitates telomerase and SHREC association with telomeres in fission yeast.

Evolutionarily conserved shelterin complex is essential for telomere maintenance in the fission yeast Schizosaccharomyces pombe. Elimination of the fission yeast shelterin subunit Ccq1 causes progressive loss of telomeres due to the inability to recruit telomerase, activates the DNA damage checkpoint, and loses heterochromatin at telomere/subtelomere regions due to reduced recruitment of the heterochromatin regulator complex Snf2/histone deacetylase-containing repressor complex (SHREC). The shelterin subunit Tpz1(TPP1) directly interacts with Ccq1 through conserved C-terminal residues in Tpz1(TPP1), and tpz1 mutants that fail to interact with Ccq1 show telomere shortening, checkpoint activation, and loss of heterochromatin. While we have previously concluded that Ccq1-Tpz1(TPP1) interaction contributes to Ccq1 accumulation and telomerase recruitment based on analysis of tpz1 mutants that fail to interact with Ccq1, another study reported that loss of Ccq1-Tpz1(TPP1) interaction does not affect accumulation of Ccq1 or telomerase. Furthermore, it remained unclear whether loss of Ccq1-Tpz1(TPP1) interaction affects SHREC accumulation at telomeres. To resolve these issues, we identified and characterized a series of ccq1 mutations that disrupt Ccq1-Tpz1(TPP1) interaction. Characterization of these ccq1 mutants established that Ccq1-Tpz1(TPP1) interaction contributes to optimal binding of the Ccq1-SHREC complex, and is critical for Rad3(ATR)/Tel1(ATM)-dependent Ccq1 Thr93 phosphorylation and telomerase recruitment.

Telomere regulation during the cell cycle in fission yeast.

The fission yeast Schizosaccharomyces pombe has emerged as a useful model organism to study telomere maintenance mechanisms. In this chapter, we provide detailed protocols for quantitative ChIP and BrdU incorporation analyses to investigate how fission yeast telomeres are regulated during the cell cycle by utilizing cdc25-22 synchronized cell cultures.

Purification and characterization of the fission yeast telomere clustering factors, Bqt1 and Bqt2

During meiosis, chromosomes adopt a bouquet arrangement, which is widely conserved among eukaryotes. This arrangement is assumed to play an important role in the normal progression of meiosis, by mediating the proper pairing of homologous chromosomes. In Schizosaccharomyces pombe, the complex of Bqt1 and Bqt2 plays a key role in telomere clustering and the subsequent bouquet arrangement of chromosomes during early meiotic prophase. Bqt1 and Bqt2 are part of a multi-protein complex that mediates the attachment of the telomere to the nuclear membrane. However, the structural details of the complex are needed to clarify the mechanism of telomere clustering. To enable biophysical studies of Bqt1 and Bqt2, we established a purification procedure for the Schizosaccharomyces japonicus Bqt1-Bqt2 complex, which is closely related to the S. pombe Bqt1–Bqt2 complex. A co-expression vector, in which one of the expressed subunits is fused to a removable SUMO tag, yielded high amounts of the proteins in the soluble fraction. The solubility of the Bqt1–Bqt2 complex after the removal of the SUMO tag was maintained by including CHAPS, a nondenaturing, zwitterionic detergent, in the purification buffers. These procedures enabled us to rapidly purify the stable Bqt1–Bqt2 complex. The co-purified Bqt1 and Bqt2 proteins formed a stable heterodimer, consistent with results from in vivo studies showing the requirement of both proteins for the bouquet arrangement. The expression and purification procedures established here will facilitate further biophysical studies of the Bqt1–Bqt2 complex.

Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast

SUMMARYThe shelterin complex plays both positive and negative roles in telomerase regulation. While shelterin prevents the checkpoint kinases ATM and ATR from fully activating DNA damage responses at telomeres, those kinases are also required for telomere maintenance. In fission yeast, cells lacking both Tel1 (ATM ortholog) and Rad3 (ATR ortholog) fail to recruit telomerase to telomeres, and survive by circularizing chromosomes. However, the critical telomere substrate(s) of Tel1ATM/Rad3ATR remained unknown. Here, we show that Tel1ATM/Rad3ATR-dependent phosphorylation of the shelterin subunit Ccq1 on Thr93 is essential for telomerase association with telomeres. In addition, we show that the telomerase subunit Est1 interacts directly with the phosphorylated Thr93 of Ccq1 to ensure telomere maintenance. The shelterin subunits Taz1, Rap1 and Poz1 (previously established inhibitors of telomerase) were also found to negatively regulate Ccq1 phosphorylation. These findings establish Tel1ATM/Rad3ATR-dependent Ccq1 Thr93 phosphorylation as a critical regulator of telomere maintenance in fission yeast.

Pot1 inactivation leads to rampant telomere resection and loss in one cell cycle

Removal of the conserved telomere protein, Pot1, confers the immediate loss of fission yeast telomeres. This drastic phenotype has established the centrality of Pot1 for telomere maintenance but prohibited elucidation of the intermediate steps leading to telomere loss. To circumvent this problem, we have generated a conditional allele, pot1–1. We show that loss of Pot1 function during G1 leads to rapid telomere erosion during the ensuing S/G2 period. Precipitous telomere loss depends upon S-phase progression and is preceded by 5′ telomeric resection. Telomere loss is accompanied by ATR- and Chk1-mediated checkpoint activation, but is not caused by checkpoint arrest.

Roles of the checkpoint sensor clamp Rad9-Rad1-Hus1 (911)-complex and the clamp loaders Rad17-RFC and Ctf18-RFC in Schizosaccharomyces pombe telomere maintenance

While telomeres must provide mechanisms to prevent DNA repair and DNA damage checkpoint factors from fusing chromosome ends and causing permanent cell cycle arrest, these factors associate with functional telomeres and play critical roles in the maintenance of telomeres. Previous studies have established that Tel1 (ATM) and Rad3 (ATR) kinases play redundant but essential roles for telomere maintenance in fission yeast. In addition, the Rad9-Rad1-Hus1 (911) and Rad17-RFC complexes work downstream of Rad3 (ATR) in fission yeast telomere maintenance. Here, we investigated how 911, Rad17-RFC and another RFC-like complex Ctf18-RFC contribute to telomere maintenance in fission yeast cells lacking Tel1 and carrying a novel hypomorphic allele of rad3 (DBD-rad3), generated by the fusion between the DNA binding domain (DBD) of the fission yeast telomere capping protein Pot1 and Rad3. Our investigations have uncovered a surprising redundancy for Rad9 and Hus1 in allowing Rad1 to contribute to telomere maintenance in DBD-rad3 tel1 cells. In addition, we found that Rad17-RFC and Ctf18-RFC carry out redundant telomere maintenance functions in DBD-rad3 tel1 cells. Since checkpoint sensor proteins are highly conserved, genetic redundancies uncovered here may be relevant to telomere maintenance and detection of DNA damage in other eukaryotes.

A Kinase-Independent Role for the Rad3ATR-Rad26ATRIP Complex in Recruitment of Tel1ATM to Telomeres in Fission Yeast

ATM and ATR are two redundant checkpoint kinases essential for the stable maintenance of telomeres in eukaryotes. Previous studies have established that MRN (Mre11-Rad50-Nbs1) and ATRIP (ATR Interacting Protein) interact with ATM and ATR, respectively, and recruit their partner kinases to sites of DNA damage. Here, we investigated how Tel1ATM and Rad3ATR recruitment to telomeres is regulated in fission yeast. Quantitative chromatin immunoprecipitation (ChIP) assays unexpectedly revealed that the MRN complex could also contribute to the recruitment of Tel1ATM to telomeres independently of the previously established Nbs1 C-terminal Tel1ATM interaction domain. Recruitment of Tel1ATM to telomeres in nbs1-c60Δ cells, which lack the C-terminal 60 amino acid Tel1ATM interaction domain of Nbs1, was dependent on Rad3ATR-Rad26ATRIP, but the kinase domain of Rad3ATR was dispensable. Thus, our results establish that the Rad3ATR-Rad26ATRIP complex contributes to the recruitment of Tel1ATM independently of Rad3ATR kinase activity, by a mechanism redundant with the Tel1ATM interaction domain of Nbs1. Furthermore, we found that the N-terminus of Nbs1 contributes to the recruitment of Rad3ATR-Rad26ATRIP to telomeres. In response to replication stress, mammalian ATR–ATRIP also contributes to ATM activation by a mechanism that is dependent on the MRN complex but independent of the C-terminal ATM interaction domain of Nbs1. Since telomere protection and DNA damage response mechanisms are very well conserved between fission yeast and mammalian cells, mammalian ATR–ATRIP may also contribute to the recruitment of ATM to telomeres and to sites of DNA damage independently of ATR kinase activity.

Roles of Heterochromatin and Telomere Proteins in Regulation of Fission Yeast Telomere Recombination and Telomerase Recruitment

When the telomerase catalytic subunit (Trt1/TERT) is deleted, a majority of fission yeast cells survives by circularizing chromosomes. Alternatively, a small minority survives by maintaining telomeric repeats through recombination among telomeres. The recombination-based telomere maintenance in trt1Delta cells is inhibited by the telomere protein Taz1. In addition, catalytically inactive full-length Trt1 (Trt1-CI) and truncated Trt1 lacking the T-motif and reverse transcriptase (RT) domain (Trt1-DeltaT/RT) can strongly inhibit recombination-based survival. Here, we investigated the effects of deleting the heterochromatin proteins Swi6 (HP1 ortholog) and Clr4 (Suv39 family of histone methyltransferases) and the telomere capping complex subunits Poz1 and Ccq1 on Taz1- and Trt1-dependent telomere recombination inhibition. The ability of Taz1 to inhibit telomere recombination did not require Swi6, Clr4, Poz1, or Ccq1. Although Swi6, Clr4, and Poz1 were dispensable for the inhibition of telomere recombination by Trt1-CI, Ccq1 was required for efficient telomere recruitment of Trt1 and Trt1-CI-dependent inhibition of telomere recombination. We also found that Swi6, Clr4, Ccq1, the checkpoint kinase Rad3 (ATR ortholog), and the telomerase regulatory subunit Est1 are all required for Trt1-DeltaT/RT to inhibit telomere recombination. However, because loss of Swi6, Clr4, Rad3, Ccq1, or Est1 did not significantly alter the recruitment efficiency of Trt1-DeltaT/RT to telomeres, these factors are likely to enhance the ability of Trt1-DeltaT/RT to inhibit recombination-based survival by contributing to the negative regulation of telomere recombination.

Reverse Transcribing the Code for Chromosome Stability

The linearity of eukaryotic chromosomes presents challenges to cells, as the presence of DNA "ends" poses problems for the DNA replication machinery and the cell's damage response systems. This year's Nobel Prize in Physiology or Medicine recognized groundbreaking studies establishing the telomere field as a crucial area of biomedical research.

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 Tel1ATM and Rad3ATR 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, Tel1ATM and Rad3ATR 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 Tel1ATM/Rad3ATR kinases and Tpz1-Ccq1 to ensure proper protection and maintenance of telomeres in fission yeast.

Antics at the telomere: uncoupled polymerases solve the end replication problem

Replicating the end of a linear chromosome poses a problem that can be solved by the combined action of the general DNA replication machinery, DNA repair factors, telomere proteins and telomerase. In this issue of The EMBO Journal , a new study by Moser et al examines the timing of replication, repair and telomere factor association with fission yeast telomeres. The study demonstrates the dynamic nature of protein binding and provides a framework for understanding how leading and lagging strand polymerases, DNA damage signalling and telomere factors cooperate during telomere replication.

Differential arrival of leading and lagging strand DNA polymerases at fission yeast telomeres

To maintain genomic integrity, telomeres must undergo switches from a protected state to an accessible state that allows telomerase recruitment. To better understand how telomere accessibility is regulated in fission yeast, we analysed cell cycle-dependent recruitment of telomere-specific proteins (telomerase Trt1, Taz1, Rap1, Pot1 and Stn1), DNA replication proteins (DNA polymerases, MCM, RPA), checkpoint protein Rad26 and DNA repair protein Nbs1 to telomeres. Quantitative chromatin immunoprecipitation studies revealed that MCM, Nbs1 and Stn1 could be recruited to telomeres in the absence of telomere replication in S-phase. In contrast, Trt1, Pot1, RPA and Rad26 failed to efficiently associate with telomeres unless telomeres are actively replicated. Unexpectedly, the leading strand DNA polymerase epsilon (Polepsilon) arrived at telomeres earlier than the lagging strand DNA polymerases alpha (Polalpha) and delta (Poldelta). Recruitment of RPA and Rad26 to telomeres matched arrival of DNA Polepsilon, whereas S-phase specific recruitment of Trt1, Pot1 and Stn1 matched arrival of DNA Polalpha. Thus, the conversion of telomere states involves an unanticipated intermediate step where lagging strand synthesis is delayed until telomerase is recruited.

TER1, the RNA subunit of fission yeast telomerase

Telomerase is the ribonucleoprotein complex that adds telomeric repeats to the ends of chromosomes. Its protein subunit TERT is highly conserved among eukaryotes, whereas the RNA subunit varies greatly in size and sequence, hindering the identification of telomerase RNAs in some important model organisms. Here we report the identification and functional characterization of TER1, the telomerase RNA component from fission yeast Schizosaccharomyces pombe. Deletion of ter1+ caused progressive shortening of telomeres and cellular senescence followed by chromosome circularization. Interactions between Est1 and Trt1, the two known protein components of fission yeast telomerase, were dependent on TER1, supporting its role as a scaffold for the assembly of protein subunits. Using a series of template mutations, we show that translocation or dissociation site variability and template-primer slippage account for the sequence heterogeneity of fission yeast telomeres.

Extended DNA Binding Site in Pot1 Broadens Sequence Specificity to Allow Recognition of Heterogeneous Fission Yeast Telomeres

The Pot1 (protection of telomeres) protein binds to single-stranded telomeric DNA and is essential for the protection of chromosome ends from degradation and end-to-end fusions. The Pot1 amino-terminal DNA binding domain, Pot1N, adopts an oligonucleotide/oligosaccharide binding fold and binds GGTTAC motifs cooperatively and with exceptionally high sequence specificity. We have now examined DNA binding to naturally occurring telomeric substrates based on the analysis of 100 cloned chromosome ends and in the context of the full-length Pot1 protein. Here, we describe several important differences between Pot1 and Pot1N with apparent consequences for chromosome end protection. Specifically, full-length Pot1.DNA complexes are more stable, and the minimal binding site for a Pot1 monomer is extended into two adjacent telomeric repeats. We provide evidence that Pot1 contains a second DNA binding motif that recognizes DNA with reduced sequence specificity compared with the domain present in Pot1N. The two DNA binding motifs cooperate, whereby the amino-terminal oligonucleotide/oligosaccharide binding fold determines the registry of binding, and the internal DNA binding motif stabilizes the complex and expands the protected region toward the 3' -end. Consistent with a role in chromosome end capping, Pot1 prevents access of telomerase to the 3'-end and protects against exonucleolytic degradation.