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Title: Mechanisms for maintaining the telomere cap in eukaryotic cells
Author: Holstein, Eva-Maria
ISNI:       0000 0004 2731 6750
Awarding Body: University of Newcastle Upon Tyne
Current Institution: University of Newcastle upon Tyne
Date of Award: 2012
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Telomeres are found at the end of linear eukaryotic chromosome ends and contribute to genetic stability. It is crucial that telomeres are not perceived and treated as doublestrand breaks by checkpoint and DNA repair proteins in order to prevent potentially lethal consequences, such as cellular ageing and cancer formation in mammalian cells (Murnane, 2006; Shin et al., 2006; Gilley et al., 2005). Hence, eukaryotic chromosome ends are masked by various telomere-binding proteins. Two crucial telomere capping proteins in Saccharomyces cerevisiae are Cdc13 and Yku70. Cdc13 forms a complex with Stn1 and Ten1 and binds specifically to single-stranded telomeric DNA (Grandin et al., 2001). Yku70 is a subunit of the Yku complex that is found at double-stranded telomeric DNA (Tuteja and Tuteja, 2000). A temperature-sensitive allele of CDC13, known as cdc13-1, or a null-mutation of YKU70 is used in this study to induce telomere uncapping and to study its consequences in vivo. This work looks into the puzzling observation that components of the nonsensemediated mRNA decay pathway (Upf1, Upf2, Upf3) suppress one form of telomere capping defect (yku70Δ), but enhance another (cdc13-1) (Addinall et al., 2011). The same effect can be seen in Ebs1, which has previously been linked to NMD (Azzalin et al., 2007). Here it is shown that Ebs1, like components of nonsense-mediated decay (NMD), regulate transcript levels of the two telomere binding proteins Stn1 and Ten1. Interestingly, increased levels of Stn1, but not Ten1, suppress the yku70Δ capping defect, but enhance the cdc13-1 capping defect, indicating that NMD and Ebs1 influence Cdc13- and Yku70-dependent telomere capping through modification of Stn1 levels. It is also shown that increase in Stn1 levels alters stochiometry of the Cdc13/Stn1/Ten1 (CST) complex. Cdc13 association to telomeres is significantly reduced at the presence of increased levels of Stn1. Inefficient telomere protection allows resection of telomeric DNA and the generation of ssDNA tracts triggers cell cycle arrest (Booth et al., 2001; Garvik et al., 1995; Lydall and Weinert, 1995; Weinert and Hartwell, 1993). It is shown that Upf2 and Ebs1 do not affect checkpoint activation in response to cdc13-1 uncapped telomeres, but are required for efficient G1 to S phase progression. Furthermore, Upf2 contributes to ssDNA generation at cdc13-1 uncapped telomeres in a parallel pathway to the exonuclease Exo1 and the checkpoint protein Rad24. Remarkably, elimination of Upf2 and Exo1 or Upf2 and Rad24 allows viability of cells in the absence of the otherwise essential telomere capping protein Cdc13.
Supervisor: Not available Sponsor: BBSRC ; ONDEX ; Wellcome Trust
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available