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Title: Analysis of anaphase inhibitors in fission yeast
Author: Sczaniecka, Matylda
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2007
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As a result of checkpoint activation, a signalling cascade is initiated and a number of complexes between the checkpoint components are formed. This leads to the inhibition of the Anaphase Promoting Complex (APC), which is the ubiquitin ligase responsible for targeting mitotic proteins: securing and cyclin B for degradation by the 26S proteasome. The complexes formed include the MCC, or Mitotic Checkpoint Complex, which in fission yeast (Schizosaccharomyces pombe) consists of Mad2, Mad3 checkpoint proteins together with the APC activator, Slp1 (the Cdc20 homologue). The MCC has been shown to bind and inhibit the APC in HeLa cells. In my PhD I focused on the interactions between the MCC and the APC, in particular on Mad3 protein. Mad3 is a conserved checkpoint component, homologous to human BubR1. It carries 2 putative KEN boxes, motifs, which typically target proteins for degradation (like D-boxes). We mutated both KEN boxes in S. pombe Mad3 and show that they are essential for Mad3 checkpoint function. One of the two motifs is also required for MCC formation, MCC/APC binding and Mad3 turnover in G1 stage of cell cycle. We argue that KEN boxes mediate the inhibitory interactions between checkpoint proteins and the APC. The formation of the MCC requires Mad2 and Mad3. These proteins are also dependent on one another for binding to the APC. We study the formation of the MCC, as well as its binding to the APC in different checkpoint mutants, trying to understand dependencies between these proteins and the requirement for kinetochore localisation. Finally, we make an attempt to study the cellular localisation of S1p1 and the APC and find that while S1p1 colocalises with a kinetochore marker, the APC subunits Cut9 and Lid1 remain distributed around the fission yeast nucleus.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available