Structural and biochemical investigations into the anaphase-promoting complex regulation during mitosis
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Embargo End Date
2025-11-29
ICR Authors
Authors
Young, R
Document Type
Thesis or Dissertation
Date
2025-05-29
Date Accepted
Abstract
The APC/C is a master regulator of the cell cycle, it is an E3 ubiquitin ligase responsible for the ubiquitination of numerous cell cycle related proteins through mitosis and G1. A key mitotic substrate of the APC/C is cyclin B1, its
timely degradation at the metaphase to anaphase transition is necessary for sister chromatid separation. Because of its pivotal role in the cell cycle, and its numerous substrates, the activity of the APC/C is tightly regulated to ensure correct progression through the cell cycle. This regulation is achieved by several different mechanisms
acting directly on the APC/C, such as association with coactivators, inhibitors and E2 enzymes as well as PTMs
and differing substrate affinities. In addition to this the inhibitors, coactivators, substrates and E2 enzymes
themselves are also regulated in a cell cycle related manner.
In this thesis I explore some aspects of APC/C regulation further. First, I investigate the regulation of the APC/C via localisation. I use a combination of EMSAs and XL-MS to identify arginine anchor motifs in both the APC/C and in cyclin B1 which localise them to nucleosomes during mitosis. I also visualise the acidic patch interaction of both cyclin B1 and the APC/C using cryo-EM. I then use protein purification to show that the APC subunit involved in nucleosome localisation is also implicated in nuclear localisation via an interaction with importin-4 and have begun to structurally determine this interaction using cryo-EM. Finally, I have investigated the regulation of the APC/C by its mitotic inhibitor the MCC, specifically looking at an interaction interface formed by the CRY box of CDC20MCC, BubR1KNOT and CDC20APC/C. Mutations to this interface do not affect complex formation but do effect APC/C inhibition. I have also used an improved cryo-EM sample preparation pipeline to make cryo-EM grids
of the APC/CMCC and am attempting to improve the current resolution of the solved structure to resolve more of the sidechain interactions to improve our understanding of APC/C inhibition by the MCC.
Citation
2025
DOI
Source Title
Publisher
Institute of Cancer Research (University Of London)
ISSN
eISSN
Collections
Research Team
Mol mech cell cycle reg