The impact of dysfunctions in telomere capping on ageing and cellular senescence

Shaheen, Noshina (2015) The impact of dysfunctions in telomere capping on ageing and cellular senescence. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

There is increased evidence of the important roles of telomere capping proteins Cdc13 and Stn1 for cell survival in budding yeast (Saccharomyces cerevisiae). Dysfunctional telomeres due to alterations in telomere capping proteins activate DNA damage response and genomic instability causing cellular senescence and eventually cell death. The main aim of this study was to investigate the roles of Cdc13 and Stn1 in ageing and cellular senescence. Based on the phenomenon of non-essentiality of CDC13 gene in the absence of EXO1 gene, CDC13 full gene deletion and its N-and C-termini disrupted variants were generated using target oriented PCR based approach and verified by automated DNA sequencing. Loss of Cdc13 and/or Stn1 or the presence of defective Cdc13 with truncated N- or C-termini, manifested heterogeneous telomeric DNA profiles with various sizes of unique telomeric bands comparable to type I and type II survivors. The mutants with telomere capping dysfunctions in the absence of Cdc13 and/or Stn1 disclosed significant differences in survival time under different metabolic conditions. The reduction in viability of the combined deletion mutants (cdc13Δ and stn1Δ) was much slower in water at 4°C compared to synthetic complete (SC) medium at 30°C, and a similar mortality rate observed at day 6 in (SC) medium was only observed at day 35 in water. The dramatic loss of viability in combined deletion mutant highlighted additive effects of the two capping proteins on chronological survival. The Stn1 deletion mutants (stn1::HIS exo1 LEU2 rad24::TRP) showed significantly reduced lifespan as compared to Cdc13 deletion mutants. However, the N- and C-truncated variants of Cdc13 manifested large differences in ageing as compared to WT, indicating different roles of the Cdc13 domains in cell survival. The strong inhibition of cellular growth of combined deletions mutants of cdc13 and stn1 (in the absence of EXO1 and RAD24 genes) in responses to genotoxic agents (UV, MMS and HU) suggest the interaction of cdc13Δ and stn1Δ cooperatively. However, the absence of Stn1 protein made cells highly resistant to H2O2-mediated oxidative stress as compared to the combined deletion and cdc13Δ mutants. Different extent of growth inhibition was observed for cdc13Δ mutants, and its N- and C-terminus truncated variants with the allele dosage effect in N/C-heterozygous diploids. In conclusion, this study highlighted the significance of Cdc13 and Stn1 in the integrity of telomeres and revealed their important roles in preventing senescence and in cellular responses to oxidative and genotoxic stress. The previously unknown role of Stn1 in chronological lifespan and in cellular responses to oxidative stress observed in this study warrants further investigations about Stn1 in genetic stability, checkpoint control and cellular senescence in the integrated scenario of ageing.