Shelterin-Like Proteins and Yku Inhibit Nucleolytic Processing of Saccharomyces cerevisiae Telomeres

Bonetti, Diego; Clerici, Michela; Anbalagan, Savani; Martina, Marina; Lucchini, Giovanna; Longhese, Maria Pia

doi:10.1371/journal.pgen.1000966

Cited by 96 publications

(159 citation statements)

References 43 publications

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“…Resection is almost completely inhibited in angiosperm plants, resulting in telomeres with no or only a few-nucleotide long G-overhangs. Studies in budding yeast demonstrated that Ku physically associates with and protects chromosome termini in nondividing cells (Bonetti et al 2010;Vodenicharov et al 2010) and that telomeric function requires direct loading of Ku onto DNA through a circular channel in its structure (Lopez et al 2011;Pfingsten et al 2012). Blunt-ended telomeres generated by the leading strand replication are perfect substrates for Ku loading, which may be occluded after resection by proteins that bind to ssDNA (Faure et al 2010).…”

Section: Discussionmentioning

confidence: 99%

Chromosome end protection by blunt-ended telomeres

Kazda¹,

Zellinger²,

Rössler³

et al. 2012

Genes Dev.

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Single-stranded telomeric DNA protrusions are considered to be evolutionarily conserved structural elements essential for chromosome end protection. Their formation at telomeres replicated by the leading strand mechanism is thought to involve poorly understood post-replicative processing of blunt ends. Unexpectedly, we found that angiosperm plants contain blunt-ended and short (1-to 3-nucleotide) G-overhang-containing telomeres that are stably retained in post-mitotic tissues, revealing a novel mechanism of chromosome end protection. The integrity of blunt-ended telomeres depends on the Ku70/80 heterodimer but not on another telomere capping protein, STN1. Curiously, Ku-depleted telomeres are fully functional. They are resected by exonuclease 1, promoting intrachromatid recombination, which may facilitate formation of an alternative capping structure. These data challenge the view that telomeres require ssDNA protrusions for forming a functional capping structure and demonstrate flexibility in solutions to the chromosome end protection problem.

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Section: Discussionmentioning

confidence: 99%

Chromosome end protection by blunt-ended telomeres

Kazda¹,

Zellinger²,

Rössler³

et al. 2012

Genes Dev.

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“…The same gel was denatured and hybridized with the end-labeled C-rich oligonucleotide for loading control. Resection of the 5= strand at the HO-induced DSB adjacent to telomeric repeat sequences was monitored as previously described (7). To monitor resection at the HO-induced DSB, SspI-and XbaI-digested genomic DNA was hybridized with a single-stranded riboprobe, which anneals to the 5= strand to a site located 248 nt from the HO cutting site.…”

Section: Strains Strain Genotypes Are Listed Inmentioning

confidence: 99%

“…Rif2 physically interacts with MRX in vitro (26) and inhibits MRXdependent 5=-end resection at telomeres (6,7). Furthermore, the lack of Rif2 enhances MRX and Tel1 association at telomeres (7,26), suggesting that Rif2 regulates nucleolytic processing of telomeres by inhibiting MRX recruitment. However, the artificial tethering of Rif2 at DNA ends leads to decreased Tel1 binding, but not MRX binding, to these ends (26), indicating that Rif2 counteracts Tel1 association to DNA ends.…”

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confidence: 99%

A Balance between Tel1 and Rif2 Activities Regulates Nucleolytic Processing and Elongation at Telomeres

et al. 2012

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Generation of G-strand overhangs at Saccharomyces cerevisiae yeast telomeres depends primarily on the MRX (Mre11-Rad50-Xrs2) complex, which is also necessary to maintain telomere length by recruiting the Tel1 kinase. MRX physically interacts with Rif2, which inhibits both resection and elongation of telomeres. We provide evidence that regulation of telomere processing and elongation relies on a balance between Tel1 and Rif2 activities. Tel1 regulates telomere nucleolytic processing by promoting MRX activity. In fact, the lack of Tel1 impairs MRX-dependent telomere resection, which is instead enhanced by the Tel1-hy909 mutant variant, which causes telomerase-dependent telomere overelongation. The Tel1-hy909 variant is more robustly associated than wild-type Tel1 to double-strand-break (DSB) ends carrying telomeric repeat sequences. Furthermore, it increases the persistence at a DSB adjacent to telomeric repeats of both MRX and Est1, which in turn likely account for the increased telomere resection and elongation in TEL1-hy909 cells. Strikingly, Rif2 is unable to negatively regulate processing and lengthening at TEL1-hy909 telomeres, indicating that the Tel1-hy909 variant overcomes the inhibitory activity exerted by Rif2 on MRX. Altogether, these findings highlight a primary role of Tel1 in overcoming Rif2-dependent negative regulation of MRX activity in telomere resection and elongation.A highly ordered nucleoprotein complex called the telomere prevents the ends of linear chromosomes from being recognized as DNA double-strand breaks (DSBs) (reviewed in reference 30). Another key function of telomeres is to compensate for the incomplete replication of chromosome ends caused by discontinuous DNA synthesis. In most eukaryotes, telomeric DNA comprises tandemly repeated G-rich sequences (TG 1-3 repeats in Saccharomyces cerevisiae yeast and T 2 AG 3 in vertebrates), ending in a single-stranded 3= overhang (G tail) (reviewed in reference 20). The addition of telomeric repeats depends on the action of telomerase, a specialized reverse transcriptase that extends the TGrich strand of chromosome ends (23). The yeast telomerase complex consists of a reverse transcriptase subunit (Est2), a template RNA (TLC1), and two accessory proteins (Est1 and Est3), which are required for telomerase activity in vivo but not in vitro.The single-stranded G tails of budding yeast telomeres are short (about 10 to 15 nucleotides [nt]) for most of the cell cycle, but their length increases transiently to 50 to 100 nucleotides in late S phase (15, 50). While telomeric G tails can be generated during lagging-strand replication by removal of the last RNA primer, the 5= C strand of the telomere generated by leadingstrand synthesis must be nucleolytically processed (resected) to generate 3= overhangs (32, 51). Cyclin-dependent kinase (Cdk1 in S. cerevisiae) activity is required for this telomeric C-strand resection (17, 49), which occurs only during S and G 2 cell cycle phases, when Cdk1 activity is high and telomeres are elongated by telomerase (36...

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“…In support of this idea, recent data show that Rif1 and Rif2 inhibit the nucleolytic processing of telomeres in G1 and G2 phases of the cell cycle. 37 However, it remains to be determined whether the observed T-Recs in rif1/2 cells indeed form outside of S phase or are formed in S phase, never dissolve, and therefore persist after S phase. (Fig.…”

Section: Telomerase In the Act: Divided We Fallmentioning

confidence: 99%

Telomerase caught in the act

et al. 2012

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T he stable linearity of eukaryotic chromosomes depends on special characteristics of their ends, the telomeres. Accurate telomere function in turn requires a sustained presence of repeated DNA elements, which are maintained by the enzyme telomerase. The telomerase holoenzyme is composed of both protein and RNA, and its functions rely on proper expression, maturation, trafficking and assembly of these components. Conflicting models for the recruitment of telomerase at telomeres have been proposed; one suggests a local activation of telomerase at short telomeres, while the other proposes that telomerase is recruited only at short telomeres. To discriminate between these models and investigate the cell cycle-dependent regulation of telomerase in living cells, a GFP reporter system to visualize the yeast telomerase RNA has been recently developed. This assay shed new light on the mechanism of recruitment of telomerase to telomeres, and it uncovered a hitherto unrecognized mechanism for restricting telomerase access to telomeres.

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Shelterin-Like Proteins and Yku Inhibit Nucleolytic Processing of Saccharomyces cerevisiae Telomeres

Cited by 96 publications

References 43 publications

Chromosome end protection by blunt-ended telomeres

Chromosome end protection by blunt-ended telomeres

A Balance between Tel1 and Rif2 Activities Regulates Nucleolytic Processing and Elongation at Telomeres

Telomerase caught in the act

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