Telomeres protect the ends of chromosomes from the activity of DNA repair machinery and provide a solution to the end-replication problem. In humans, the core protein complex located at telomeres is known as shelterin and consists of six protein subunits. Although variation is seen in the telomeric complex between species, in fission yeast the complex has notable similarities to that of humans. Separately to shelterin, the CST complex (Cdc13/Stn1/Ten1) is conserved in budding yeast, plants and mammals and is thought to negatively regulate telomerase, in addition to being required for telomere protection. However, unlike Stn1 and Ten1, Cdc13 has not yet been identified in fission yeast. Poz1 is a bridging molecule equivalent to TIN2 in human shelterin, which links the Taz1-Rap1 and the Pot1-Tpz1-Ccq1 sub-complexes, respectively bound to double- and single-stranded DNA at telomeres. Poz1 is required for the regulation of telomerase activity, and it has been hypothesised that it might do so by playing a structural role in the switching of telomeres from an open to a closed state. In this study, a reverse-2-hybrid approach was used to generate Poz1 alleles unable to interact with Rap1 or Tpz1 specifically. These alleles were subjected to phenotypic and biochemical analysis which indicated that neither individual interaction is sufficient to maintain telomere homeostasis. With telomere lengths similar to a Poz1 deletion, it is proposed that negative regulation cannot occur without the ability to form a closed complex. Given that Cdc13 is currently the only missing component in fission yeast, a second study was initiated aiming to identify a homologue by yeast-2-hybrid screening of a cDNA library, using Stn1 and Ten1 as baits. However, this approach did not yield any positive candidates. In an alternative approach, Stn1 temperature-sensitive (ts) alleles were generated and characterised. These were used to screen a genomic library for suppressors of the Stn1 ts phenotype. Several candidates were identified that require further examination while the ts allele analysis indicated that telomeres are lost in their entirety at non-permissive temperatures and that survivors of this process did so by chromosome circularisation, similar to Pot1 mutants.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:606054 |
| Date | January 2014 |
| Creators | Ahmed, Jubed Omee |
| Publisher | University of Sussex |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://sro.sussex.ac.uk/id/eprint/48855/ |
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