Telomeres are specialized nucleoprotein complexes that distinguish the natural ends of linear chromosomes from intrachromosomal double-strand breaks. In fact, telomeres are protected from DNA damage checkpoints, homologous recombination or end-to-end fusions that normally promote repair of intrachromosomal DNA breaks. When chromosome end protection fails, dysfunctional telomeres are targeted by the DNA repair and recombination apparatus, whose outcomes range from the generation of chromosomal abnormalities, general hallmarks for human cancer cells, to permanent cell cycle arrest and cell death. While several studies address the consequences of telomere dysfunctions, the mechanisms by which telomere protection is achieved remain to be determined. During my PhD, I contributed to investigate this issue by analyzing the role of evolutionarily conserved telomeric proteins in protecting budding yeast telomeres from degradation. In particular, the data obtained during the first year of my PhD show that the shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also the Yku complex prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres. In fact, while Yku plays the major role in preventing initiation, Rif2 and Rap1 act primarily by limiting extensive resection. In particular, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX (Mre11-Rad50-Xrs2) access to telomeres, which are also protected from the Exo1 nuclease by Yku. The MRX complex is also necessary to maintain telomere length by recruiting the Tel1 kinase, and Rif2 was recently shown to interact with the C-terminus of Xrs2. As also Tel1 binds the same portion of Xrs2, it has been proposed that Rif2 and Tel1 might compete with each other for binding to MRX, with Rif2 preventing Xrs2 interaction with Tel1. This issue was explored during the second part of my PhD by taking advantage of the TEL1-hy909 mutant allele, previously identified as a dominant suppressor of the hypersensitivity to genotoxic agents and checkpoint defects of Mec1-deficient cells (Baldo et al., 2008). The data obtained by this analysis provide evidence that regulation of telomere processing and elongation relies on a balance between Tel1 and Rif2 activities. In particular, Tel1 appears to regulate 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. Our data indicate that 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 of both the MRX complex and the telomerase subunit Est1 at a DSB adjacent to telomeric repeats, 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.

(2013). Roles of shelterin-like proteins and yku in saccharomyces cerevisiae telomere homeostasis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).

Roles of shelterin-like proteins and yku in saccharomyces cerevisiae telomere homeostasis

MARTINA, MARINA
2013

Abstract

Telomeres are specialized nucleoprotein complexes that distinguish the natural ends of linear chromosomes from intrachromosomal double-strand breaks. In fact, telomeres are protected from DNA damage checkpoints, homologous recombination or end-to-end fusions that normally promote repair of intrachromosomal DNA breaks. When chromosome end protection fails, dysfunctional telomeres are targeted by the DNA repair and recombination apparatus, whose outcomes range from the generation of chromosomal abnormalities, general hallmarks for human cancer cells, to permanent cell cycle arrest and cell death. While several studies address the consequences of telomere dysfunctions, the mechanisms by which telomere protection is achieved remain to be determined. During my PhD, I contributed to investigate this issue by analyzing the role of evolutionarily conserved telomeric proteins in protecting budding yeast telomeres from degradation. In particular, the data obtained during the first year of my PhD show that the shelterin-like proteins Rif1, Rif2, and Rap1 inhibit nucleolytic processing at both de novo and native telomeres during G1 and G2 cell cycle phases, with Rif2 and Rap1 showing the strongest effects. Also the Yku complex prevents telomere resection in G1, independently of its role in non-homologous end joining. Yku and the shelterin-like proteins have additive effects in inhibiting DNA degradation at G1 de novo telomeres. In fact, while Yku plays the major role in preventing initiation, Rif2 and Rap1 act primarily by limiting extensive resection. In particular, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX (Mre11-Rad50-Xrs2) access to telomeres, which are also protected from the Exo1 nuclease by Yku. The MRX complex is also necessary to maintain telomere length by recruiting the Tel1 kinase, and Rif2 was recently shown to interact with the C-terminus of Xrs2. As also Tel1 binds the same portion of Xrs2, it has been proposed that Rif2 and Tel1 might compete with each other for binding to MRX, with Rif2 preventing Xrs2 interaction with Tel1. This issue was explored during the second part of my PhD by taking advantage of the TEL1-hy909 mutant allele, previously identified as a dominant suppressor of the hypersensitivity to genotoxic agents and checkpoint defects of Mec1-deficient cells (Baldo et al., 2008). The data obtained by this analysis provide evidence that regulation of telomere processing and elongation relies on a balance between Tel1 and Rif2 activities. In particular, Tel1 appears to regulate 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. Our data indicate that 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 of both the MRX complex and the telomerase subunit Est1 at a DSB adjacent to telomeric repeats, 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.
LUCCHINI, GIOVANNA
Rif2, Tel1, telomere, yeast
BIO/18 - GENETICA
English
10-gen-2013
BIOLOGIA - 48R
25
2011/2012
open
(2013). Roles of shelterin-like proteins and yku in saccharomyces cerevisiae telomere homeostasis. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/41884
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