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Title: The checkpoint role of Cdc18 in fission yeast
Author: Fersht, N.
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 2007
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The highly conserved eukaryotic checkpoints keep tight control over cell cycle progression, arresting the cell in response to incomplete DNA replication or DNA damage. In fission yeast, Rad3 (functional homologue of ATM, mutated in ataxia telangiectasia, and structural homologue of ATR, ataxia telangiectasia and rad3 related) is necessary for activation of both replication and damage checkpoints. However, despite the identification of many checkpoint genes, the actual sequence of upstream events leading to Rad3 activation remains unclear. The aim of my project was to identify and characterise the Rad3- dependent DNA damage/perturbed replication sensors and checkpoint activators. A genetic screen was carried out in fission yeast, using the working hypothesis that overexpression of these sensors/checkpoint activators would ectopically induce a Rad3-dependent block over mitosis in the absence of DNA damage or disturbed replication. The screen identified several genes of which the DNA replication initiation factor Cdc18/CDC6 had the strongest and most reproducible phenotype. Cdc18 is essential to prevent mitosis during S phase. I chose to concentrate on characterisation of the Rad3-dependent checkpoint role of Cdc18. The actual level of Cdc18 is important for producing the Rad3-dependent cell cycle block. A stabilised Cdc18 protein, mutated at the conserved CDK (cyclin dependent kinase) consensus sites also caused a Rad3-dependent cell cycle arrest, and was more stable and easier to manipulate than the screen- derived clone. Genetic crosses demonstrated Cdc18 acts early on in the checkpoint pathway, and through Crb2/Chk1. There was no gross DNA damage or detectable replication intermediates in the presence of elevated or stabilised Cdc18 levels. I also found that artificial depletion of Cdc18 during an S phase block results in loss of the checkpoint but not the replication structures, uncoupling the maintenance of replication forks from the maintenance of the mitotic block. An unexpected consequence of Cdc18 stabilisation was an increase in the size and variability of chromosome III on pulsed field gel electrophoresis. This localised to an expansion of Sfi1 restriction fragments containing the rDNA repeats. In conclusion, Cdc18 stabilisation activates a Rad3-dependent checkpoint in the absence of apparent re-replication, which is associated with an expansion of the rDNA repeats on chromosome III. Two models are proposed. In the first, Cdc18 induces low level genome wide replication, that is undetectable but sufficient for checkpoint activation. This leads to increased recombination with unequal crossover events in the rDNA repeats on chromosome III, with subsequent repeat expansion. In the second, the increased levels of Cdc18 directly activate the cell cycle checkpoint independently of the concurrent expansion of chromosome III.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available