Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. In this work, we investigated the role of key telomeric proteins in protecting Saccharomyces cerevisiae telomeres from degradation. We 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 Yku 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 particular, while Yku plays the major role in preventing initiation, Rif2 and Rap1 act primarily by limiting extensive resection. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases. Since Rif1 plays a very minor role in protecting wild type telomeres from degradation, we further investigated whether Rif1 participates in telomere protection in combination with other capping activities, like those exerted by the CST complex (Cdc13-Stn1-Ten1). We found that, unlike RIF2 deletion, the lack of RIF1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, lethality in cdc13 rif1Δ cells is partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. Like CDC13, RIF1 also genetically interacts with the Polα-primase complex, which is involved in the fill-in of the telomeric complementary strand. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex.

(2013). Role of saccharomyces cerevisiae Rif1 and Rif2 proteins in protection of telomeres. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2013).

Role of saccharomyces cerevisiae Rif1 and Rif2 proteins in protection of telomeres

ANBALAGAN, SAVANI
2013-02-07

Abstract

Eukaryotic cells distinguish their chromosome ends from accidental DNA double-strand breaks (DSBs) by packaging them into protective structures called telomeres that prevent DNA repair/recombination activities. In this work, we investigated the role of key telomeric proteins in protecting Saccharomyces cerevisiae telomeres from degradation. We 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 Yku 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 particular, while Yku plays the major role in preventing initiation, Rif2 and Rap1 act primarily by limiting extensive resection. Finally, Rap1 and Rif2 prevent telomere degradation by inhibiting MRX access to telomeres, which are also protected from the Exo1 nuclease by Yku. Thus, chromosome end degradation is controlled by telomeric proteins that specifically inhibit the action of different nucleases. Since Rif1 plays a very minor role in protecting wild type telomeres from degradation, we further investigated whether Rif1 participates in telomere protection in combination with other capping activities, like those exerted by the CST complex (Cdc13-Stn1-Ten1). We found that, unlike RIF2 deletion, the lack of RIF1 is lethal for stn1ΔC cells and causes a dramatic reduction in viability of cdc13-1 and cdc13-5 mutants. Both cdc13-1 rif1Δ and cdc13-5 rif1Δ cells display very high amounts of telomeric single-stranded DNA and DNA damage checkpoint activation, indicating that severe defects in telomere integrity cause their loss of viability. In agreement with this hypothesis, lethality in cdc13 rif1Δ cells is partially counteracted by the lack of the Exo1 nuclease, which is involved in telomeric single-stranded DNA generation. Like CDC13, RIF1 also genetically interacts with the Polα-primase complex, which is involved in the fill-in of the telomeric complementary strand. Thus, these data highlight a novel role for Rif1 in assisting the essential telomere protection function of the CST complex.
LONGHESE, MARIA PIA
Telomere protection, Telomere capping, Yeast
BIO/18 - GENETICA
English
BIOTECNOLOGIE INDUSTRIALI - 15R
25
2011/2012
(2013). Role of saccharomyces cerevisiae Rif1 and Rif2 proteins in protection of telomeres. (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: http://hdl.handle.net/10281/43717
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