Open Access is an initiative that aims to make scientific research freely available to all. To date our community has made over 100 million downloads. It’s based on principles of collaboration, unobstructed discovery, and, most importantly, scientific progression. As PhD students, we found it difficult to access the research we needed, so we decided to create a new Open Access publisher that levels the playing field for scientists across the world. How? By making research easy to access, and puts the academic needs of the researchers before the business interests of publishers.
We are a community of more than 103,000 authors and editors from 3,291 institutions spanning 160 countries, including Nobel Prize winners and some of the world’s most-cited researchers. Publishing on IntechOpen allows authors to earn citations and find new collaborators, meaning more people see your work not only from your own field of study, but from other related fields too.
To purchase hard copies of this book, please contact the representative in India:
CBS Publishers & Distributors Pvt. Ltd.
www.cbspd.com
|
customercare@cbspd.com
Telomeres are specialized structures essential for genomic stability in eukaryotic cells. Inducible systems causing telomere shortening or telomere formation from short tracts of telomere repeats were developed in the late 1990s in Saccharomyces cerevisiae and have been adapted to investigate multiple aspects of telomere biology. In the formation system, an internal tract of telomere repeats is placed next to an inducible double-strand break. Inducing the break converts the telomere tract into a functional telomere whose fate can be followed kinetically and allows one to assay elongation, protein recruitment, and the DNA damage checkpoint activation. This work was extended to Schizosaccharomyces pombe, as it shares some features of telomeric chromatin with mammalian cells that are missing in S. cerevisiae. The S. pombe system has revealed novel aspects of telomeric chromatin formation and similarities with S. cerevisiae. This chapter will review these past discoveries in different yeast model organisms, and what they reveal about telomere physiology that may well be conserved in mammals.
Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic Foundation, USA
Kurt W. Runge
Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic Foundation, USA
Department of Genetics and Genome Sciences, Case Western Reserve University, USA
*Address all correspondence to: audryj@ccf.org
1. Introduction
1.1 Context
Telomeres are the physical ends of linear eukaryotic chromosomes. The chromosomal end structure of most telomeres is composed of G-rich DNA repeats bound by specific proteins (Table 1 and Figure 1). One of the major functions of this nucleoprotein complex is to prevent chromosome fusions and the recognition of the ends as a double-strand break (DSB) [1, 2]. Due to the semi-conservative replication and nuclease activities occurring at the chromosome ends, the telomeres shorten at each cell division, a process called the “end replication problem” [3, 4]. To counteract this gradual shortening, telomerase can elongate short telomeres at each division [5, 6]. Telomerase is expressed constitutively in unicellular eukaryotic organisms such as yeasts, but not in most of the somatic cells of humans [7]. The gradual erosion of telomere repeats leads to loss of telomere binding proteins and chromosome end deprotection, which activates the DNA damage response (DDR). The DDR can cause replicative senescence or apoptosis, acting as an anti-proliferative barrier [8, 9]. The dysfunction of this barrier can generate genomic instability, chromosomal aberrations and initiate tumorigenesis or oncogenic transformation of the cells [10].
S. cerevisiae
S. pombe
H. sapiens
Regulates telomerase
Major functions at telomeres
Refs
Rap1
Rap1
RAP1
−
Inhibits NHEJ, plays a role in telomere heterochromatin. Only in Sc: binds telomeric dsDNA
Major telomere proteins and functions of S. cerevisiae, S. pombe, and H. sapiens—Sc: S. cerevisiae, Sp: S. pombe, Hs: H. sapiens, NHEJ: non-homologous-end-joining.
dsDNA: double-stranded DNA. -np-: not present. ssDNA: single-stranded DNA.
“+” means positively regulates telomerase. “−” means negatively regulates telomerase.
Figure 1.
Telomere complexes conservation from yeasts to humans (A) S. cerevisiae telomere complex. (B) S. pombe telomere complex. (C) Humans telomere complex (also named “shelterin”).
Telomere length is defined as the number of short G-rich repeats at chromosome ends. Organisms have a required minimum number of repeats, or telomere repeat tracts, to recruit enough telomere binding proteins to accomplish telomere function [11, 12, 13, 14, 15]. Some proteins within the telomere protein complex recruit telomerase to allow elongation of the telomeres (positively regulating telomerase), while others prevent telomerase from continuously adding repeats (negatively regulating telomerase) (Tables 1 and 2).
Major telomerase components of S. cerevisiae, S. pombe, and H. sapiens.
Telomere length is linked to aging, as it was shown that late generation mice with limited telomerase (heterozygotes for telomerase RNA mTR+/−) have short telomeres and revealed a decrease of tissue renewal capacity [16]. In contrast, mice overexpressing telomerase have an extended lifespan [17]. In humans, a correlation between telomere length, aging, and age-related diseases has also been shown (reviewed in [18, 19]). For instance, in dyskeratosis congenita or its most severe form: Hoyeraal-Hreidarsson syndrome, patients have abnormally short telomeres and shorter lifespan [20]. Most of the known mutations associated are found in telomere proteins (TIN2), telomerase (hTR and hTERT) or the telomerase-associated factor as dyskerin (DKC1), which stabilizes the telomerase RNA (hTR) (Table 2) [18]. Moreover, shortened telomere length was observed in other premature aging disorders such as Down’s syndrome leading to accelerated aging, or Cockayne syndrome in which neurological degeneration is observed (aging-disorders linked to a telomere phenotype reviewed in [21]). In Down’s syndrome, the genetic defect observed is mostly a trisomy of chromosome 21, which increased the biological age of tissues, whereas in Cockayne syndrome, mutations are described in the majority in Cockayne syndrome group B protein (CSB) involved in telomere length maintenance with TRF1 and TRF2 [21]. Prematurely shortened telomeres can also lead to loss of telomere capping function, which in turn can cause telomeres to behave as DNA breaks and undergo recombination to promote genomic instability leading to cell death or tumorigenesis [22]. Telomerase defects have also been associated with cancer, and telomerase promoter mutations are prevalent in multiple cancer types [23], where the presence of telomerase activity allows extended growth of cancer cells [24].
Telomeres are also able to convert adjacent chromatin into heterochromatin, a compact and transcriptionally silent form of chromatin with important roles in chromosome biology, for example, in development and mammalian female X-chromosome inactivation and development of cancers [25, 26, 27]. This function, the inhibition of the DDR, and how the cells distinguish a DSB from a telomere or heterochromatin regulation at the chromosome ends are still not fully understood. Elucidation of these mechanisms will provide an understanding of some of the molecular mechanisms associated with aging and the initiation of tumorigenesis.
1.2 Telomere complexes from yeasts to humans
The yeast model systems have the advantage of a small eukaryotic genome that can be easily altered due to high levels of homologous recombination [28, 29, 30, 31]. The terminal telomere repeat tracts of Saccharomyces cerevisiae and Schizosaccharomyces pombe follow the scale of these smaller genomes with lengths of ~0.3 kb [12, 32]. In contrast, telomeres size in humans is between 10 and 30 kb, but with measurement limitations due to a highly variable length either at the time of birth [33, 34], in different tissues [35] or among others between women and men [36]. Therefore, yeasts are significant model organisms for dissecting the molecular genetics of basic telomere biology (reviewed in [37, 38]).
Telomeres have also been studied in several budding yeasts somewhat related to S. cerevisiae [39, 40]. A striking difference between S. cerevisiae and these organisms is that while S. cerevisiae has a short heterogeneous telomere repeat sequence (abbreviated as TG1–3), several other species of Saccharomyces, Candida, and Kluyveromyces lactis have longer, homogeneous repeats [39, 40]. These longer repeats allowed the cloning of the gene for telomerase RNA in K. lactis and Saccharomyces castellii (now referred to as Naumovozyma castellii) and the study of highly related telomere binding proteins that co-evolved with the different telomere sequences [39, 41, 42, 43, 44, 45]. Work in N. castellii showed that the Rap1 protein of this yeast can bind to both the double-stranded telomere repeats and the junction between the double and single strands DNA, suggesting a level of coordination between Rap1 and the single-strand telomere repeat binding protein Cdc13 [46, 47]. N. castellii may also provide insight into the regulation of telomerase synthesis of the 3′ overhang as this yeast synthesizes a specific 70 nucleotides 3′ overhang in late S-phase [48].
Mutation of the telomerase RNA in K. lactis allowed the synthesis of mutant telomere repeats which revealed surprising aspects of telomerase-dependent and telomerase-independent telomere elongation. Some mutant telomere repeats gave rise to hyper-extended telomeres in a phenomenon called “runaway telomere elongation,” which was thought to be related to the loss of telomere binding proteins that negatively regulate telomerase [42, 49]. Similar hyper-elongation phenotypes are known in the more “popular” yeasts, S. cerevisiae and S. pombe, caused by loss of Rif1 and Rif2 from budding yeast or Taz1 or Rap1 from S. pombe (Table 1). K. lactis mutant telomere repeats have also been used to follow a telomerase-independent mode of recombinational telomere elongation (RTE). In this model, shortened telomeres become highly recombinogenic, and the formation of double-stranded circular DNAs made of telomere repeats (t-circles) allows a “rolling circle” replication mechanism to produce telomere repeats that can be incorporated into short chromosomal telomeres [45, 50] This model is supported by further work indicating that a single t-circle may be a sufficient source for the elongation of all chromosomal telomeres [51, 52]. Results from these studies indicate that K. lactis telomeres may become very short prior to RTE and provide a model for human cancers that replicate telomeres by a recombinational mechanism termed ALT for alternative lengthening of telomeres [53]. Therefore, these budding yeasts have provided insights into the mechanisms of telomerase-dependent and -independent telomere elongation. However, both of them lack a rapidly inducible telomere formation system, which has the potential to follow some of the unique processes observed in these yeasts in real time.
The fission yeast S. pombe is significantly different from S. cerevisiae, K. lactis, and N. castellii, having diverged from the budding yeasts early in fungal evolution [54]. A consequence of this divergence is that S. pombe has conserved many features of telomere protein and chromatin structure that were lost from the budding yeasts. S. pombe has the same genome size as S. cerevisiae but only three chromosomes compared to 16 for the same size genome, and early studies confirmed that S. pombe was amenable to the same molecular genetic manipulations as S. cerevisiae [28] including telomere formation [32, 55], but had differences in telomere repeat sequence and the structure of the repeated subtelomeric elements adjacent to chromosome ends [32]. The human telomere complex, also called “shelterin,” is more similar to the telomere complex of S. pombe than to S. cerevisiae (Tables 1 and 2; Figure 1) (reviewed in [56, 57]). Rap1 does not bind directly to the double-strand telomeric DNA in humans or S. pombe, but is recruited to the telomeres by its interaction with double-stranded DNA binding proteins, that is, Taz1 in S. pombe and TRF1 and TRF2 in humans. In S. pombe, Taz1 and Rap1 inhibit NHEJ, playing a role in the silencing of subtelomere regions and negatively regulating telomerase [58, 59, 60, 61]. At the single strand, Pot1 protects the 3′ overhang, as in humans, and is bound by Tpz1 [62, 63]. Tpz1, the ortholog of human TPP1, plays central roles in telomerase recruitment (via its interaction with Ccq1 [64, 65, 66, 67]), telomerase activation (via its interaction with the catalytic subunit of the telomerase [68]) and the termination of telomere elongation (via its SUMOylation [69]). Poz1, the ortholog of human TIN2, links the double-strand DNA binding complex of Taz1-Rap1 and Rif1 to the single-strand DNA binding complex of Pot1-Tpz1-Ccq1 (Figure 1). The entire complex negatively regulates telomerase as loss of Poz1 or Rap1 results in elongation of the terminal telomere repeat sequence tracts [70, 71]. In addition to telomere proteins, S. pombe has also conserved heterochromatin-mediated transcriptional silencing via di- and tri-methylation of lysine 9 of histone H3 (H3K9me2 and H3K9me3), which is absent in S. cerevisiae [72, 73].
Inducible systems that form telomeres from short tracts of telomere repeats were first developed in the late 1990s in S. cerevisiae [98]. The system modified inducible DSB systems, which consists of the rapid expression of a restriction enzyme or endonuclease allowing a cut at a specific engineered locus (Figure 2A and B). These systems have variants in many organisms (reviewed in [99]) with relevance for fundamental understanding of DSB repair mechanisms [100] and therapeutic response to DSBs, as in radiotherapies [101]. In yeast, two site-specific endonucleases, HO and I-SceI were used, with a similar kinetics of cleavage [102, 103]. However, I-SceI was preferred in early mammalian work [104].
Figure 2.
Double-strand break (DSB) and telomere formation systems (A) restriction enzyme or endonuclease with no natural sites in the genome is produced in cells from a rapidly inducible promoter. (B) Inducible DSB system: a unique cut site (red triangle) is engineered next to a selectable marker gene. After induction, the endonuclease produces a DSB which leads to the degradation of both 5′ and 3′ strands on each side of the cut site (indicated by short black lines) and to the marker degradation, conducting to cell growth inhibition. (C) Inducible telomere formation system: the cut here exposes telomere repeats (black triangles) to form a new functional telomere that is stable and elongated. If the distal chromosomal DNA (3′ to the cut site) is dispensable, the new functional telomere allows normal cell growth. Modified and adapted from Wang et al. [107].
The telomere formation system integrates telomeric DNA repeats next to an engineered cut site, and the newly exposed telomere tract is converted into a functional, stable telomere without causing growth inhibition (Figure 2C) [98, 105, 106, 107]. In contrast, an induced DSB in the middle of a chromosome leads to DNA degradation and growth inhibition [108]. A variant of this system involving an inducible recombinase and specific recognition sites was also made to target one specific telomere in budding yeast and artificially shorten it [89, 109, 110, 111].
The S. cerevisiae telomere formation system proved to be a highly useful tool, but was limited by the biology of this yeast species. By translating this system to S. pombe, some of these issues, such as the presence of H3K9me2 and 3 modifications and a high level of conservation of the telomeric complexes with humans were addressed (Figure 1B and C) [56, 70, 72, 73]. The recent development of the S. pombe telomere formation system thus opens new avenues to study telomeric chromatin regulation and telomere formation.
Telomere formation systems allow a real-time study of telomere formation, elongation or heterochromatin spreading from the newly induced end. It also gives us the opportunities to examine the effects of different mutations in telomere proteins or to test the protein requirements for how telomeres are distinguished from DSBs. This non-exhaustive list highlights the significant advantages of these systems compared to steady-state experiments, as studies introducing mutations into cells with existing telomeres can only monitor telomeres after they have reached their equilibrium state. Formation systems can also study the initiation of mechanisms associated with telomeres, such as heterochromatinization of the nearby sequences or DNA damage checkpoint inhibition. Technically, these systems allow multiple experiments such as ChIP for protein recruitment at the break, Southern blotting to follow the de novo telomere elongation or DNA degradation, cell morphology observation (as large budded cells in S. cerevisiae or elongated cells in S. pombe—characteristic of a G2/M arrest), or western blotting for Chk1 phosphorylation (checkpoint activation).
2.1 Inducible telomere systems in S. cerevisiae
The first inducible telomere formation system in S. cerevisiae used the HO endonuclease placed under the control of a galactose-inducible promoter (in a MATa-inc allele strain which contains a point mutation to avoid an unwanted HO cut at the MAT locus [112]) and to the HO recognition sequence placed next to an 80 bp tract of telomere repeats (Figure 3A) [98].
Figure 3.
Inducible telomere formation system in S. cerevisiae (A) schematic representation of the induction of the HO endonuclease (under the control of GAL1 promoter) in presence of galactose and modified chromosome VII-L containing the telomere cassette. The telomere cassette containing the ADE2 marker, the HO recognition site, and the LYS2 marker was inserted at the ADH4 locus at 20 kb from the telomere end (this 20 kb contains no essential genes) [98]. This insertion placed the HO recognition site at 13 kb from the distal telomere end. CEN: centromere. TEL: telomere (terminal black triangles). TG1–3: telomere repeats sequence. (B) Variants of telomeres cassettes. The telomere cassette at the chromosome V-R containing the TRP1 marker was inserted at the YER188W locus (there are no essential genes from this locus to the chromosome end) [77]. Numbers represent the telomere tract length in base pairs (bp). CA1–3: telomere repeats on the other side of the break in the opposite orientation represented in dark red. Modified and adapted from Ribeyre and Shore [77].
The telomere cassette containing the ADE2 marker, the HO recognition site, and the LYS2 marker, was inserted at the ADH4 locus at the left arm of the chromosome VII (VII-L) at 20 kb from the telomere end (this 20 kb contains no essential genes) (Figure 3A). The LYS2 gene here serves as a marker for loss of the non-essential distal DNA and indirectly measure the HO cut efficiency by comparing Lys+ and Lys− cells.
The Diede and Gottsheing studies [98, 113] highlighted the efficacy of the system: a rapid and efficient cutting exposed a new stable telomere end that was elongated by telomerase. They also described a cell cycle-regulated elongation and the requirement of the DNA primase and DNA polymerases α and δ for the de novo telomere elongation [98]. Additionally, they also revealed the involvement of the MRX (Mre11-Rad50-Xrs2) complex and its exonuclease activity at the new telomere end for its elongation and Cdc13 loading [113]. It is worth noting that short formed telomeres in different mutant strains that have a functional chromosome ends do not require MRX for elongation [114], highlighting how a broken end forming a telomere may differ from an existing end that requires elongation. Finally, they showed that the interaction of Ku (YKu70/YKu80) and telomerase RNA (TLC1) promotes telomere addition at the newly formed chromosome end [115].
Michelson et al. showed that even if the telomere formation system has the characteristics of a DSB, the cells respond differently when the DSB is next to a telomeric tract [116]. The de novo telomere end and degrading DNA fragment (Figure 1C) does not induce a normal checkpoint arrest, giving rise to a “telomere anti-checkpoint” activity.
The Shore lab followed up these results by creating new variants of the systems (Figure 3B) with some variations of the telomere tract length, or the addition of inverted telomere tract directly after the HO cut site (distal DNA fragment) [77]. Similarly, a telomere cassette was added at a different locus in the right arm of the chromosome V (V-R) with TRP1 as the only proximal marker (Figure 3B) [77]. These variants were used to show that the telomere anti-checkpoint activity required the telomere proteins: Rif1 and Rif2. These results imply that a telomeric tract on one side of a break influences the recruitment of DNA damage checkpoint proteins on the other side [77].
An alternative inducible telomere elongation system has been constructed where an existing telomere with a functional chromosome end loses internal telomere repeats, so the resulting telomere tract is much shorter than normal [89, 109, 110, 111]. The first one used the site-specific recombinase Flp1 under a galactose-inducible promoter and two FRT (Flp1-Recognition Target) sites inserted at the ADH4 locus on chromosome VII-L (Figure 4A) [109, 110, 117]. The system is based on the Flp1 recombinase excising a tract between two FRT sites and leaving only the distal telomere DNA sequence next to the terminal FRT site. Because the FLP1 gene is encoded on the S. cerevisiae endogenous plasmid called the “2-micron circle,” only cir° strains that lack this plasmid can be used with the FRT system. The system was later improved by using the loxP sites instead of FRT, and the Cre-recombinase, allowing its use in any strains, at the chromosome VII-L [111] or chromosome V-R, with an unchanged or short telomere induction (Figure 4B and C) [89, 118]. The constructs contain 16 inverted Rap1-binding sites, a loxP site, and a terminal telomere tract. Arrays of Rap1 binding sites at this position are considered by the cell to be a part of the terminal telomere tract, so the internal repeat tract distal to the loxP site is shorter than a normal telomere [119, 120, 121]. In these systems, after recombination, the remaining terminal tract is about 90 bp or 1/3 of the normal telomere length (Figure 4B) [89, 109, 110, 111, 117, 118]. These constructs were used to show a cell cycle restriction of telomere elongation in late S phase and a progressive telomerase inhibition upon telomere elongation [109, 110]. Additionally, an early replication of a short telomere was linked to an increase association of telomerase [111, 117] and highlighted two distinct roles of Stn1 in telomere capping and telomerase inhibition [89].
Figure 4.
S. cerevisiae systems to generate a single shortened telomere (A) schematic representation of the inducible recombinase-based system to generate a single shortened telomere at the chromosome VII-L (ADH4 locus). FRT: Flp1-recognition target site. The galactose-mediated induction allows the expression of Flp1 which induced homologous recombination through the FRT sites to generate an excised circle (containing the indicated elements) and a short telomere. Numbers represent the telomere tract length in base pairs (bp). Note that the terminal tract length is variable at individual telomeres due to lengthening and shortening at each cell division, and the numbers presented represent the average of the population [109, 110, 117]. (B) Variant system using loxP site and galactose inducible Cre-recombinase expression at the same locus (ADH4, chromosome VII-L) [111] or at the chromosome V-R end (YER188W locus) [89, 118]. (C) The control chromosome V-R construct that does not excise telomere repeats [89]. Modified and adapted from Marcand et al. and Puglisi et al. [89, 109].
2.2 Inducible telomere systems in S. pombe
More recently, the telomere formation system was extended to S. pombe. The telomeric complex of the fission yeast has a high level of conservation with humans, both structurally with a “bridged organization” of telomere proteins [70] and in how telomerase is regulated (Figure 1B and C) (reviewed in [56]). This yeast also exhibits the H3K9me2 and -3 modifications present in multicellular eukaryotes that is absent in S. cerevisiae opening new perspectives of translational studies in telomeric chromatin regulations [72, 73].
The building of an efficient system took more time in S. pombe, as a rapidly inducible promoter was not available in this system. A system with the HO endonuclease using the nmt1 promoter had been used but required 24 hours for full induction that would complicate kinetic analysis [122]. Two systems that could rapidly induce a site-specific nuclease that did not cut in the S. pombe genome were published in 2011 and 2012, but were inappropriate for a telomere formation system because they either prevented the use of the ura4+ marker, which is important for monitoring telomere-associated heterochromatin [123, 124, 125], or special strains [126]. We, therefore, developed a telomere system that was using the anhydrotetracycline (ahTET)-inducible promoter and an I-SceI enzyme with preferred S. pombe codons [127] coupled to two NLS (Nuclear Localization Signals) to ensure a rapid and efficient cut [107] (Figure 5A). I-SceI has no endogenous site in the fission yeast genome [128], so the only DSB induced is at the engineered I-SceI site in the telomere formation system (called the proto-telomere cassette in S. pombe).
Figure 5.
The S. pombe inducible telomere formation system (A) the I-SceI endonuclease was expressed from a TetR controlled promoter (CaMV35S). In the absence of ahTET (left part), TetR represses the expression of I-SceI, and the addition of ahTET (9uM) into the medium induces expression (right part). (B) The 48 and 0 bp proto-telomeres cassettes are presented here and consist of a ura4+ gene followed by either a 48 bp of telomere tract (black triangles) or no telomere sequences for the 0 bp version, the I-SceI cut site (red triangle) and the hygromycin resistance gene (hph+) at the distal part of the construct. The cassettes were introduced at the chromosome II-R, 3′ to the gal1+ locus. CEN: centromere. TEL: telomere (terminal black triangles). The number represents the telomere tract length in base pairs (bp). Modified and adapted from Wang et al. [107].
Two constructs were then created containing either 48 or 0 bp of telomere repeat sequence, an I-SceI cut site, and two flanking selectable markers: the ura4+ gene and the hygromycin resistance gene (hph+)(Figure 5B) [107]. The cassettes were placed at the right arm of the chromosome II (II-R), 3′ to the gal1+ locus and, as in the S. cerevisiae systems, the region between this locus and the chromosome end was dispensable (without essential genes).
The S. pombe telomere formation system revealed several novel aspects of telomere function [107]. First, the DSB next to the telomere repeat tract immediately acquires telomere function: the end is immediately stable and not degraded (even in a strain lacking telomerase activity), does not undergo recombination, and the DNA damage response is somehow blocked. Second, the heterochromatin domain associated with telomeres formed in two distinct phases. The first consists of heterochromatin spreading from the telomere toward the interior of the chromosome over ~3 kb in the first cell division (about 5 hours). The newly formed telomere is elongated to wild type lengths during this time by telomerase. Heterochromatin spreading over ~10 kb continues for the next eight generations (population doublings) after the terminal telomere repeat tracts are at their equilibrium lengths. It is worth noting that the clr4 deletion, which blocks heterochromatin formation, had no effect on telomere elongation [107], indicating that heterochromatin is independent of telomerase-mediated elongation as it is for chromosome end stability as observed in S. cerevisiae [129, 130]. In a second phase studied 34–87 population doublings after telomere formation, the established heterochromatin domain was surprisingly dynamic, with significant expansions and contraction of the heterochromatin mark H3K9me2 over the 35 kb domain that was monitored. Thus, different lineages from the same telomere formation event had different heterochromatin domains at different times after formation. These lineages were monitored at the single cell level by integrating the ade6+ colony color marker at different distances from the proto-telomere and plating single cells on inducing medium to follow the spreading of heterochromatin to silence the color marker at different stages of colony growth [107]. A marker close to the newly formed telomere initially showed expression before it was extinguished after several population doublings and remained extinguished as cells continued to grow. In contrast, markers placed further away from the telomere switched between the transcribed and repressed state in subsequent population doublings. These relative kinetics of telomere-associated functions would not have been observable using a telomere formed by cellular transformation, which requires ~30 generations of growth between telomere formation of a single cell to the generation of a sufficient number of cells to analyze the heterochromatin domain.
It is worth noting that this work and many other S. pombe heterochromatin studies follow the histone modification H3K9me2, but the H3K9me3 mark is also associated with heterochromatin. Recent work by the Moazed lab showed that the H3K9me2 mark is associated with very low gene activity, and its conversion to H3K9me3 extinguished detectable transcription [131]. This model can explain the level of H3K9me2 near the newly formed telomere, which peaked about 3–13 kb from the new telomere in different experiments [107]. Presumably, H3K9me3 modification replaced H3K9me2 closer to the new telomere where expression of the ura4+ and ade6+ genes was undetectable [107]. Thus, the inducible telomere formation system may also prove useful in studying how the transition between these chromatin marks is regulated.
The inducible telomere formation system first developed in S. cerevisiae has provided several important results in the understanding of telomere elongation, in the cell cycle regulation of telomerase, in DNA checkpoint inhibition induced by a telomere end, and in the role of specific telomere proteins (Table 3) [77, 89, 98, 109, 110, 113, 115, 116, 117, 118]. The new S. pombe system has allowed one to follow the establishment of telomere-end protection functions and the formation and dynamics of heterochromatin (Table 3) [107].
Organism
System description
Used to show
S. cerevisiae
HO endonuclease
Galactose inducible promoter
Specific cut site at chromosome VII-L or V-R to create a new telomere end
New telomere stable and elongation by telomerase [98].
DNA primase and DNA polymerases α and δ are required of the de novo telomere elongation [98].
Involvement of the MRX complex and its exonuclease activity at the new telomere end for its elongation and the Cdc13 loading [113].
Ku complex interacts with TLC1 (telomerase RNA) to promote telomere addition at the newly formed chromosome end [115].
The de novo telomere end and degrading DNA fragment does not induce a checkpoint arrest [116].
S. cerevisiae
FLP1 recombinase
Galactose inducible promoter
FRT sites inserted at chromosome VII-L to artificially shorten a telomere end
Progressive telomerase inhibition upon telomere elongation [109].
Cell cycle restriction of telomere elongation in late S phase [110].
Telomerase is preferentially associated with short telomeres [117].
S. cerevisiae
Cre recombinase
Galactose inducible promoter
LoxP sites inserted at chromosome VII-L or V-R to artificially shorten a telomere end
Early replication of a short telomere linked to an increased association of telomerase [111].
Two distinct roles of Stn1 in telomere capping and telomerase inhibition [89].
S. pombe
I-SceI endonuclease
Tetracycline (TetR) controlled promoter (CaMV35S)
Specific cut site at chromosome II-R to create a new telomere end
New telomere-end stable and elongated by telomerase [107].
Gradual heterochromatin formation which remains dynamic after the new end reaches its equilibrium length [107].
Table 3.
Summary of telomere formation systems in different yeast species.
The ability to monitor the relative kinetics of different telomere-associated processes of replication, end protection, and chromatin domain formation has provided insights that could not be obtained using steady-state experiments with established telomeres. As telomere dysfunctions contribute to genomic instability or chromosome aberrations in human cancers, aging disorders, or specific pathologies such as dyskeratosis congenita [10, 19, 20, 21, 23, 24], telomere formation studies have the potential to identify defects related to these diseases. Heterochromatin also plays an important role in chromosome biology [25, 26, 27], and inducible telomeres and heterochromatin domains may shed light on these metazoan processes.
A key component of these telomere formation systems is the rapidly inducible DSB to expose the new physical chromosome end with adjacent telomere repeats. Inducible DSB systems using I-SceI or CRISPR/Cas9 have been used in mammalian cells to reveal differences in DNA repair at internal sites compared to subtelomeric ones [132, 133, 134, 135, 136, 137]. An issue with the CRISPR/Cas9 approach is that it is a two-component system consisting of a guide RNA to target the endonuclease to a precise site, so how rapidly inducible this system is compared to the single component I-SceI nuclease needs to be carefully tested. Using I-SceI, an inducible telomere system in mouse cells to measure telomere repeat addition over 48 hours was developed [105]. While not used kinetically, the system allowed the assay of the effect of the DNA damage kinase ATM on the addition of new telomere repeats by telomerase. The events discovered in the S. cerevisiae and S. pombe telomere formation systems will provide important models for testing in this and other mammalian telomere formation systems.
This work was supported by grants from the National Science Foundation and the National Institutes of Health (both USA) to KWR. The funding agencies had no influence on the content of this chapter.
1.Muller HJ. The remaking of chromosomes. The Collect Net. 1938;3:181-198
2.McClintock B. The stability of broken ends of chromosomes in Zea Mays. Genetics. 1941;26(2):234-282
3.Watson JD. Origin of concatemeric T7 DNA. Nature New Biology. 1972;239(94):197-201
4.Olovnikov AM. A theory of marginotomy. The incomplete copying of template margin in enzymic synthesis of polynucleotides and biological significance of the phenomenon. Journal of Theoretical Biology. 1973;41(1):181-190
5.Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in tetrahymena extracts. Cell. 1985;43(2):405-413
6.Hemann MT, Strong MA, Hao LY, Greider CW. The shortest telomere, not average telomere length, is critical for cell viability and chromosome stability. Cell. 2001;107(1):67-77
7.Collins K, Mitchell JR. Telomerase in the human organism. Oncogene. 2002;21(4):564-579
8.Cosme-Blanco W, Shen M-F, Lazar AJF, Pathak S, Lozano G, Multani AS, et al. Telomere dysfunction suppresses spontaneous tumorigenesis in vivo by initiating p53-dependent cellular senescence. EMBO Reports. 2007;8(5):497-503
9.Feldser DM, Greider CW. Short telomeres limit tumor progression in vivo by inducing senescence. Cancer Cell. 2007;11(5):461-469
10.Cesare AJ, Karlseder J. A three-state model of telomere control over human proliferative boundaries. Current Opinion in Cell Biology. 2012;24(6):731-738
12.Wang SS, Zakian VA. Sequencing of Saccharomyces telomeres cloned using T4 DNA polymerase reveals two domains. Molecular and Cellular Biology. 1990;10(8):4415-4419
13.de Lange T, Shiue L, Myers RM, Cox DR, Naylor SL, Killery AM, et al. Structure and variability of human chromosome ends. Molecular and Cellular Biology. 1990;10(2):518-527
14.Sfeir A, de Lange T. Removal of shelterin reveals the telomere end-protection problem. Science. 2012;336(6081):593-597
15.de Lange T. Protection of mammalian telomeres. Oncogene. 2002;21(4):532-540
16.Hao L-Y, Armanios M, Strong MA, Karim B, Feldser DM, Huso D, et al. Short telomeres, even in the presence of telomerase, limit tissue renewal capacity. Cell. 2005;123(6):1121-1131
17.Tomás-Loba A, Flores I, Fernández-Marcos PJ, Cayuela ML, Maraver A, Tejera A, et al. Telomerase reverse transcriptase delays aging in cancer-resistant mice. Cell. 2008;135(4):609-622
18.Fasching CL. Telomere length measurement as a clinical biomarker of aging and disease. Critical Reviews in Clinical Laboratory Sciences. 2018;55(7):443-465
20.Kirwan M, Dokal I. Dyskeratosis congenita, stem cells and telomeres. Biochimica et Biophysica Acta. 2009;1792(4):371-379
21.Turner KJ, Vasu V, Griffin DK. Telomere biology and human phenotype. Cell. 2019;8(1):pii:E73
22.Blasco MA, Lee HW, Hande MP, Samper E, Lansdorp PM, DePinho RA, et al. Telomere shortening and tumor formation by mouse cells lacking telomerase RNA. Cell. 1997;91(1):25-34
23.Heidenreich B, Rachakonda PS, Hemminki K, Kumar R. TERT promoter mutations in cancer development. Current Opinion in Genetics & Development. 2014;24:30-37
24.Shay JW, Wright WE. Role of telomeres and telomerase in cancer. Seminars in Cancer Biology. 2011;21(6):349-353
25.Chaligné R, Heard E. X-chromosome inactivation in development and cancer. FEBS Letters. 2014;588(15):2514-2522
26.Lunyak VV, Rosenfeld MG. Epigenetic regulation of stem cell fate. Human Molecular Genetics. 2008;17(R1):R28-R36
27.Sharma S, Kelly TK, Jones PA. Epigenetics in cancer. Carcinogenesis. 2010;31(1):27-36
28.Simanis V. The molecular biology of Schizosaccharomyces pombe: Genetics, genomics and beyond. Journal of Cell Science. 2004;117(17):3712-3712
29.Hoekstra MF. In: Jones EW, Pringle JR, Broach JR, editors. The Molecular and Cellular Biology of the Yeast Saccharomyces. Vol. 1: Genome Dynamics, Protein Synthesis, and Energetics, Trends in Genetics. Cold Spring Harbor USA, New York: Laboratory Press (Monograph 21A); 1992
30.Newman A. In: Jones EW, Pringle JR, Broach JR, editors. The Molecular and Cellular Biology of the Yeast Saccharomyces. Vol. 2: Gene Expression, Trends in Cell Biology. Cold Spring Harbor USA, New York: Laboratory Press (Monograph 21B); 1994
31.Fantes PA. In: Jones EW, Pringle JR, Broach JR, editors. The Molecular and Cellular Biology of the Yeast Saccharomyces. Vol. 3: Cell Cycle and Cell Biology, Trends in Genetics. Cold Spring Harbor USA, New York: Laboratory Press (Monograph 21C); 1998
32.Sugawara NF, thesis PD. DNA Sequences at the Telomeres of the Fission Yeast S. pombe. Cambridge, Massachusetts, USA: Harvard University; 1988
33.Okuda K, Bardeguez A, Gardner JP, Rodriguez P, Ganesh V, Kimura M, et al. Telomere length in the newborn. Pediatric Research. 2002;52(3):377-381
34.Vasu V, Turner KJ, George S, Greenall J, Slijepcevic P, Griffin DK. Preterm infants have significantly longer telomeres than their term born counterparts. PLoS One. 2017;12(6):e0180082
35.Friedrich U, Griese E, Schwab M, Fritz P, Thon K, Klotz U. Telomere length in different tissues of elderly patients. Mechanisms of Ageing and Development. 2000;119(3):89-99
36.Nordfjäll K, Eliasson M, Stegmayr B, Melander O, Nilsson P, Roos G. Telomere length is associated with obesity parameters but with a gender difference. Obesity. 2008;16(12):2682-2689
37.Bianchi A, Shore D. How telomerase reaches its end: Mechanism of telomerase regulation by the telomeric complex. Molecular Cell. 2008;31(2):153-165
38.Churikov D, Corda Y, Luciano P, Géli V. Cdc13 at a crossroads of telomerase action. Frontiers in Oncology. 2013;3:39
39.McEachern MJ, Blackburn EH. A conserved sequence motif within the exceptionally diverse telomeric sequences of budding yeasts. Proceedings of the National Academy of Sciences of the United States of America. 1994;91(8):3453-3457
40.Cohn M, McEachern MJ, Blackburn EH. Telomeric sequence diversity within the genus Saccharomyces. Current Genetics. 1998;33(2):83-91
41.Cohn M, Blackburn EH. Telomerase in yeast. Science. 1995;269(5222):396-400
42.Krauskopf A, Blackburn EH. Control of telomere growth by interactions of RAP1 with the most distal telomeric repeats. Nature. 1996;383(6598):354-357
43.Gustafsson C, Rhodin Edsö J, Cohn M. Rap1 binds single-stranded DNA at telomeric double- and single-stranded junctions and competes with Cdc13 protein. The Journal of Biological Chemistry. 2011;286(52):45174-45185
44.Rhodin Edsö J, Tati R, Cohn M. Highly sequence-specific binding is retained within the DNA-binding domain of the Saccharomyces castellii Cdc13 telomere-binding protein. FEMS Yeast Research. 2008;8(8):1289-1302
45.Basenko EY, Cesare AJ, Iyer S, Griffith JD, McEachern MJ. Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination. Nucleic Acids Research. 2010;38(1):182-189
46.Runnberg R, Narayanan S, Cohn M. Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends. Scientific Reports. 2017;7(1):8729
47.Rhodin Edsö J, Gustafsson C, Cohn M. Single- and double-stranded DNA binding proteins act in concert to conserve a telomeric DNA core sequence. Genome Integrity. 2011;2(1):2
48.Fridholm H, Astromskas E, Cohn M. Telomerase-dependent generation of 70-nt-long telomeric single-stranded 3′ overhangs in yeast. Nucleic Acids Research. 2013;41(1):242-252
50.McEachern MJ, Iyer S. Short telomeres in yeast are highly recombinogenic. Molecular Cell. 2001;7(4):695-704
51.Natarajan S, Groff-Vindman C, McEachern MJ. Factors influencing the recombinational expansion and spread of telomeric tandem arrays in Kluyveromyces lactis. Eukaryotic Cell. 2003;2(5):1115-1127
52.Natarajan S, McEachern MJ. Recombinational telomere elongation promoted by DNA circles. Molecular and Cellular Biology. 2002;22(13):4512-4521
53.Xu J, McEachern MJ. Long telomeres produced by telomerase-resistant recombination are established from a single source and are subject to extreme sequence scrambling. PLoS Genetics. 2012;8(11):e1003017
54.Hayles J, Nurse P. Introduction to fission yeast as a model system. Cold Spring Harbor Protocols. 2018;2018(5):pdb.top079749
55.Guerrini AM, Ascenzioni F, Tribioli C, Donini P. Transformation of Saccharomyces cerevisiae and Schizosaccharomyces pombe with linear plasmids containing 2 micron sequences. The EMBO Journal. 1985;4(6):1569-1573
56.Nandakumar J, Cech TR. Finding the end: Recruitment of telomerase to telomeres. Nature Reviews Molecular Cell Biology. 2013;14(2):69-82
57.de Lange T. Shelterin-mediated telomere protection. Annual Review of Genetics. 2018;52:223-247
58.Ferreira MG, Cooper JP. The fission yeast Taz1 protein protects chromosomes from Ku-dependent end-to-end fusions. Molecular Cell. 2001;7(1):55-63
59.Miller KM, Rog O, Cooper JP. Semi-conservative DNA replication through telomeres requires Taz1. Nature. 2006;440(7085):824-828
60.Kanoh J, Ishikawa F. spRap1 and spRif1, recruited to telomeres by Taz1, are essential for telomere function in fission yeast. Current Biology. 2001;11(20):1624-1630
61.Miller KM, Ferreira MG, Cooper JP. Taz1, Rap1 and Rif1 act both interdependently and independently to maintain telomeres. The EMBO Journal. 2005;24(17):3128-3135
62.Baumann P. Pot1, the putative telomere end-binding protein in fission yeast and humans. Science. 2001;292(5519):1171-1175
63.Denchi EL, de Lange T. Protection of telomeres through independent control of ATM and ATR by TRF2 and POT1. Nature. 2007;448(7157):1068-1071
64.Moser BA, Chang Y-T, Kosti J, Nakamura TM. Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast. Nature Structural & Molecular Biology. 2011;18(12):1408-1413
65.Chang Y-T, Moser BA, Nakamura TM. Fission yeast shelterin regulates DNA polymerases and Rad3ATR kinase to limit telomere extension. PLoS Genetics. 2013;9(11):e1003936
67.Webb CJ, Zakian VA. Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase-telomere association. Genes & Development. 2012;26(1):82-91
68.Armstrong CA, Pearson SR, Amelina H, Moiseeva V, Tomita K. Telomerase activation after recruitment in fission yeast. Current Biology. 2014;24(17):2006-2011
69.Garg M, Gurung RL, Mansoubi S, Ahmed JO, Dave A, Watts FZ, et al. Tpz1TPP1 SUMOylation reveals evolutionary conservation of SUMO-dependent Stn1 telomere association. EMBO Reports. 2014;15(8):871-877
70.Kim J-K, Liu J, Hu X, Yu C, Roskamp K, Sankaran B, et al. Structural basis for Shelterin bridge assembly. Molecular Cell. 2017;68(4):698-714.e5
71.Harland JL, Chang Y-T, Moser BA, Nakamura TM. Tpz1-Ccq1 and Tpz1-Poz1 interactions within fission yeast shelterin modulate Ccq1 Thr93 phosphorylation and telomerase recruitment. PLoS Genetics. 2014;10(10):e1004708
72.Allshire RC, Ekwall K. Epigenetic regulation of chromatin states in Schizosaccharomyces pombe. Cold Spring Harbor Perspectives in Biology. 2015;7(7):a018770
73.Martienssen R, Moazed D. RNAi and heterochromatin assembly. Cold Spring Harbor Perspectives in Biology. 2015;7(8):a019323
74.Li B, de Lange T. Rap1 affects the length and heterogeneity of human telomeres. Molecular Biology of the Cell. 2003;14(12):5060-5068
75.Pardo B, Marcand S. Rap1 prevents telomere fusions by nonhomologous end joining. The EMBO Journal. 2005;24(17):3117-3127
76.Wahlin J. Saccharomyces cerevisiae RAP1 binds to telomeric sequences with spatial flexibility. Nucleic Acids Research. 2000;28(12):2292-2301
77.Ribeyre C, Shore D. Anticheckpoint pathways at telomeres in yeast. Nature Structural & Molecular Biology. 2012;19(3):307-313
78.Silverman J. Human Rif1, ortholog of a yeast telomeric protein, is regulated by ATM and 53BP1 and functions in the S-phase checkpoint. Genes & Development. 2004;18(17):2108-2119
79.Dehé P-M, Rog O, Ferreira MG, Greenwood J, Cooper JP. Taz1 enforces cell-cycle regulation of telomere synthesis. Molecular Cell. 2012;46(6):797-808
80.Deng Z, Norseen J, Wiedmer A, Riethman H, Lieberman PM. TERRA RNA binding to TRF2 facilitates heterochromatin formation and ORC recruitment at telomeres. Molecular Cell. 2009;35(4):403-413
81.Roy R, Meier B, McAinsh AD, Feldmann HM, Jackson SP. Separation-of-function mutants of yeast Ku80 reveal a Yku80p-Sir4p interaction involved in Telomeric silencing. The Journal of Biological Chemistry. 2004;279(1):86-94
82.Zappulla DC, Goodrich KJ, Arthur JR, Gurski LA, Denham EM, Stellwagen AE, et al. Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP. RNA. 2011;17(2):298-311
83.Celli GB, Denchi EL, de Lange T. Ku70 stimulates fusion of dysfunctional telomeres yet protects chromosome ends from homologous recombination. Nature Cell Biology. 2006;8(8):885-890
84.Rai R, Zheng H, He H, Luo Y, Multani A, Carpenter PB, et al. The function of classical and alternative non-homologous end-joining pathways in the fusion of dysfunctional telomeres. The EMBO Journal. 2010;29(15):2598-2610
85.Nandakumar J, Bell CF, Weidenfeld I, Zaug AJ, Leinwand LA, Cech TR. The TEL patch of telomere protein TPP1 mediates telomerase recruitment and processivity. Nature. 2012;492(7428):285-289
86.Pennock E, Buckley K, Lundblad V. Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell. 2001;104(3):387-396
87.Li S, Makovets S, Matsuguchi T, Blethrow JD, Shokat KM, Blackburn EH. Cdk1-dependent phosphorylation of Cdc13 coordinates telomere elongation during cell-cycle progression. Cell. 2009;136(1):50-61
88.Hang LE, Liu X, Cheung I, Yang Y, Zhao X. SUMOylation regulates telomere length homeostasis by targeting Cdc13. Nature Structural & Molecular Biology. 2011;18(8):920-926
89.Puglisi A, Bianchi A, Lemmens L, Damay P, Shore D. Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition. The EMBO Journal. 2008;27(17):2328-2339
90.Chen L-Y, Redon S, Lingner J. The human CST complex is a terminator of telomerase activity. Nature. 2012;488(7412):540-544
91.Reichenbach P, Höss M, Azzalin CM, Nabholz M, Bucher P, Lingner J. A human homolog of yeast Est1 associates with telomerase and uncaps chromosome ends when overexpressed. Current Biology. 2003;13(7):568-574
92.Qi H, Zakian VA. The Saccharomyces telomere-binding protein Cdc13p interacts with both the catalytic subunit of DNA polymerase alpha and the telomerase-associated est1 protein. Genes & Development. 2000;14(14):1777-1788
93.Nakamura TM. Telomerase catalytic subunit homologs from fission yeast and human. Science. 1997;277(5328):955-959
94.Hughes TR, Evans SK, Weilbaecher RG, Lundblad V. The Est3 protein is a subunit of yeast telomerase. Current Biology. 2000;10(13):809-812
95.Singer M, Gottschling D. TLC1: Template RNA component of Saccharomyces cerevisiae telomerase. Science. 1994;266(5184):404-409
96.Webb CJ, Zakian VA. Identification and characterization of the Schizosaccharomyces pombe TER1 telomerase RNA. Nature Structural & Molecular Biology. 2008;15(1):34-42
97.Leonardi J, Box JA, Bunch JT, Baumann P. TER1, the RNA subunit of fission yeast telomerase. Nature Structural & Molecular Biology. 2008;15(1):26-33
98.Diede SJ, Gottschling DE. Telomerase-mediated telomere addition in vivo requires DNA primase and DNA polymerases alpha and delta. Cell. 1999;99(7):723-733
99.Jasin M, Haber JE. The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair. DNA Repair. 2016;44:6-16
100.Janssen A, Breuer GA, Brinkman EK, van der Meulen AI, Borden SV, van Steensel B, et al. A single double-strand break system reveals repair dynamics and mechanisms in heterochromatin and euchromatin. Genes & Development. 2016;30(14):1645-1657
101.Mladenova V, Mladenov E, Iliakis G. Novel biological approaches for testing the contributions of single DSBs and DSB clusters to the biological effects of high LET radiation. Frontiers in Oncology. 2016;6:163
102.Plessis A, Perrin A, Haber JE, Dujon B. Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics. 1992;130(3):451-460
103.Haber JE. In vivo biochemistry: Physical monitoring of recombination induced by site-specific endonucleases. BioEssays. 1995;17(7):609-620
104.Rouet P, Smih F, Jasin M. Introduction of double-strand breaks into the genome of mouse cells by expression of a rare-cutting endonuclease. Molecular and Cellular Biology. 1994;14(12):8096-8106
105.Lee SS, Bohrson C, Pike AM, Wheelan SJ, Greider CW. ATM kinase is required for telomere elongation in mouse and human cells. Cell Reports. 2015;13(8):1623-1632
106.Ribeyre C, Shore D. Regulation of telomere addition at DNA double-strand breaks. Chromosoma. 2013;122(3):159-173
107.Wang J, Eisenstatt JR, Audry J, Cornelius K, Shaughnessy M, Berkner KL, et al. A heterochromatin domain forms gradually at a new telomere and is dynamic at stable telomeres. Molecular and Cellular Biology. 2018;38(15):pii: e00393-17
108.Harrison JC, Haber JE. Surviving the breakup: The DNA damage checkpoint. Annual Review of Genetics. 2006;40(1):209-235
109.Marcand S, Brevet V, Gilson E. Progressive cis-inhibition of telomerase upon telomere elongation. The EMBO Journal. 1999;18(12):3509-3519
110.Marcand S, Brevet V, Mann C, Gilson E. Cell cycle restriction of telomere elongation. Current Biology. 2000;10(8):487-490
111.Bianchi A, Shore D. Early replication of short telomeres in budding yeast. Cell. 2007;128(6):1051-1062
112.Sweetser DB, Hough H, Whelden JF, Arbuckle M, Nickoloff JA. Fine-resolution mapping of spontaneous and double-strand break-induced gene conversion tracts in Saccharomyces cerevisiae reveals reversible mitotic conversion polarity. Molecular and Cellular Biology. 1994;14(6):3863-3875
113.Diede SJ, Gottschling DE. Exonuclease activity is required for sequence addition and Cdc13p loading at a de novo telomere. Current Biology. 2001;11(17):1336-1340
114.Tsukamoto Y, Taggart AKP, Zakian VA. The role of the Mre11-Rad50-Xrs2 complex in telomerase- mediated lengthening of Saccharomyces cerevisiae telomeres. Current Biology. 2001;11(17):1328-1335
115.Stellwagen AE, Haimberger ZW, Veatch JR, Gottschling DE. Ku interacts with telomerase RNA to promote telomere addition at native and broken chromosome ends. Genes & Development. 2003;17(19):2384-2395
116.Michelson RJ, Rosenstein S, Weinert T. A telomeric repeat sequence adjacent to a DNA double-stranded break produces an anticheckpoint. Genes & Development. 2005;19(21):2546-2559
117.Sabourin M, Tuzon CT, Zakian VA. Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae. Molecular Cell. 2007;27(4):550-561
118.Bianchi A, Shore D. Increased association of telomerase with short telomeres in yeast. Genes & Development. 2007;21(14):1726-1730
119.Grossi S, Bianchi A, Damay P, Shore D. Telomere formation by Rap1p binding site arrays reveals end-specific length regulation requirements and active telomeric recombination. Molecular and Cellular Biology. 2001;21(23):8117-8128
120.Marcand S, Gilson E, Shore D. A protein-counting mechanism for telomere length regulation in yeast. Science. 1997;275(5302):986-990
121.Ray A, Runge KW. The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction. Molecular and Cellular Biology. 1999;19(1):31-45
122.Du L-L, Nakamura TM, Russell P. Histone modification-dependent and -independent pathways for recruitment of checkpoint protein Crb2 to double-strand breaks. Genes & Development. 2006;20(12):1583-1596
123.Watson AT, Werler P, Carr AM. Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus. Gene. 2011;484(1-2):75-85
124.Nimmo ERR, Cranston G, Allshire RCC. Telomere-associated chromosome breakage in fission yeast results in variegated expression of adjacent genes. The EMBO Journal. 1994;13(16):3801-3811
125.Thon G, Bjerling P, Bünner CM, Verhein-Hansen J. Expression-state boundaries in the mating-type region of fission yeast. Genetics. 2002;161(2):611-622
126.Sunder S, Greeson-Lott NT, Runge KW, Sanders SL. A new method to efficiently induce a site-specific double-strand break in the fission yeast Schizosaccharomyces pombe. Yeast. 2012;29(7):275-291
127.Forsburg SL. Codon usage table for Schizosaccharomyces pombe. Yeast. 1994;10(8):1045-1047
128.Wood V, Gwilliam R, Rajandream M-A, Lyne M, Lyne R, Stewart A, et al. The genome sequence of Schizosaccharomyces pombe. Nature. 2002;415(6874):871-880
129.Castillo AG, Pidoux AL, Catania S, Durand-Dubief M, Choi ES, Hamilton G, et al. Telomeric repeats facilitate CENP-A(Cnp1) incorporation via telomere binding proteins. PLoS One. 2013;8(7):e69673
130.Wellinger RJ, Zakian VA. Everything you ever wanted to know about Saccharomyces cerevisiae telomeres: Beginning to end. Genetics. 2012;191(4):1073-1105
131.Jih G, Iglesias N, Currie MA, Bhanu NV, Paulo JA, Gygi SP, et al. Unique roles for histone H3K9me states in RNAi and heritable silencing of transcription. Nature. 2017;547(7664):463-467
132.Gao Q , Reynolds GE, Wilcox A, Miller D, Cheung P, Artandi SE, et al. Telomerase-dependent and -independent chromosome healing in mouse embryonic stem cells. DNA Repair. 2008;7(8):1233-1249
133.Muraki K, Han L, Miller D, Murnane JP. The role of ATM in the deficiency in nonhomologous end-joining near telomeres in a human cancer cell line. PLoS Genetics. 2013;9(3):e1003386
134.Alcaraz Silva B, Jones TJ, Murnane JP. Differences in the recruitment of DNA repair proteins at subtelomeric and interstitial I-SceI endonuclease-induced DNA double-strand breaks. DNA Repair. 2017;49:1-8
135.Doksani Y, de Lange T. Telomere-internal double-Strand breaks are repaired by homologous recombination and PARP1/Lig3-dependent end-joining. Cell Reports. 2016;17(6):1646-1656
136.Mao P, Liu J, Zhang Z, Zhang H, Liu H, Gao S, et al. Homologous recombination-dependent repair of telomeric DSBs in proliferating human cells. Nature Communications. 2016;7(1):12154
137.Liddiard K, Ruis B, Takasugi T, Harvey A, Ashelford KE, Hendrickson EA, et al. Sister chromatid telomere fusions, but not NHEJ-mediated inter-chromosomal telomere fusions, occur independently of DNA ligases 3 and 4. Genome Research. 2016;26(5):588-600
Written By
Julien Audry and Kurt W. Runge
Submitted: 21 January 2019Reviewed: 03 April 2019Published: 05 June 2019
Open access peer-reviewed chapter
