Journal articles: 'Telomere protection, Telomere capping, Yeast'

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Bryan, Tracy M. "G-Quadruplexes at Telomeres: Friend or Foe?" Molecules 25, no. 16 (August 13, 2020): 3686. http://dx.doi.org/10.3390/molecules25163686.

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Telomeres are DNA-protein complexes that cap and protect the ends of linear chromosomes. In almost all species, telomeric DNA has a G/C strand bias, and the short tandem repeats of the G-rich strand have the capacity to form into secondary structures in vitro, such as four-stranded G-quadruplexes. This has long prompted speculation that G-quadruplexes play a positive role in telomere biology, resulting in selection for G-rich tandem telomere repeats during evolution. There is some evidence that G-quadruplexes at telomeres may play a protective capping role, at least in yeast, and that they may positively affect telomere maintenance by either the enzyme telomerase or by recombination-based mechanisms. On the other hand, G-quadruplex formation in telomeric DNA, as elsewhere in the genome, can form an impediment to DNA replication and a source of genome instability. This review summarizes recent evidence for the in vivo existence of G-quadruplexes at telomeres, with a focus on human telomeres, and highlights some of the many unanswered questions regarding the location, form, and functions of these structures.

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Singh, Sunitha M., Olga Steinberg-Neifach, I. Saira Mian, and Neal F. Lue. "Analysis of Telomerase in Candida albicans: Potential Role in Telomere End Protection." Eukaryotic Cell 1, no. 6 (December 2002): 967–77. http://dx.doi.org/10.1128/ec.1.6.967-977.2002.

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ABSTRACT Telomerase is a ribonucleoprotein reverse transcriptase responsible for the maintenance of one strand of telomere terminal repeats. Analysis of the telomerase complex in the budding yeast Saccharomyces cerevisiae has revealed the presence of one catalytic protein subunit (Est2p/TERT) and at least two noncatalytic components (Est1p and Est3p). The genome of the pathogenic yeast Candida albicans contains putative orthologues of all three telomerase components. Disruption of each homologue resulted in significant but distinct telomere dysfunction in Candida. Similar to S. cerevisiae, the Candida EST3 disruption strain exhibits progressive telomere loss over many generations, at a rate that is consistent with incomplete replication. In contrast, telomeres in both the Candida TERT and EST1 disruption strains can contract rapidly, followed by partial or nearly complete recovery, suggesting a defect in telomere “capping.” We propose that these two telomerase subunits may participate in the protection of chromosomal ends in Candida. Analysis of telomerase-mediated primer extension in vitro indicates that only the TERT protein is absolutely essential for enzyme activity. Our results support the conservation of telomerase protein components beyond the catalytic subunit but reveal species-specific phenotypic alterations in response to loss of individual telomerase component. We also identify potential homologues of Est1p in phylogenetically diverse organisms. The Est1p sequence family possesses a conserved N-terminal domain predicted to be structurally related to tetratricopeptide repeat-containing proteins.

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Churikov, Dmitri, Chao Wei, and Carolyn M. Price. "Vertebrate POT1 Restricts G-Overhang Length and Prevents Activation of a Telomeric DNA Damage Checkpoint but Is Dispensable for Overhang Protection." Molecular and Cellular Biology 26, no. 18 (September 15, 2006): 6971–82. http://dx.doi.org/10.1128/mcb.01011-06.

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ABSTRACT Although vertebrate POT1 is thought to play a role in both telomere capping and length regulation, its function has proved difficult to analyze. We therefore generated a conditional cell line that lacks wild-type POT1 but expresses an estrogen receptor-POT1 fusion. The cells grow normally in tamoxifen, but drug removal causes loss of POT1 from the telomere, rapid cell cycle arrest, and eventual cell death. The arrested cells have a 4N DNA content, and addition of caffeine causes immediate entry into mitosis, suggesting a G2 arrest due to an ATM- and/or ATR-mediated checkpoint. γH2AX accumulates at telomeres, indicating a telomeric DNA damage response, the likely cause of the checkpoint. However, POT1 loss does not cause degradation of the G-strand overhang. Instead, the amount of G overhang increases two- to threefold. Some cells eventually escape the cell cycle arrest and enter mitosis. They rarely exhibit telomere fusions but show severe chromosome segregation defects due to centrosome amplification. Our data indicate that vertebrate POT1 is required for telomere capping but that it functions quite differently from TRF2. Instead of being required for G-overhang protection, POT1 is required to suppress a telomeric DNA damage response. Our results also indicate significant functional similarities between POT1 and Cdc13 from budding yeast (Saccharomyces cerevisiae).

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Xu, Ling, Ruben C. Petreaca, Hovik J. Gasparyan, Stephanie Vu, and Constance I. Nugent. "TEN1 Is Essential for CDC13-Mediated Telomere Capping." Genetics 183, no. 3 (September 14, 2009): 793–810. http://dx.doi.org/10.1534/genetics.109.108894.

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Telomere binding proteins protect chromosome ends from degradation and mask chromosome termini from checkpoint surveillance. In Saccharomyces cerevisiae, Cdc13 binds single-stranded G-rich telomere repeats, maintaining telomere integrity and length. Two additional proteins, Ten1 and Stn1, interact with Cdc13 but their contributions to telomere integrity are not well defined. Ten1 is known to prevent accumulation of aberrant single-stranded telomere DNA; whether this results from defective end protection or defective telomere replication is unclear. Here we report our analysis of a new group of ten1 temperature-sensitive (ts) mutants. At permissive temperatures, ten1-ts strains display greatly elongated telomeres. After shift to nonpermissive conditions, however, ten1-ts mutants accumulate extensive telomeric single-stranded DNA. Cdk1 activity is required to generate these single-stranded regions, and deleting the EXO1 nuclease partially suppresses ten1-ts growth defects. This is similar to cdc13-1 mutants, suggesting ten1-ts strains are defective for end protection. Moreover, like Cdc13, our analysis reveals Ten1 promotes de novo telomere addition. Interestingly, in ten1-ts strains at high temperatures, telomeric single-stranded DNA and Rad52-YFP repair foci are strongly induced despite Cdc13 remaining associated with telomeres, revealing Cdc13 telomere binding is not sufficient for end protection. Finally, unlike cdc13-1 mutants, ten1-ts strains display strong synthetic interactions with mutations in the POLα complex. These results emphasize that Cdc13 relies on Ten1 to execute its essential function, but leave open the possibility that Ten1 has a Cdc13-independent role in DNA replication.

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Lamm, Noa, Shhadeh Bsoul, Majdi M. Kabaha, and Yehuda Tzfati. "“Poisoning” yeast telomeres distinguishes between redundant telomere capping pathways." Chromosoma 121, no. 6 (October 6, 2012): 613–27. http://dx.doi.org/10.1007/s00412-012-0385-6.

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DuBois, Michelle L., Zara W. Haimberger, Martin W. McIntosh, and Daniel E. Gottschling. "A Quantitative Assay for Telomere Protection in Saccharomyces cerevisiae." Genetics 161, no. 3 (July 1, 2002): 995–1013. http://dx.doi.org/10.1093/genetics/161.3.995.

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Abstract Telomeres are the protective ends of linear chromosomes. Telomeric components have been identified and described by their abilities to bind telomeric DNA, affect telomere repeat length, participate in telomeric DNA replication, or modulate transcriptional silencing of telomere-adjacent genes; however, their roles in chromosome end protection are not as well defined. We have developed a genetic, quantitative assay in Saccharomyces cerevisiae to measure whether various telomeric components protect chromosome ends from homologous recombination. This “chromosomal cap” assay has revealed that the telomeric end-binding proteins, Cdc13p and Ku, both protect the chromosome end from homologous recombination, as does the ATM-related kinase, Tel1p. We propose that Cdc13p and Ku structurally inhibit recombination at telomeres and that Tel1p regulates the chromosomal cap, acting through Cdc13p. Analysis with recombination mutants indicated that telomeric homologous recombination events proceeded by different mechanisms, depending on which capping component was compromised. Furthermore, we found that neither telomere repeat length nor telomeric silencing correlated with chromosomal capping efficiency. This capping assay provides a sensitive in vivo approach for identifying the components of chromosome ends and the mechanisms by which they are protected.

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Dubruille, R., and B. Loppin. "Protection of Drosophila chromosome ends through minimal telomere capping." Journal of Cell Science 128, no. 10 (April 23, 2015): 1969–81. http://dx.doi.org/10.1242/jcs.167825.

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Larrivée, Michel, and Raymund J. Wellinger. "Telomerase- and capping-independent yeast survivors with alternate telomere states." Nature Cell Biology 8, no. 7 (June 11, 2006): 741–47. http://dx.doi.org/10.1038/ncb1429.

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Kibe, Tatsuya, Yuuki Ono, Koichiro Sato, and Masaru Ueno. "Fission Yeast Taz1 and RPA Are Synergistically Required to Prevent Rapid Telomere Loss." Molecular Biology of the Cell 18, no. 6 (June 2007): 2378–87. http://dx.doi.org/10.1091/mbc.e06-12-1084.

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The telomere complex must allow nucleases and helicases to process chromosome ends to make them substrates for telomerase, while preventing these same activities from disrupting chromosome end-protection. Replication protein A (RPA) binds to single-stranded DNA and is required for DNA replication, recombination, repair, and telomere maintenance. In fission yeast, the telomere binding protein Taz1 protects telomeres and negatively regulates telomerase. Here, we show that taz1-d rad11-D223Y double mutants lose their telomeric DNA, indicating that RPA (Rad11) and Taz1 are synergistically required to prevent telomere loss. Telomere loss in the taz1-d rad11-D223Y double mutants was suppressed by additional mutation of the helicase domain in a RecQ helicase (Rqh1), or by overexpression of Pot1, a single-strand telomere binding protein that is essential for protection of chromosome ends. From our results, we propose that in the absence of Taz1 and functional RPA, Pot1 cannot function properly and the helicase activity of Rqh1 promotes telomere loss. Our results suggest that controlling the activity of Rqh1 at telomeres is critical for the prevention of genomic instability.

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Ueno, Masaru. "Exploring Genetic Interactions with Telomere Protection Gene pot1 in Fission Yeast." Biomolecules 13, no. 2 (February 15, 2023): 370. http://dx.doi.org/10.3390/biom13020370.

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The regulation of telomere length has a significant impact on cancer risk and aging in humans. Circular chromosomes are found in humans and are often unstable during mitosis, resulting in genome instability. Some types of cancer have a high frequency of a circular chromosome. Fission yeast is a good model for studying the formation and stability of circular chromosomes as deletion of pot1 (encoding a telomere protection protein) results in rapid telomere degradation and chromosome fusion. Pot1 binds to single-stranded telomere DNA and is conserved from fission yeast to humans. Loss of pot1 leads to viable strains in which all three fission yeast chromosomes become circular. In this review, I will introduce pot1 genetic interactions as these inform on processes such as the degradation of uncapped telomeres, chromosome fusion, and maintenance of circular chromosomes. Therefore, exploring genes that genetically interact with pot1 contributes to finding new genes and/or new functions of genes related to the maintenance of telomeres and/or circular chromosomes.

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Jaco, Isabel, Purificación Muñoz, Fermín Goytisolo, Joanna Wesoly, Susan Bailey, Guillermo Taccioli, and María A. Blasco. "Role of Mammalian Rad54 in Telomere Length Maintenance." Molecular and Cellular Biology 23, no. 16 (August 15, 2003): 5572–80. http://dx.doi.org/10.1128/mcb.23.16.5572-5580.2003.

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ABSTRACT The homologous recombination (HR) DNA repair pathway participates in telomere length maintenance in yeast but its putative role at mammalian telomeres is unknown. Mammalian Rad54 is part of the HR machinery, and Rad54-deficient mice show a reduced HR capability. Here, we show that Rad54-deficient mice also show significantly shorter telomeres than wild-type controls, indicating that Rad54 activity plays an essential role in telomere length maintenance in mammals. Rad54 deficiency also resulted in an increased frequency of end-to-end chromosome fusions involving telomeres compared to the controls, suggesting a putative role of Rad54 in telomere capping. Finally, the study of mice doubly deficient for Rad54 and DNA-PKcs showed that telomere fusions due to DNA-PKcs deficiency were not rescued in the absence of Rad54, suggesting that they are not mediated by Rad54 activity.

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Puglisi, Andrea, Alessandro Bianchi, Laure Lemmens, Pascal Damay, and David Shore. "Distinct roles for yeast Stn1 in telomere capping and telomerase inhibition." EMBO Journal 27, no. 17 (August 14, 2008): 2328–39. http://dx.doi.org/10.1038/emboj.2008.158.

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Vodenicharov, Momchil D., Nancy Laterreur, and Raymund J. Wellinger. "Telomere capping in non-dividing yeast cells requires Yku and Rap1." EMBO Journal 29, no. 17 (July 13, 2010): 3007–19. http://dx.doi.org/10.1038/emboj.2010.155.

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Iyer, Shilpa, Ashley D. Chadha, and Michael J. McEachern. "A Mutation in the STN1 Gene Triggers an Alternative Lengthening of Telomere-Like Runaway Recombinational Telomere Elongation and Rapid Deletion in Yeast." Molecular and Cellular Biology 25, no. 18 (September 15, 2005): 8064–73. http://dx.doi.org/10.1128/mcb.25.18.8064-8073.2005.

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

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Underwood, Dana H., Coleen Carroll, and Michael J. McEachern. "Genetic Dissection of the Kluyveromyces lactis Telomere and Evidence for Telomere Capping Defects in TER1 Mutants with Long Telomeres." Eukaryotic Cell 3, no. 2 (April 2004): 369–84. http://dx.doi.org/10.1128/ec.3.2.369-384.2004.

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ABSTRACT In the yeast Kluyveromyces lactis, the telomeres are composed of perfect 25-bp repeats copied from a 30-nucleotide RNA template defined by 5-nucleotide terminal repeats. A genetic dissection of the K. lactis telomere was performed by using mutant telomerase RNA (TER1) alleles to incorporate mutated telomeric repeats. This analysis has shown that each telomeric repeat contains several functional regions, some of which may physically overlap. Mutations in the terminal repeats of the template RNA typically lead to telomere shortening, as do mutations in the right side of the Rap1p binding site. Mutations in the left half of the Rap1p binding site, however, lead to the immediate formation of long telomeres. When mutated, the region immediately 3′ of the Rap1p binding site on the TG-rich strand of the telomere leads to telomeres that are initially short but eventually undergo extreme telomere elongation. Mutations between this region and the 3′ terminal repeat cause elevated recombination despite the presence of telomeres of nearly wild-type length. Mutants with highly elongated telomeres were further characterized and exhibit signs of telomere capping defects, including elevated levels of subtelomeric recombination and the formation of extrachromosomal and single-stranded telomeric DNA. Lengthening caused by some Rap1 binding site mutations can be suppressed by high-copy-number RAP1. Mutated telomeric repeats from a delayed elongation mutant are shown to be defective at regulating telomere length in cells with wild-type telomerase, indicating that the telomeric repeats are defective at telomere length regulation.

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Hirano, Yukinori, and Katsunori Sugimoto. "Cdc13 Telomere Capping Decreases Mec1 Association but Does Not Affect Tel1 Association with DNA Ends." Molecular Biology of the Cell 18, no. 6 (June 2007): 2026–36. http://dx.doi.org/10.1091/mbc.e06-12-1074.

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Chromosome ends, known as telomeres, have to be distinguished from DNA breaks that activate DNA damage checkpoint. Two large protein kinases, ataxia-teleangiectasia mutated (ATM) and ATM-Rad3-related (ATR), control not only checkpoint activation but also telomere length. In budding yeast, Mec1 and Tel1 correspond to ATR and ATM, respectively. Here, we show that Cdc13-dependent telomere capping attenuates Mec1 association with DNA ends. The telomeric TG repeat sequence inhibits DNA degradation and decreases Mec1 accumulation at the DNA end. The TG-mediated degradation block requires binding of multiple Cdc13 proteins. The Mre11–Rad50-Xrs2 complex and Exo1 contribute to DNA degradation at DNA ends. Although the TG sequence impedes Exo1 association with DNA ends, it allows Mre11 association. Moreover, the TG sequence does not affect Tel1 association with the DNA end. Our results suggest that the Cdc13 telomere cap coordinates Mec1 and Tel1 accumulation rather than simply covering the DNA ends at telomeres.

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Zahid, Syed, Sarah Aloe, Jeanette H. Sutherland, William K. Holloman, and Neal F. Lue. "Ustilago maydis telomere protein Pot1 harbors an extra N-terminal OB fold and regulates homology-directed DNA repair factors in a dichotomous and context-dependent manner." PLOS Genetics 18, no. 5 (May 19, 2022): e1010182. http://dx.doi.org/10.1371/journal.pgen.1010182.

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The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the structure and activities of Pot1 in Ustilago maydis, a fungal model that recapitulates key features of mammalian telomere regulation. Compared to the well-characterized primate and fission yeast Pot1 orthologs, UmPot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. UmPot1 binds directly to Rad51 and regulates the latter’s strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere maintenance. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1-deficient cells were suppressed, and cell viability was restored by the deletion of genes encoding Rad51 or Brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection.

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Anbalagan, Savani, Diego Bonetti, Giovanna Lucchini, and Maria Pia Longhese. "Rif1 Supports the Function of the CST Complex in Yeast Telomere Capping." PLoS Genetics 7, no. 3 (March 17, 2011): e1002024. http://dx.doi.org/10.1371/journal.pgen.1002024.

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Grossi, Simona, Alessandro Bianchi, Pascal Damay, and David Shore. "Telomere Formation by Rap1p Binding Site Arrays Reveals End-Specific Length Regulation Requirements and Active Telomeric Recombination." Molecular and Cellular Biology 21, no. 23 (December 1, 2001): 8117–28. http://dx.doi.org/10.1128/mcb.21.23.8117-8128.2001.

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ABSTRACT Rap1p, the major telomere repeat binding protein in yeast, has been implicated in both de novo telomere formation and telomere length regulation. To characterize the role of Rap1p in these processes in more detail, we studied the generation of telomeres in vivo from linear DNA substrates containing defined arrays of Rap1p binding sites. Consistent with previous work, our results indicate that synthetic Rap1p binding sites within the internal half of a telomeric array are recognized as an integral part of the telomere complex in an orientation-independent manner that is largely insensitive to the precise spacing between adjacent sites. By extending the lengths of these constructs, we found that several different Rap1p site arrays could never be found at the very distal end of a telomere, even when correctly oriented. Instead, these synthetic arrays were always followed by a short (≈100-bp) “cap” of genuine TG repeat sequence, indicating a remarkably strict sequence requirement for an end-specific function(s) of the telomere. Despite this fact, even misoriented Rap1p site arrays promote telomere formation when they are placed at the distal end of a telomere-healing substrate, provided that at least a single correctly oriented site is present within the array. Surprisingly, these heterogeneous arrays of Rap1p binding sites generate telomeres through a RAD52-dependent fusion resolution reaction that results in an inversion of the original array. Our results provide new insights into the nature of telomere end capping and reveal one way by which recombination can resolve a defect in this process.

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Longhese, Maria Pia, Vera Paciotti, Holger Neecke, and Giovanna Lucchini. "Checkpoint Proteins Influence Telomeric Silencing and Length Maintenance in Budding Yeast." Genetics 155, no. 4 (August 1, 2000): 1577–91. http://dx.doi.org/10.1093/genetics/155.4.1577.

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AbstractA complex network of surveillance mechanisms, called checkpoints, interrupts cell cycle progression when damage to the genome is detected or when cells fail to complete DNA replication, thus ensuring genetic integrity. In budding yeast, components of the DNA damage checkpoint regulatory network include the RAD9, RAD17, RAD24, MEC3, DDC1, RAD53, and MEC1 genes that are proposed to be involved in different aspects of DNA metabolism. We provide evidence that some DNA damage checkpoint components play a role in maintaining telomere integrity. In fact, rad53 mutants specifically enhance repression of telomere-proximal transcription via the Sir-mediated pathway, suggesting that Rad53 might be required for proper chromatin structure at telomeres. Moreover, Rad53, Mec1, Ddc1, and Rad17 are necessary for telomere length maintenance, since mutations in all of these genes cause a decrease in telomere size. The telomeric shortening in rad53 and mec1 mutants is further enhanced in the absence of SIR genes, suggesting that Rad53/Mec1 and Sir proteins contribute to chromosome end protection by different pathways. The finding that telomere shortening, but not increased telomeric repression of gene expression in rad53 mutants, can be suppressed by increasing dNTP synthetic capacity in these strains suggests that transcriptional silencing and telomere integrity involve separable functions of Rad53.

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Ray, Alo, and Kurt W. Runge. "The C Terminus of the Major Yeast Telomere Binding Protein Rap1p Enhances Telomere Formation." Molecular and Cellular Biology 18, no. 3 (March 1, 1998): 1284–95. http://dx.doi.org/10.1128/mcb.18.3.1284.

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ABSTRACT The telomeres of most organisms consist of short repeated sequences that can be elongated by telomerase, a reverse transcriptase complex that contains its own RNA template for the synthesis of telomere repeats. In Saccharomyces cerevisiae, the RAP1gene encodes the major telomere binding protein Rap1p. Here we use a quantitative telomere formation assay to demonstrate that Rap1p C termini can enhance telomere formation more than 30-fold when they are located at internal sites. This stimulation is distinct from protection from degradation. Enhancement of formation required the gene for telomerase RNA but not Sir1p, Sir2p, Sir3p, Sir4p, Tel1p, or the Rif1p binding site in the Rap1p C terminus. Our data suggest that Rap1p C termini enhance telomere formation by attracting or increasing the activity of telomerase near telomeres. Earlier work suggests that Rap1p molecules at the chromosome terminus inhibit the elongation of long telomeres by blocking the access of telomerase. Our results suggest a model where a balance between internal Rap1p increasing telomerase activity and Rap1p at the termini of long telomeres controlling telomerase access maintains telomeres at a constant length.

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Bechard, Laura H., Bilge D. Butuner, George J. Peterson, Will McRae, Zeki Topcu, and Michael J. McEachern. "Mutant Telomeric Repeats in Yeast Can Disrupt the Negative Regulation of Recombination-Mediated Telomere Maintenance and Create an Alternative Lengthening of Telomeres-Like Phenotype." Molecular and Cellular Biology 29, no. 3 (November 24, 2008): 626–39. http://dx.doi.org/10.1128/mcb.00423-08.

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ABSTRACT Some human cancers maintain telomeres using alternative lengthening of telomeres (ALT), a process thought to be due to recombination. In Kluyveromyces lactis mutants lacking telomerase, recombinational telomere elongation (RTE) is induced at short telomeres but is suppressed once telomeres are moderately elongated by RTE. Recent work has shown that certain telomere capping defects can trigger a different type of RTE that results in much more extensive telomere elongation that is reminiscent of human ALT cells. In this study, we generated telomeres composed of either of two types of mutant telomeric repeats, Acc and SnaB, that each alter the binding site for the telomeric protein Rap1. We show here that arrays of both types of mutant repeats present basally on a telomere were defective in negatively regulating telomere length in the presence of telomerase. Similarly, when each type of mutant repeat was spread to all chromosome ends in cells lacking telomerase, they led to the formation of telomeres produced by RTE that were much longer than those seen in cells with only wild-type telomeric repeats. The Acc repeats produced the more severe defect in both types of telomere maintenance, consistent with their more severe Rap1 binding defect. Curiously, although telomerase deletion mutants with telomeres composed of Acc repeats invariably showed extreme telomere elongation, they often also initially showed persistent very short telomeres with few or no Acc repeats. We suggest that these result from futile cycles of recombinational elongation and truncation of the Acc repeats from the telomeres. The presence of extensive 3′ overhangs at mutant telomeres suggests that Rap1 may normally be involved in controlling 5′ end degradation.

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Bryan, Christopher, Cory Rice, Michael Harkisheimer, David Schultz, and Emmanuel Skordalakes. "Structure of the Human Telomeric Stn1-Ten1 Complex." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1587. http://dx.doi.org/10.1107/s2053273314084125.

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The telomeric CST complex plays a central role in chromosome end capping and replication in budding yeast, and homologues of CST were identified recently in higher eukaryotes. The human CST (Ctc1, hStn1, hTen1) has been shown to play a role in telomere maintenance, but the extent of conservation across species has been in question because of low sequence identity (below 10% for Ctc1, the core subunit of the CST complex) and data suggesting subtle differences in function between complexes. We solved the high-resolution crystal structure of the human Stn1-Ten1 complex, which revealed striking structural similarity between the yeast and human CST complexes. We also showed using southern blots and fluorescence in situ hybridization experiments that disruption of the hStn1-Ten1 binding interface in vivo produces elongated telomeres and telomere defects in accordance with what has been previously observed for the yeast CST complex. Our results support structural and functional conservation of telomeric CST across species.

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Nakamura, Toru M., Bettina A. Moser, and Paul Russell. "Telomere Binding of Checkpoint Sensor and DNA Repair Proteins Contributes to Maintenance of Functional Fission Yeast Telomeres." Genetics 161, no. 4 (August 1, 2002): 1437–52. http://dx.doi.org/10.1093/genetics/161.4.1437.

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Abstract Telomeres, the ends of linear chromosomes, are DNA double-strand ends that do not trigger a cell cycle arrest and yet require checkpoint and DNA repair proteins for maintenance. Genetic and biochemical studies in the fission yeast Schizosaccharomyces pombe were undertaken to understand how checkpoint and DNA repair proteins contribute to telomere maintenance. On the basis of telomere lengths of mutant combinations of various checkpoint-related proteins (Rad1, Rad3, Rad9, Rad17, Rad26, Hus1, Crb2, Chk1, Cds1), Tel1, a telomere-binding protein (Taz1), and DNA repair proteins (Ku70, Rad32), we conclude that Rad3/Rad26 and Tel1/Rad32 represent two pathways required to maintain telomeres and prevent chromosome circularization. Rad1/Rad9/Hus1/Rad17 and Ku70 are two additional epistasis groups, which act in the Rad3/Rad26 pathway. However, Rad3/Rad26 must have additional target(s), as cells lacking Tel1/Rad32, Rad1/Rad9/Hus1/Rad17, and Ku70 groups did not circularize chromosomes. Cells lacking Rad3/Rad26 and Tel1/Rad32 senesced faster than a telomerase trt1Δ mutant, suggesting that these pathways may contribute to telomere protection. Deletion of taz1 did not suppress chromosome circularization in cells lacking Rad3/Rad26 and Tel1/Rad32, also suggesting that two pathways protect telomeres. Chromatin immunoprecipitation analyses found that Rad3, Rad1, Rad9, Hus1, Rad17, Rad32, and Ku70 associate with telomeres. Thus, checkpoint sensor and DNA repair proteins contribute to telomere maintenance and protection through their association with telomeres.

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Maser, Richard S., Kwok-Kin Wong, Erguen Sahin, Huili Xia, Maria Naylor, H. Mason Hedberg, Steven E. Artandi, and Ronald A. DePinho. "DNA-Dependent Protein Kinase Catalytic Subunit Is Not Required for Dysfunctional Telomere Fusion and Checkpoint Response in the Telomerase-Deficient Mouse." Molecular and Cellular Biology 27, no. 6 (December 4, 2006): 2253–65. http://dx.doi.org/10.1128/mcb.01354-06.

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ABSTRACT Telomeres are key structural elements for the protection and maintenance of linear chromosomes, and they function to prevent recognition of chromosomal ends as DNA double-stranded breaks. Loss of telomere capping function brought about by telomerase deficiency and gradual erosion of telomere ends or by experimental disruption of higher-order telomere structure culminates in the fusion of defective telomeres and/or the activation of DNA damage checkpoints. Previous work has implicated the nonhomologous end-joining (NHEJ) DNA repair pathway as a critical mediator of these biological processes. Here, employing the telomerase-deficient mouse model, we tested whether the NHEJ component DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was required for fusion of eroded/dysfunctional telomere ends and the telomere checkpoint responses. In late-generation mTerc − / − DNA-PKcs − / − cells and tissues, chromosomal end-to-end fusions and anaphase bridges were readily evident. Notably, nullizygosity for DNA Ligase4 (Lig4)—an additional crucial NHEJ component—was also permissive for chromosome fusions in mTerc − / − cells, indicating that, in contrast to results seen with experimental disruption of telomere structure, telomere dysfunction in the context of gradual telomere erosion can engage additional DNA repair pathways. Furthermore, we found that DNA-PKcs deficiency does not reduce apoptosis, tissue atrophy, or p53 activation in late-generation mTerc − / − tissues but rather moderately exacerbates germ cell apoptosis and testicular degeneration. Thus, our studies indicate that the NHEJ components, DNA-PKcs and Lig4, are not required for fusion of critically shortened telomeric ends and that DNA-PKcs is not required for sensing and executing the telomere checkpoint response, findings consistent with the consensus view of the limited role of DNA-PKcs in DNA damage signaling in general.

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Caslini, Corrado, and Jay L. Hess. "MLL Modulates Telomere Length in Mammalian Cells." Blood 108, no. 11 (November 1, 2006): 2209. http://dx.doi.org/10.1182/blood.v108.11.2209.2209.

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Abstract In mammalian cells, the telomeric complex at the end of chromosomes consists of several thousand copies of the exanucleotide TTAGGG and associated proteins attached to the nuclear matrix. Chromatin modifying enzymes involved in histone H3/lysine 9 and histone H4/lysine 20 trimethylation, and DNA methylation are known to preserve the telomere heterochromatic structure, length and capping function. Loss of these heterochromatic marks leads to telomere lengthening, most likely through the negative regulation of telomerase or alternative lengthening of telomeres (ALT) mechanisms. The MLL protein is a chromatin modifying enzyme with histone H3/lysine 4 methyltransferase activity, which maintains active transcriptional state of target genes in a large multiprotein complex. Analogously, the yeast’s MLL homologous protein Set1 is part of a multiprotein complex required for maintenance of target genes expression. In addition, Set1 deletion mutants show disruption of telomeric silencing along with telomere shortening or lengthening, respectively in budding and fission yeast. This raised the question of whether MLL, like Set1, plays a role in epigenetic maintenance of telomeric heterochromatin. Here, using chromatin immunoprecipitation (ChIP) analysis, we show that MLL associates with the heterochromatic complex at telomeres of primary and transformed human cell lines. ChIP analysis of cell lines conditionally expressing Flag-tagged MLL chimeric proteins and deletion mutants shows the amino terminus of MLL, which confers association to the nuclear matrix, is responsible for targeting to the telomeric complex. MLL associates with the telomeres of telomerase and ALT positive cell lines in amount that is proportional to the telomere length, as revealed by Southern blot terminal restriction fragment analysis. Moreover, immunoprecipitation analysis evidenced the association of MLL with the terminal-repeat binding factor TRF2, a protein known to play a key role in telomere capping, and indirect immunofluorescence analysis showed MLL and TRF2 colocalization at ALT-associated PML nuclear bodies. In search for possible biological functions of MLL at the telomeric complex, we found abnormally longer telomeres in Mll-null mouse embryonic stem (ES) cells and fibroblasts (MEFs) than in wild-type control cells. In Mll-null MEFs, a significant telomere shortening was obtained by stable reexpression of an MLL allele. In addition, we found that in aging human cells the MLL binding to the telomeric complex is abrogated by the progressive telomere shortening due to telomere attrition, suggesting a possible involvement of MLL in signaling for replicative senescence.

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Hofr, Ctirad, Pavla Šultesová, Michal Zimmermann, Iva Mozgová, Petra Procházková Schrumpfová, Michaela Wimmerová, and Jiří Fajkus. "Single-Myb-histone proteins from Arabidopsis thaliana: a quantitative study of telomere-binding specificity and kinetics." Biochemical Journal 419, no. 1 (March 13, 2009): 221–30. http://dx.doi.org/10.1042/bj20082195.

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Proteins that bind telomeric DNA modulate the structure of chromosome ends and control telomere function and maintenance. It has been shown that AtTRB (Arabidopsis thaliana telomere-repeat-binding factor) proteins from the SMH (single-Myb-histone) family selectively bind double-stranded telomeric DNA and interact with the telomeric protein AtPOT1b (A. thaliana protection of telomeres 1b), which is involved in telomere capping. In the present study, we performed the first quantitative DNA-binding study of this plant-specific family of proteins. Interactions of full-length proteins AtTRB1 and AtTRB3 with telomeric DNA were analysed by electrophoretic mobility-shift assay, fluorescence anisotropy and surface plasmon resonance to reveal their binding stoichiometry and kinetics. Kinetic analyses at different salt conditions enabled us to estimate the electrostatic component of binding and explain different affinities of the two proteins to telomeric DNA. On the basis of available data, a putative model explaining the binding stoichiometry and the protein arrangement on telomeric DNA is presented.

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Pan, Lili, Katie Hildebrand, Cian Stutz, Nicolas Thomä, and Peter Baumann. "Minishelterins separate telomere length regulation and end protection in fission yeast." Genes & Development 29, no. 11 (June 1, 2015): 1164–74. http://dx.doi.org/10.1101/gad.261123.115.

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Moser, Bettina A., Lakxmi Subramanian, Lyne Khair, Ya-Ting Chang, and Toru M. Nakamura. "Fission Yeast Tel1ATM and Rad3ATR Promote Telomere Protection and Telomerase Recruitment." PLoS Genetics 5, no. 8 (August 28, 2009): e1000622. http://dx.doi.org/10.1371/journal.pgen.1000622.

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Gasparyan, H. J., L. Xu, R. C. Petreaca, A. E. Rex, V. Y. Small, N. S. Bhogal, J. A. Julius, et al. "Yeast telomere capping protein Stn1 overrides DNA replication control through the S phase checkpoint." Proceedings of the National Academy of Sciences 106, no. 7 (January 26, 2009): 2206–11. http://dx.doi.org/10.1073/pnas.0812605106.

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Bunch, Jeremy T., Nancy S. Bae, Jessica Leonardi, and Peter Baumann. "Distinct Requirements for Pot1 in Limiting Telomere Length and Maintaining Chromosome Stability." Molecular and Cellular Biology 25, no. 13 (July 1, 2005): 5567–78. http://dx.doi.org/10.1128/mcb.25.13.5567-5578.2005.

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ABSTRACT The fission yeast Pot1 (protection of telomeres) protein binds to the single-stranded extensions at the ends of telomeres, where its presence is critical for the maintenance of linear chromosomes. Homologs of Pot1 have been identified in a wide variety of eukaryotes, including plants, animals, and humans. We now show that Pot1 plays dual roles in telomere length regulation and chromosome end protection. Using a series of Pot1 truncation mutants, we have defined distinct areas of the protein required for chromosome stability and for limiting access to telomere ends by telomerase. We provide evidence that a large portion of Pot1, including the N-terminal DNA binding domain and amino acids close to the C terminus, is essential for its protective function. C-terminal Pot1 fragments were found to exert a dominant-negative effect by displacing endogenous Pot1 from telomeres. Reducing telomere-bound Pot1 in this manner resulted in dramatic lengthening of the telomere tract. Upon further reduction of Pot1 at telomeres, the opposite phenotype was observed: loss of telomeric DNA and chromosome end fusions. Our results demonstrate that cells must carefully regulate the amount of telomere-bound Pot1 to differentiate between allowing access to telomerase and catastrophic loss of telomeres.

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Itriago, Humberto, Rishi K. Jaiswal, Susanne Philipp, and Marita Cohn. "The telomeric 5′ end nucleotide is regulated in the budding yeast Naumovozyma castellii." Nucleic Acids Research 50, no. 1 (December 15, 2021): 281–92. http://dx.doi.org/10.1093/nar/gkab1229.

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Abstract The junction between the double-stranded and single-stranded telomeric DNA (ds–ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5′ end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5′ end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5′ end, indicating that not all ds-ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds–ss junction ensures the protection of both 5′ ends and 3′ overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.

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Palacios, Jose A., Daniel Herranz, Maria Luigia De Bonis, Susana Velasco, Manuel Serrano, and Maria A. Blasco. "SIRT1 contributes to telomere maintenance and augments global homologous recombination." Journal of Cell Biology 191, no. 7 (December 27, 2010): 1299–313. http://dx.doi.org/10.1083/jcb.201005160.

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Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1super) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies.

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Yu, Eun Young, Wei-Feng Yen, Olga Steinberg-Neifach, and Neal F. Lue. "Rap1 in Candida albicans: an Unusual Structural Organization and a Critical Function in Suppressing Telomere Recombination." Molecular and Cellular Biology 30, no. 5 (December 14, 2009): 1254–68. http://dx.doi.org/10.1128/mcb.00986-09.

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ABSTRACT Rap1 (repressor activator protein 1) is a conserved multifunctional protein initially identified as a transcriptional regulator of ribosomal protein genes in Saccharomyces cerevisiae but subsequently shown to play diverse functions at multiple chromosomal loci, including telomeres. The function of Rap1 appears to be evolutionarily plastic, especially in the budding yeast lineages. We report here our biochemical and molecular genetic characterizations of Candida albicans Rap1, which exhibits an unusual, miniaturized domain organization in comparison to the S. cerevisiae homologue. We show that in contrast to S. cerevisiae, C. albicans RAP1 is not essential for cell viability but is critical for maintaining normal telomere length and structure. The rap1 null mutant exhibits drastic telomere-length dysregulation and accumulates high levels of telomere circles, which can be largely attributed to aberrant recombination activities at telomeres. Analysis of combination mutants indicates that Rap1 and other telomere proteins mediate overlapping but nonredundant roles in telomere protection. Consistent with the telomere phenotypes of the mutant, C. albicans Rap1 is localized to telomeres in vivo and recognizes the unusual telomere repeat unit with high affinity and sequence specificity in vitro. The DNA-binding Myb domain of C. albicans Rap1 is sufficient to suppress most of the telomere aberrations observed in the null mutant. Notably, we were unable to detect specific binding of C. albicans Rap1 to gene promoters in vivo or in vitro, suggesting that its functions are more circumscribed in this organism. Our findings provide insights on the evolution and mechanistic plasticity of a widely conserved and functionally critical telomere component.

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Yang, Qin, Yun-Ling Zheng, and Curtis C. Harris. "POT1 and TRF2 Cooperate To Maintain Telomeric Integrity." Molecular and Cellular Biology 25, no. 3 (February 1, 2005): 1070–80. http://dx.doi.org/10.1128/mcb.25.3.1070-1080.2005.

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ABSTRACT Mammalian telomeric DNA contains duplex TTAGGG repeats and single-stranded overhangs. POT1 (protection of telomeres 1) is a telomere-specific single-stranded DNA-binding protein, highly conserved in eukaryotes. The biological function of human POT1 is not well understood. In the present study, we demonstrate that POT1 plays a key role in telomeric end protection. The reduction of POT1 by RNA interference led to the loss of telomeric single-stranded overhangs and induced apoptosis, chromosomal instability, and senescence in cells. POT1 and TRF2 interacted with each other to form a complex with telomeric DNA. A dominant negative TRF2, TRF2ΔBΔM, bound to POT1 and prevented it from binding to telomeres. POT1 overexpression protected against TRF2ΔBΔM-induced loss of telomeric single-stranded overhangs, chromosomal instability, and senescence. These results demonstrate that POT1 and TRF2 share in part in the same pathway for telomere capping and suggest that POT1 binds to the telomeric single-stranded DNA in the D-loop and cooperates with TRF2 in t-loop maintenance.

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Maddar, Haggar, Nir Ratzkovsky, and Anat Krauskopf. "Role for Telomere Cap Structure in Meiosis." Molecular Biology of the Cell 12, no. 10 (October 2001): 3191–203. http://dx.doi.org/10.1091/mbc.12.10.3191.

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Telomeres, the natural ends of eukaryotic chromosomes, are essential for the protection of chromosomes from end-to-end fusions, recombination, and shortening. Here we explore their role in the process of meiotic division in the budding yeast, Kluyveromyces lactis. Telomerase RNA mutants that cause unusually long telomeres with deregulated structure led to severely defective meiosis. The severity of the meiotic phenotype of two mutants correlated with the degree of loss of binding of the telomere binding protein Rap1p. We show that telomere size and the extent of potential Rap1p binding to the entire telomere are irrelevant to the process of meiosis. Moreover, we demonstrate that extreme difference in telomere size between two homologous chromosomes is compatible with the normal function of telomeres during meiosis. In contrast, the structure of the most terminal telomeric repeats is critical for normal meiosis. Our results demonstrate that telomeres play a critical role during meiotic division and that their terminal cap structure is essential for this role.

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Ge, Yunhui, Zhenfang Wu, Hongwen Chen, Qinglu Zhong, Shaohua Shi, Guohui Li, Jian Wu, and Ming Lei. "Structural insights into telomere protection and homeostasis regulation by yeast CST complex." Nature Structural & Molecular Biology 27, no. 8 (July 13, 2020): 752–62. http://dx.doi.org/10.1038/s41594-020-0459-8.

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Baumann, Peter, and Thomas R. Cech. "Protection of Telomeres by the Ku Protein in Fission Yeast." Molecular Biology of the Cell 11, no. 10 (October 2000): 3265–75. http://dx.doi.org/10.1091/mbc.11.10.3265.

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Schizosaccharomyces pombe cells survive loss of telomeres by a unique pathway of chromosome circularization. Factors potentially involved in this survival mechanism include the heterodimeric Ku protein and ligase IV, both of which are involved in the repair of DNA double-strand breaks in mammalian cells. Furthermore, Ku plays a role in telomere maintenance as well as in DNA double-strand break repair in Saccharomyces cerevisiae. We have identified Ku and ligase IV homologues in S. pombe and analyzed their functions during normal growth and in cells undergoing senescence. In the absence of either a Ku subunit (pku70 +) or ligase IV (lig4 +), nonhomologous DNA end-joining was severely reduced. Lack of functional Ku led to shorter but stable telomeres and caused striking rearrangements of telomere-associated sequences, indicating a function for Ku in inhibiting recombinational activities near chromosome ends. In contrast to S. cerevisiae, concurrent deletion ofpku70 + and the gene for the catalytic subunit of telomerase (trt1 +) was not lethal, allowing for the first time the dissection of the roles of Ku during senescence. Our results support a model in which Ku protects chromosome termini from nucleolytic and recombinational activities but is not involved in the formation of chromosome end fusions during senescence. The conclusion that nonhomologous end-joining is not required for chromosome circularization was further supported by analysis of survivors in strains lacking the genes for bothtrt1 + and lig4 +.

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Baumann, Peter, Elaine Podell, and Thomas R. Cech. "Human Pot1 (Protection of Telomeres) Protein: Cytolocalization, Gene Structure, and Alternative Splicing." Molecular and Cellular Biology 22, no. 22 (November 15, 2002): 8079–87. http://dx.doi.org/10.1128/mcb.22.22.8079-8087.2002.

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ABSTRACT Fission yeast Pot1 (protection of telomeres) is a single-stranded telomeric DNA binding protein with a critical role in ensuring chromosome stability. A putative human homolog (hPot1) was previously identified, based on moderate sequence similarity with fission yeast Pot1 and telomere end-binding proteins from ciliated protozoa. Using indirect immunofluorescence, we show here that epitope-tagged hPot1 localizes to telomeres in interphase nuclei of human cells, consistent with a direct role in telomere end protection. The hPOT1 gene contains 22 exons, most of which are present in all cDNAs examined. However, four exons are subject to exon skipping in some transcripts, giving rise to five splice variants. Four of these are ubiquitously expressed, whereas the fifth appears to be specific to leukocytes. The resultant proteins vary significantly in their ability to form complexes with single-stranded telomeric DNA as judged by electrophoretic mobility shift assays. In addition to these splice variants, the Pot1 family is expanded by the identification of six more genes from diverse species. Pot1-like proteins have now been found in plants, animals, yeasts, and microsporidia.

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McNees, Carolyn J., Agueda M. Tejera, Paula Martínez, Matilde Murga, Francisca Mulero, Oscar Fernandez-Capetillo, and Maria A. Blasco. "ATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase." Journal of Cell Biology 188, no. 5 (March 8, 2010): 639–52. http://dx.doi.org/10.1083/jcb.200908136.

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Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability.

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Dickey, Thayne H., and Deborah S. Wuttke. "The telomeric protein Pot1 from Schizosaccharomyces pombe binds ssDNA in two modes with differing 3′ end availability." Nucleic Acids Research 42, no. 15 (July 29, 2014): 9656–65. http://dx.doi.org/10.1093/nar/gku680.

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Abstract Telomere protection and length regulation are important processes for aging, cancer and several other diseases. At the heart of these processes lies the single-stranded DNA (ssDNA)-binding protein Pot1, a component of the telomere maintenance complex shelterin, which is present in species ranging from fission yeast to humans. Pot1 contains a dual OB-fold DNA-binding domain (DBD) that fully confers its high affinity for telomeric ssDNA. Studies of S. pombe Pot1-DBD and its individual OB-fold domains revealed a complex non-additive behavior of the two OB-folds in the context of the complete Pot1 protein. This behavior includes the use of multiple distinct binding modes and an ability to form higher order complexes. Here we use NMR and biochemical techniques to investigate the structural features of the complete Pot1-DBD. These experiments reveal one binding mode characterized by only subtle alternations to the individual OB-fold subdomain structures, resulting in an inaccessible 3′ end of the ssDNA. The second binding mode, which has equivalent affinity, interacts differently with the 3′ end, rendering it available for interaction with other proteins. These findings suggest a structural switch that contributes to telomere end-protection and length regulation.

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Liu, Jinqiang, Xichan Hu, Kehan Bao, Jin-Kwang Kim, Catherine Zhang, Songtao Jia, and Feng Qiao. "The cooperative assembly of shelterin bridge provides a kinetic gateway that controls telomere length homeostasis." Nucleic Acids Research 49, no. 14 (July 13, 2021): 8110–19. http://dx.doi.org/10.1093/nar/gkab550.

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Abstract Shelterin is a six-protein complex that coats chromosome ends to ensure their proper protection and maintenance. Similar to the human shelterin, fission yeast shelterin is composed of telomeric double- and single-stranded DNA-binding proteins, Taz1 and Pot1, respectively, bridged by Rap1, Poz1 and Tpz1. The assembly of the proteinaceous Tpz1-Poz1-Rap1 complex occurs cooperatively and disruption of this shelterin bridge leads to unregulated telomere elongation. However, how this biophysical property of bridge assembly is integrated into shelterin function is not known. Here, utilizing synthetic bridges with a range of binding properties, we find that synthetic shelterin bridge lacking cooperativity requires a linker pair that matches the native bridge in complex lifespan but has dramatically higher affinity. We find that cooperative assembly confers kinetic properties on the shelterin bridge allowing disassembly to function as a molecular timer, regulating the duration of the telomere open state, and consequently telomere lengthening to achieve a defined species-specific length range.

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Sui, Jiang-Dong, Zheng Tang, Benjamin P. C. Chen, Ping Huang, Meng-Qi Yang, Nuo-Han Wang, Hao-Nan Yang, et al. "Protein Phosphatase 2A–Dependent Mitotic hnRNPA1 Dephosphorylation and TERRA Formation Facilitate Telomere Capping." Molecular Cancer Research 20, no. 4 (December 21, 2021): 583–95. http://dx.doi.org/10.1158/1541-7786.mcr-21-0581.

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Abstract The heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), telomeric repeat-containing RNA (TERRA), and protection of telomeres 1 (POT1) have been reported to orchestrate to displace replication protein A (RPA) from telomeric overhangs, ensuring orderly telomere replication and capping. Our previous studies further demonstrated that DNA-dependent protein kinase catalytic subunit (DNA-PKcs)-dependent hnRNPA1 phosphorylation plays a crucial role in the promotion of hnRNPA1 binding to telomeric overhangs and RPA displacement during G2–M phases. However, it is unclear that how the subsequent exchange between hnRNPA1 and POT1 is orchestrated. Here we report that the protein phosphatase 2A (PP2A) depends on its scaffold subunit, which is called PPP2R1A, to interact with and dephosphorylate hnRNPA1 in the late M phase. Furthermore, PP2A-mediated hnRNPA1 dephosphorylation and TERRA accumulation act in concert to promote the hnRNPA1-to-POT1 switch on telomeric single-stranded DNA. Consequently, defective PPP2R1A results in ataxia telangiectasia and Rad3-related (ATR)-mediated DNA damage response at telomeres as well as induction of fragile telomeres. Combined inhibition of ATR and PP2A induces entry into a catastrophic mitosis and leads to synthetic lethality of tumor cells. In addition, PPP2R1A levels correlate with clinical stages and prognosis of multiple types of cancers. Taken together, our results indicate that PP2A is critical for telomere maintenance. Implications: This study demonstrates that the PP2A-dependent hnRNPA1 dephosphorylation and TERRA accumulation facilitates the formation of the protective capping structure of newly replicated telomeres, thus exerting essential oncogenic role in tumorigenesis.

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Negrini, S., V. Ribaud, A. Bianchi, and D. Shore. "DNA breaks are masked by multiple Rap1 binding in yeast: implications for telomere capping and telomerase regulation." Genes & Development 21, no. 3 (February 1, 2007): 292–302. http://dx.doi.org/10.1101/gad.400907.

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Dubruille, Raphaëlle, Gabriel A. B. Marais, and Benjamin Loppin. "Repeated Evolution of Testis-Specific New Genes: The Case of Telomere-Capping Genes in Drosophila." International Journal of Evolutionary Biology 2012 (July 11, 2012): 1–11. http://dx.doi.org/10.1155/2012/708980.

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Comparative genome analysis has allowed the identification of various mechanisms involved in gene birth. However, understanding the evolutionary forces driving new gene origination still represents a major challenge. In particular, an intriguing and not yet fully understood trend has emerged from the study of new genes: many of them show a testis-specific expression pattern, which has remained poorly understood. Here we review the case of such a new gene, which involves a telomere-capping gene family in Drosophila. hiphop and its testis-specific paralog K81 are critical for the protection of chromosome ends in somatic cells and male gametes, respectively. Two independent functional studies recently proposed that these genes evolved under a reproductive-subfunctionalization regime. The 2011 release of new Drosophila genome sequences from the melanogaster group of species allowed us to deepen our phylogenetic analysis of the hiphop/K81 family. This work reveals an unsuspected dynamic of gene birth and death within the group, with recurrent duplication events through retroposition mechanisms. Finally, we discuss the plausibility of different evolutionary scenarios that could explain the diversification of this gene family.

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Grandin, Nathalie, and Michel Charbonneau. "The Rad51 Pathway of Telomerase-Independent Maintenance of Telomeres Can Amplify TG1-3 Sequences in yku and cdc13 Mutants of Saccharomyces cerevisiae." Molecular and Cellular Biology 23, no. 11 (June 1, 2003): 3721–34. http://dx.doi.org/10.1128/mcb.23.11.3721-3734.2003.

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ABSTRACT In the yeast Saccharomyces cerevisiae, Cdc13, Yku, and telomerase define three parallel pathways for telomere end protection that prevent chromosome instability and death by senescence. We report here that cdc13-1 yku70Δ mutants generated telomere deprotection-resistant cells that, in contrast with telomerase-negative senescent cells, did not display classical crisis events. cdc13-1 yku70Δ cells survived telomere deprotection by exclusively amplifying TG1-3 repeats (type II recombination). In a background lacking telomerase (tlc1Δ), this process predominated over type I recombination (amplification of subtelomeric Y′ sequences). Strikingly, inactivation of the Rad50/Rad59 pathway (which is normally required for type II recombination) in cdc13-1 yku70Δ or yku70Δ tlc1Δ mutants, but also in cdc13-1 YKU70+ tlc1Δ mutants, still permitted type II recombination, but this process was now entirely dependent on the Rad51 pathway. In addition, delayed senescence was observed in cdc13-1 yku70Δ rad51Δ and cdc13-1 tlc1Δ rad51Δ cells. These results demonstrate that in wild-type cells, masking by Cdc13 and Yku prevents the Rad51 pathway from amplifying telomeric TG1-3 sequences. They also suggest that Rad51 is more efficient than Rad50 in amplifying the sequences left uncovered by the absence of Cdc13 or Yku70.

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Ngo, Hien-Ping, and David Lydall. "Survival and Growth of Yeast without Telomere Capping by Cdc13 in the Absence of Sgs1, Exo1, and Rad9." PLoS Genetics 6, no. 8 (August 19, 2010): e1001072. http://dx.doi.org/10.1371/journal.pgen.1001072.

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Malyavko, Alexander N., and Olga A. Dontsova. "The telomeric Cdc13 protein from yeast Hansenula polymorpha." Acta Naturae 12, no. 1 (April 16, 2020): 84–88. http://dx.doi.org/10.32607/actanaturae.10944.

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Telomeres are special structures at the ends of chromosomes that play an important role in the protection of the genetic material. Telomere composition is very diverse; noticeable differences can often be observed even among closely related species. Here, we identify the homolog of telomeric protein Cdc13 in the thermotolerant yeast Hansenula polymorpha. We show that it can specifically bind single-stranded telomeric DNA, as well as interact with the Stn1 protein. In addition, we have uncovered an interaction between Cdc13 and TERT (one of the core components of the telomerase complex), which suggests that Cdc13 is potentially involved in telomerase recruitment to telomeres in H. polymorpha.

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DA SILVEIRA, RITA DE CÁSSIA VIVEIROS, MARCELO SANTOS DA SILVA, VINÍCIUS SANTANA NUNES, ARINA MARINA PEREZ, and MARIA ISABEL NOGUEIRA CANO. "The natural absence of RPA1N domain did not impair Leishmania amazonensis RPA-1 participation in DNA damage response and telomere protection." Parasitology 140, no. 4 (February 7, 2013): 547–59. http://dx.doi.org/10.1017/s0031182012002028.

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SUMMARYWe have previously shown that the subunit 1 of Leishmania amazonensis RPA (LaRPA-1) alone binds the G-rich telomeric strand and is structurally different from other RPA-1. It is analogous to telomere end-binding proteins described in model eukaryotes whose homologues were not identified in the protozoan´s genome. Here we show that LaRPA-1 is involved with damage response and telomere protection although it lacks the RPA1N domain involved with the binding with multiple checkpoint proteins. We induced DNA double-strand breaks (DSBs) in Leishmania using phleomycin. Damage was confirmed by TUNEL-positive nuclei and triggered a G1/S cell cycle arrest that was accompanied by nuclear accumulation of LaRPA-1 and RAD51 in the S phase of hydroxyurea-synchronized parasites. DSBs also increased the levels of RAD51 in non-synchronized parasites and of LaRPA-1 and RAD51 in the S phase of synchronized cells. More LaRPA-1 appeared immunoprecipitating telomeres in vivo and associated in a complex containing RAD51, although this interaction needs more investigation. RAD51 apparently co-localized with few telomeric clusters but it did not immunoprecipitate telomeric DNA. These findings suggest that LaRPA-1 and RAD51 work together in response to DNA DSBs and at telomeres, upon damage, LaRPA-1 works probably to prevent loss of single-stranded DNA and to assume a capping function.

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Bertuch, Alison A., and Victoria Lundblad. "The Ku Heterodimer Performs Separable Activities at Double-Strand Breaks and Chromosome Termini." Molecular and Cellular Biology 23, no. 22 (November 15, 2003): 8202–15. http://dx.doi.org/10.1128/mcb.23.22.8202-8215.2003.

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ABSTRACT The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80tel alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80tel alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.

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Journal articles on the topic 'Telomere protection, Telomere capping, Yeast'

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