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Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping.

Dewar JM, Lydall D - EMBO J. (2010)

Bottom Line: Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1.However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination.Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK.

ABSTRACT
Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.

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Exo1 and nuclear, helicase activity of Pif1 prevent telomere maintenance following inactivation of Cdc13. (A) Cartoon of yeast telomeres, indicating the fragments detected by telomere Southern blots using Y′ probe. Arrows represent XhoI cut sites. (B) Genomic DNA was prepared from two independent CDC13+ (+) or cdc13-1 (TS) strains, grown at 23 or 36°C, digested with XhoI and Southern blotted to detect telomeric Y′ and terminal fragments. Blots were reprobed to detect CDC15 as a loading control. Also see Supplementary Figure S4. (C) cdc13-1 exo1Δ mutants defective in nuclear Pif1 (pif1-m2), mitochondrial Pif1 (pif1-m1) or carrying the helicase-deficient allele of Pif1 (pif1-hd) were serially diluted across agar plates and grown at the temperatures indicated for 3 days.
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f2: Exo1 and nuclear, helicase activity of Pif1 prevent telomere maintenance following inactivation of Cdc13. (A) Cartoon of yeast telomeres, indicating the fragments detected by telomere Southern blots using Y′ probe. Arrows represent XhoI cut sites. (B) Genomic DNA was prepared from two independent CDC13+ (+) or cdc13-1 (TS) strains, grown at 23 or 36°C, digested with XhoI and Southern blotted to detect telomeric Y′ and terminal fragments. Blots were reprobed to detect CDC15 as a loading control. Also see Supplementary Figure S4. (C) cdc13-1 exo1Δ mutants defective in nuclear Pif1 (pif1-m2), mitochondrial Pif1 (pif1-m1) or carrying the helicase-deficient allele of Pif1 (pif1-hd) were serially diluted across agar plates and grown at the temperatures indicated for 3 days.

Mentions: Yeast cells can overcome the requirement for Cdc13 by altering telomere structure, as observed in rare variants, which can be selected for after inactivation of telomerase or after attenuation of nuclease/checkpoint activities at uncapped telomeres (Larrivee and Wellinger, 2006; Zubko and Lydall, 2006). To test whether elimination of Pif1 and Exo1 caused alterations in telomere structure that could explain the growth of cdc13-1 cells at 36°C, we performed Southern blots to examine telomere structure, probing for Y′ sequences (Figure 2B), which are components of the majority of yeast telomeres (Supplementary Figure S3A and B). The Y′ probe contained G-rich sequences and weakly cross-hybridized to telomeres that did not contain Y′ sequences, so we also probed for TG repeat sequences to detect telomeres that lacked Y′ elements (Supplementary Figure S4).


Pif1- and Exo1-dependent nucleases coordinate checkpoint activation following telomere uncapping.

Dewar JM, Lydall D - EMBO J. (2010)

Exo1 and nuclear, helicase activity of Pif1 prevent telomere maintenance following inactivation of Cdc13. (A) Cartoon of yeast telomeres, indicating the fragments detected by telomere Southern blots using Y′ probe. Arrows represent XhoI cut sites. (B) Genomic DNA was prepared from two independent CDC13+ (+) or cdc13-1 (TS) strains, grown at 23 or 36°C, digested with XhoI and Southern blotted to detect telomeric Y′ and terminal fragments. Blots were reprobed to detect CDC15 as a loading control. Also see Supplementary Figure S4. (C) cdc13-1 exo1Δ mutants defective in nuclear Pif1 (pif1-m2), mitochondrial Pif1 (pif1-m1) or carrying the helicase-deficient allele of Pif1 (pif1-hd) were serially diluted across agar plates and grown at the temperatures indicated for 3 days.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3020640&req=5

f2: Exo1 and nuclear, helicase activity of Pif1 prevent telomere maintenance following inactivation of Cdc13. (A) Cartoon of yeast telomeres, indicating the fragments detected by telomere Southern blots using Y′ probe. Arrows represent XhoI cut sites. (B) Genomic DNA was prepared from two independent CDC13+ (+) or cdc13-1 (TS) strains, grown at 23 or 36°C, digested with XhoI and Southern blotted to detect telomeric Y′ and terminal fragments. Blots were reprobed to detect CDC15 as a loading control. Also see Supplementary Figure S4. (C) cdc13-1 exo1Δ mutants defective in nuclear Pif1 (pif1-m2), mitochondrial Pif1 (pif1-m1) or carrying the helicase-deficient allele of Pif1 (pif1-hd) were serially diluted across agar plates and grown at the temperatures indicated for 3 days.
Mentions: Yeast cells can overcome the requirement for Cdc13 by altering telomere structure, as observed in rare variants, which can be selected for after inactivation of telomerase or after attenuation of nuclease/checkpoint activities at uncapped telomeres (Larrivee and Wellinger, 2006; Zubko and Lydall, 2006). To test whether elimination of Pif1 and Exo1 caused alterations in telomere structure that could explain the growth of cdc13-1 cells at 36°C, we performed Southern blots to examine telomere structure, probing for Y′ sequences (Figure 2B), which are components of the majority of yeast telomeres (Supplementary Figure S3A and B). The Y′ probe contained G-rich sequences and weakly cross-hybridized to telomeres that did not contain Y′ sequences, so we also probed for TG repeat sequences to detect telomeres that lacked Y′ elements (Supplementary Figure S4).

Bottom Line: Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1.However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination.Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrated Systems Biology of Ageing and Nutrition, Institute for Ageing and Health, Newcastle upon Tyne, Tyne-and-Wear, UK.

ABSTRACT
Essential telomere 'capping' proteins act as a safeguard against ageing and cancer by inhibiting the DNA damage response (DDR) and regulating telomerase recruitment, thus distinguishing telomeres from double-strand breaks (DSBs). Uncapped telomeres and unrepaired DSBs can both stimulate a potent DDR, leading to cell cycle arrest and cell death. Using the cdc13-1 mutation to conditionally 'uncap' telomeres in budding yeast, we show that the telomere capping protein Cdc13 protects telomeres from the activity of the helicase Pif1 and the exonuclease Exo1. Our data support a two-stage model for the DDR at uncapped telomeres; Pif1 and Exo1 resect telomeric DNA <5 kb from the chromosome end, stimulating weak checkpoint activation; resection is extended >5 kb by Exo1 and full checkpoint activation occurs. Cdc13 is also crucial for telomerase recruitment. However, cells lacking Cdc13, Pif1 and Exo1, do not senesce and maintain their telomeres in a manner dependent upon telomerase, Ku and homologous recombination. Thus, attenuation of the DDR at uncapped telomeres can circumvent the need for otherwise-essential telomere capping proteins.

Show MeSH
Related in: MedlinePlus