Yeast Telomeres Research Articles

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.

Computing the Length of the Shortest Telomere in the Nucleus

The telomere length can either be shortened or elongated by an enzyme called telomerase after each cell division. Interestingly, the shortest telomere is involved in controlling the ability of a cell to divide. Yet, its dynamics remains elusive. We present here a stochastic approach where we model this dynamics using a Markov jump process. We solve the forward Fokker-Planck equation to obtain the steady state distribution and the statistical moments of telomere lengths. We focus specifically on the shortest one and we estimate its length difference with the second shortest telomere. After extracting key parameters such as elongation and shortening dynamics from experimental data, we compute the length of telomeres in yeast and obtain as a possible prediction the minimum concentration of telomerase required to ensure a proper cell division.

Dna2 Is Involved in CA Strand Resection and Nascent Lagging Strand Completion at Native Yeast Telomeres

Post-replicational telomere end processing involves both extension by telomerase and resection to produce 3'-GT-overhangs that extend beyond the complementary 5'-CA-rich strand. Resection must be carefully controlled to maintain telomere length. At short de novo telomeres generated artificially by HO endonuclease in the G2 phase, we show that dna2-defective strains are impaired in both telomere elongation and sequential 5'-CA resection. At native telomeres in dna2 mutants, GT-overhangs do clearly elongate during late S phase but are shorter than in wild type, suggesting a role for Dna2 in 5'-CA resection but also indicating significant redundancy with other nucleases. Surprisingly, elimination of Mre11 nuclease or Exo1, which are complementary to Dna2 in resection of internal double strand breaks, does not lead to further shortening of GT-overhangs in dna2 mutants. A second step in end processing involves filling in of the CA-strand to maintain appropriate telomere length. We show that Dna2 is required for normal telomeric CA-strand fill-in. Yeast dna2 mutants, like mutants in DNA ligase 1 (cdc9), accumulate low molecular weight, nascent lagging strand DNA replication intermediates at telomeres. Based on this and other results, we propose that FEN1 is not sufficient and that either Dna2 or Exo1 is required to supplement FEN1 in maturing lagging strands at telomeres. Telomeres may be among the subset of genomic locations where Dna2 helicase/nuclease is essential for the two-nuclease pathway of primer processing on lagging strands.

The tenacious recognition of yeast telomere sequence by Cdc13 is fully exerted by a single OB-fold domain

Cdc13, the telomere end-binding protein from Saccharomyces cerevisiae, is a multidomain protein that specifically binds telomeric single-stranded DNA (ssDNA) with exquisitely high affinity to coordinate telomere maintenance. Recent structural and genetic data have led to the proposal that Cdc13 is the paralog of RPA70 within a telomere-specific RPA complex. Our understanding of Cdc13 structure and biochemistry has been largely restricted to studies of individual domains, precluding analysis of how each domain influences the activity of the others. To better facilitate a comparison to RPA70, we evaluated the ssDNA binding of full-length S. cerevisiae Cdc13 to its minimal substrate, Tel11. We found that, unlike RPA70 and the other known telomere end-binding proteins, the core Cdc13 ssDNA-binding activity is wholly contained within a single tight-binding oligosaccharide/oligonucleotide/oligopeptide binding (OB)-fold. Because two OB-folds are implicated in dimerization, we also evaluated the relationship between dimerization and ssDNA-binding activity and found that the two activities are independent. We also find that Cdc13 binding exhibits positive cooperativity that is independent of dimerization. This study reveals that, while Cdc13 and RPA70 share similar domain topologies, the corresponding domains have evolved different and specialized functions.

TERRA –A Calling Card for Telomerase

Transcription of telomeric DNA has long been thought to inhibit telomerase. Using live cell imaging, in this issue, Cusanelli et al. (2013) now reveal a different scenario: telomeric RNA is preferentially generated at short telomeres and delivers telomerase to the chromosome end from which the transcript originated.

Telomeric Noncoding RNA TERRA Is Induced by Telomere Shortening to Nucleate Telomerase Molecules at Short Telomeres

Elongation of a short telomere depends on the action of multiple telomerase molecules, which are visible as telomerase RNA foci or clusters associated with telomeres in yeast and mammalian cells. How several telomerase molecules act on a single short telomere is unknown. Herein, we report that the telomeric noncoding RNA TERRA is involved in the nucleation of telomerase molecules into clusters prior to their recruitment at a short telomere. We find that telomere shortening induces TERRA expression, leading to the accumulation of TERRA molecules into a nuclear focus. Simultaneous time-lapse imaging of telomerase RNA and TERRA reveals spontaneous events of telomerase nucleation on TERRA foci in early S phase, generating TERRA-telomerase clusters. This cluster is subsequently recruited to the short telomere from which TERRA transcripts originate during S phase. We propose that telomere shortening induces noncoding RNA expression to coordinate the recruitment and activity of telomerase molecules at short telomeres.

Rif1 and Rif2 Shape Telomere Function and Architecture through Multivalent Rap1 Interactions

Yeast telomeres comprise irregular TG₁₋₃ DNA repeats bound by the general transcription factor Rap1. Rif1 and Rif2, along with Rap1, form the telosome, a protective cap that inhibits telomerase, counteracts SIR-mediated transcriptional silencing, and prevents inadvertent recognition of telomeres as DNA double-strand breaks. We provide a molecular, biochemical, and functional dissection of the protein backbone at the core of the yeast telosome. The X-ray structures of Rif1 and Rif2 bound to the Rap1 C-terminal domain and that of the Rif1 C terminus are presented. Both Rif1 and Rif2 have separable and independent Rap1-binding epitopes, allowing Rap1 binding over large distances (42-110 Å). We identify tetramerization (Rif1) and polymerization (Rif2) modules that, in conjunction with the long-range binding, give rise to a higher-order architecture that interlinks Rap1 units. This molecular Velcro relies on Rif1 and Rif2 to recruit and stabilize Rap1 on telomeric arrays and is required for telomere homeostasis in vivo.

Tel1 and Rad51 are involved in the maintenance of telomeres with capping deficiency

Vertebrate-like T2AG3 telomeres in tlc1-h yeast consist of short double-stranded regions and long single-stranded overhang (G-tails) and, although based on Tbf1-capping activity, they are capping deficient. Consistent with this idea, we observe Y’ amplification because of homologous recombination, even in the presence of an active telomerase. In these cells, Y’ amplification occurs by different pathways: in Tel1+ tlc1h cells, it is Rad51-dependent, whereas in the absence of Tel1, it depends on Rad50. Generation of telomeric G-tail, which is cell cycle regulated, depends on the MRX (Mre11-Rad50-Xrs2) complex in tlc1h cells or is MRX-independent in tlc1h tel1Δ mutants. Unexpectedly, we observe telomere elongation in tlc1h lacking Rad51 that seems to act as a telomerase competitor for binding to telomeric G-tails. Overall, our results show that Tel1 and Rad51 have multiple roles in the maintenance of vertebrate-like telomeres in yeast, supporting the idea that they may participate to evolutionary conserved telomere protection mechanism/s acting at uncapped telomeres.

Spatial telomere organization and clustering in yeast Saccharomyces cerevisiae nucleus is generated by a random dynamics of aggregation–dissociation

Spatial and temporal behavior of chromosomes and their regulatory proteins is a key control mechanism in genomic function. This is exemplified by the clustering of the 32 budding yeast telomeres that form foci in which silencing factors concentrate. To uncover the determinants of telomere distribution, we compare live-cell imaging with a stochastic model of telomere dynamics that we developed. We show that random encounters alone are inadequate to produce the clustering observed in vivo. In contrast, telomere dynamics observed in vivo in both haploid and diploid cells follows a process of dissociation-aggregation. We determine the time that two telomeres spend in the same cluster for the telomere distribution observed in cells expressing different levels of the silencing factor Sir3 protein, limiting for telomere clustering. We conclude that telomere clusters, their dynamics, and their nuclear distribution result from random motion, aggregation, and dissociation of telomeric regions, specifically determined by the amount of Sir3.

Yeast telomere maintenance is globally controlled by programmed ribosomal frameshifting and the nonsense-mediated mRNA decay pathway

We have previously shown that ~10% of all eukaryotic mRNAs contain potential programmed -1 ribosomal frameshifting (-1 PRF) signals and that some function as mRNA destabilizing elements through the Nonsense-Mediated mRNA Decay (NMD) pathway by directing translating ribosomes to premature termination codons. Here, the connection between -1 PRF, NMD and telomere end maintenance are explored. Functional -1 PRF signals were identified in the mRNAs encoding two components of yeast telomerase, EST1 and EST2, and in mRNAs encoding proteins involved in recruiting telomerase to chromosome ends, STN1 and CDC13. All of these elements responded to mutants and drugs previously known to stimulate or inhibit -1 PRF, further supporting the hypothesis that they promote -1 PRF through the canonical mechanism. All affected the steady-state abundance of a reporter mRNA and the wide range of -1 PRF efficiencies promoted by these elements enabled the determination of an inverse logarithmic relationship between -1 PRF efficiency and mRNA accumulation. Steady-state abundances of the endogenous EST1, EST2, STN1 and CDC13 mRNAs were similarly inversely proportional to changes in -1 PRF efficiency promoted by mutants and drugs, supporting the hypothesis that expression of these genes is post-transcriptionally controlled by -1 PRF under native conditions. Overexpression of EST2 by ablation of -1 PRF signals or inhibition of NMD promoted formation of shorter telomeres and accumulation of large budded cells at the G2/M boundary. A model is presented describing how limitation and maintenance of correct stoichiometries of telomerase components by -1 PRF is used to maintain yeast telomere length.

Global control of DNA replication timing by the budding yeast telomere protein Rif1

Results We showed previously that telomere TG-repeat tract length exerts an epigenetic effect in cis on the activity of nearby subtelomeric replication origins, such that a shortened telomere will replicate earlier [1]. Here we show that deletion of the RIF1 gene, which encodes a telomere-specific Rap1-interacting protein involved in telomere length regulation and telomere “capping” [2-5], also leads to premature replication of two different subtelomeric regions examined. A similar effect of RIF1 deletion on other subtelomeric regions has recently been described [6]. We show here that the effect of RIF1 deletion is epistatic to loss of Tel1 or Mec1 (ATM and ATR kinases), does not affect the intra-S phase checkpoint, and operates through a different pathway than the silencing protein Sir3. Deletion of a normally dormant telomere-proximal replication origin exerts a similar effect on replication timing as does deletion of RIF1, and these two effects are additive. Strikingly, deletion of RIF1 partially suppresses temperature-sensitive mutations in a number of essential genes that encode regulators of DNA replication initiation, without affecting the levels of the relevant gene products, several of which are present in limiting amounts.

Telomeres are guardians of chromosomal integrity

Telomeres are DNA-protein complexes located at the ends of linear chromosomes in most eukaryotic cells. Telomeres are vital components of chromosome integrity and cell proliferation. They protect internally located genes from degradation due to incomplete DNA replication, and they also mask natural DNA ends from being recognized as DNA breaks. While being guardians of genome integrity, telomeres can also be vulnerable to damage and inappropriate repair. Dysfunction in telomere maintenance can lead to serious illnesses in humans, including cancer. Cancers achieve immortalization through telomere elongation either through the activation of specialized enzyme telomerase or Alternative Lengthening of Telomeres (ALT) pathway. This review highlights the structure and the function of telomeres in yeast and humans, discusses telomere binding proteins and their roles in telomere maintenance, and briefly describes the implications of telomere dysfunction on human health.

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.

A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice

DNA double-strand break (DSB) repair pathway choice is governed by the opposing activities of 53BP1 and BRCA1. 53BP1 stimulates nonhomologous end joining (NHEJ), whereas BRCA1 promotes end resection and homologous recombination (HR). Here we show that 53BP1 is an inhibitor of BRCA1 accumulation at DSB sites, specifically in the G1 phase of the cell cycle. ATM-dependent phosphorylation of 53BP1 physically recruits RIF1 to DSB sites, and we identify RIF1 as the critical effector of 53BP1 during DSB repair. Remarkably, RIF1 accumulation at DSB sites is strongly antagonized by BRCA1 and its interacting partner CtIP. Lastly, we show that depletion of RIF1 is able to restore end resection and RAD51 loading in BRCA1-depleted cells. This work therefore identifies a cell cycle-regulated circuit, underpinned by RIF1 and BRCA1, that governs DSB repair pathway choice to ensure that NHEJ dominates in G1 and HR is favored from S phase onward.

OB Fold Contributes to Telomere Maintenance

The essential Cdc13 protein is part of the trimeric CST complex that confers genome stability by binding to and protecting yeast telomeres. In this issue of Structure, Mason and colleagues characterize an OB fold domain of Cdc13 (named OB2) and propose that homo-dimerization of OB2 is required for proper assembly of the CST complex and telomere maintenance.

The passionate life of Simon Chan

This issue features a collaborative paper [1] between the Ian Korf and the Simon Chan laboratories describing the evolution of centromeric repeats in animal and plant genomes. Simon Chan passed away on August 22 2012 at the age of 38. Although the ongoing work of his coworkers will result in multiple additional papers carrying his name, this is the last paper he actively worked on. Simon was born in Auckland, New Zealand. He graduated with a degree in biochemistry from the University of Auckland and earned a Ph.D. from UCSF, where he studied yeast telomeres under the guidance of Nobel laureate Elizabeth Blackburn. He did his postdoctoral research in the laboratory of Steve Jacobsen at UCLA working on RNA-directed DNA methylation and gene silencing. In 2006, Simon joined the faculty in the Department of Plant Biology at UC Davis. At that time, Simon took his passion for chromosomal biology and epigenetics into a new area: centromeric function and inheritance. At UC Davis, Simon and his postdoctoral associate, Ravi Maruthlalacham, labored for three years studying the function of centromeric histone H3 (CENH3). Their approach was to complement a nullimorphic cenh3 mutant of Arabidopsis with genes encoding versions of CENH3 with varying degrees of alteration. They found that if a plant with an altered CENH3 was crossed to the wild type, many F1s were sterile. Chromosome mounts from these individuals revealed five chromosomes instead of ten, the expected diploid number. The hypothesis explaining these results had far-reaching implications: centromeres organized by a modified CENH3 were stable in crosses between similarly modified plants. However, when confronted in the zygote with centromeres determined by wild-type CENH3 the centromeres with the altered CENH3 lost out, causing the loss of the connected chromosomes. The resulting haploids could be easily converted to di-haploids (doubled haploids), enabling the establishment of pure inbreds in a single generation. This discovery, published in Nature in 2010 [2], has basic and applied importance. For example, in a 2011 Science article [3], the Chan lab collaborated with Raphael Mercier and Imran Siddiqi to demonstrate the production of clonal plants through seeds in a process mimicking apomixis. This was done by crossing the haploid inducer line with a plant that bypassed meiotic recombination and reduction. In 2012, articles in PNAS [4] and Nature Genetics [5] illustrated, respectively, the use of CENH3-mediated genome elimination in the production of recombinant inbred lines and in molecular breeding. This research showed that it was possible to produce plants with a designated chromosomal composition using CENH3-mediated breeding. Simon's scientific contribution was recognized in 2011 with the prestigious Howard Hughes Medical Institute Award- Gordon and Betty Moore Foundation Investigatorship. Simon would have been well loved and respected even if his scientific career had not been as successful. His passionate, kind and friendly demeanor made him a popular teacher and colleague. He was also refreshingly informal. One of the pictures we treasure features Simon wearing a T-shirt and jeans, surrounded by others in business attire, explaining haploid induction to Bill Gates [6]. He was multi-talented: he played several instruments at nearly professional level, was broadly read, assembled his bicyles from used parts, cracked any puzzle, and had an encyclopic knowledge of both the trivial and the relevant. His friends remember with fondness his leadership in far-ranging culinary explorations, which he carefully recorded and annotated. His scientific promise and greater-than-life personality left a big vacuum in the lives of those who interacted with him, and beyond. We miss his scientific advice, his infectious smile, his stylish haircut, his colorful sneakers, his Hawaiian shirts, his tales and insights, his nodding off during seminars and his penetrating questions at the end. With no interest in material gains and personal wealth, he was passionate and intense about science, was dedicated to environmental sustainability and to making his inventions available to the developing world. He lived life to its fullest and made no compromise. Shortly before his death, he travelled to Africa to fulfill what he saw as his collaborative duties to local plant breeders and laboratories, which are aiming to apply Simon's findings to improve African staple crops. Simon had assembled an excellent team of junior scientists whose ongoing work is providing exciting insights into the fundamental questions raised by CENH3-mediated genomic elimination. In his last moments of consciousness during terminal hospitalization he would eagerly ask for updates. We hope that wherever he is, he is able to follow developments of his scientific discoveries. We, his colleagues and friends, are thankful that we could be associated with him even if the time was short.

Identification of two telomere-proximal fission yeast DNA replication origins constrained by nearby cis-acting sequences to replicate in late S phase

Telomeres of the fission yeast,  Schizosaccharomyces pombe , are known to replicate in late S phase, but the reasons for this late replication are not fully understood. We have identified two closely-spaced DNA replication origins, 5.5 to 8 kb upstream from the telomere itself. These are the most telomere-proximal of all the replication origins in the fission yeast genome. When located by themselves in circular plasmids, these origins fired in early S phase, but if flanking sequences closer to the telomere were included in the circular plasmid, then replication was restrained to late S phase – except in cells lacking the replication-checkpoint kinase, Cds1. We conclude that checkpoint-dependent late replication of telomere-associated sequences is dependent on nearby cis-acting sequences, not on proximity to the physical end of a linear chromosome.

Identification of two telomere-proximal fission yeast DNA replication origins constrained by nearby cis-acting sequences to replicate in late S phase

Telomeres of the fission yeast, Schizosaccharomyces pombe, are known to replicate in late S phase, but the reasons for this late replication are not fully understood. We have identified two closely-spaced DNA replication origins, 5.5 to 8 kb upstream from the telomere itself. These are the most telomere-proximal of all the replication origins in the fission yeast genome. When located by themselves in circular plasmids, these origins fired in early S phase, but if flanking sequences closer to the telomere were included in the circular plasmid, then replication was restrained to late S phase – except in cells lacking the replication-checkpoint kinase, Cds1. We conclude that checkpoint-dependent late replication of telomere-associated sequences is dependent on nearby cis-acting sequences, not on proximity to the physical end of a linear chromosome.

Human CST Has Independent Functions during Telomere Duplex Replication and C-Strand Fill-In

Human CST (CTC1-STN1-TEN1) is an RPA-like complex that is needed for efficient replication through the telomere duplex and genome-wide replication restart after fork stalling. Here, we show that STN1/CST has a second function in telomere replication during G-overhang maturation. Analysis of overhang structure after STN1 depletion revealed normal kinetics for telomerase-mediated extension in S phase but a delay in subsequent overhang shortening. This delay resulted from a defect in C-strand fill-in. Short telomeres exhibited the fill-in defect but normal telomere duplex replication, indicating that STN1/CST functions independently in these processes. Our work also indicates that the requirement for STN1/CST in telomere duplex replication correlates with increasing telomere length and replication stress. Our results provide direct evidence that STN1/CST participates in C-strand fill-in. They also demonstrate that STN1/CST participates in two mechanistically separate steps during telomere replication and identify CST as a replication factor that solves diverse replication-associated problems.

“Poisoning” yeast telomeres distinguishes between redundant telomere capping pathways

In most eukaryotes, telomeres are composed of tandem arrays of species-specific DNA repeats ending with a G-rich 3' overhang. In budding yeast, Cdc13 binds this overhang and recruits Ten1-Stn1 and the telomerase protein Est1 to protect (cap) and elongate the telomeres, respectively. To dissect and study the various pathways employed to cap and maintain the telomere end, we engineered telomerase to incorporate Tetrahymena telomeric repeats (G₄T₂) onto the telomeres of the budding yeast Kluyveromyces lactis. These heterologous repeats caused telomere-telomere fusions, cell cycle arrest at G2/M, and severely reduced viability--the hallmarks of telomere uncapping. Fusing Cdc13 or Est1 to universal minicircle sequence binding protein (UMSBP), a small protein that binds the single-stranded G₄T₂ repeats, rescued the cell viability and restored telomere capping, but not telomerase-mediated telomere maintenance. Surprisingly, Cdc13-UMSBP-mediated telomere capping was dependent on the homologous recombination factor Rad52, while Est1-UMSBP was not. Thus, our results distinguish between two, redundant, telomere capping pathways.