Molecular Mechanisms of Mitotic Checkpoint Complex Assembly onto Kinetochores
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Authors
Fischer, Elyse
Advisors
Barford, David
Date
2022-05-21Awarding Institution
University of Cambridge
Qualification
Doctor of Philosophy (PhD)
Type
Thesis
Metadata
Show full item recordCitation
Fischer, E. (2022). Molecular Mechanisms of Mitotic Checkpoint Complex Assembly onto Kinetochores (Doctoral thesis). https://doi.org/10.17863/CAM.80226
Abstract
During metaphase, in response to improper kinetochore-microtubule attachments, the spindle
assembly checkpoint (SAC), activates the mitotic checkpoint complex (MCC), to inhibit the
E3 ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C). Inhibition of the
APC/C then delays premature chromosome segregation by preventing APC/C-mediated
degradation of two key cell cycle regulators, cyclin B and securin. The MCC is composed of
BubR1, Cdc20 and Mad2, and while their assembly is an intrinsically very slow process, in
cells it is catalytically activated. Recent work points towards hierarchical recruitments of
SAC proteins onto the outer kinetochore by means of a Mps1-dependent phosphorylation
cascade, which creates a catalytic platform for MCC assembly.
This thesis investigates several mechanisms of catalytic MCC assembly in humans using a
combination of biochemical assays and structural biology. Chapter 3 uses X-ray
crystallography and NMR spectroscopy to explore the structure and function of the Bub1-
Mad1 complex, including how sequential phosphorylation of the Bub1 CD1 domain by Cdk1
and Mps1 promotes kinetochore targeting of the Mad1:C-Mad2 complex. Chapter 4
investigates how Mad1 C-terminal phosphorylation by Mps1 promotes juxtaposition of SAC
proteins for MCC assembly. This includes using NMR to gain detailed structural insights into
how phosphorylation of Mad1 promotes its interaction with both the N-terminus of Cdc20, as
well as a region within Bub1 just C-terminal to its CD1 domain. Chapter 5 investigates the
structure of the Mad1:C-Mad2:O-Mad2 complex by cryo-EM and reveals a mechanism of
Mad1CTD fold-over which has import implications for MCC assembly. Chapter 6 sets the
premise for ongoing work on the molecular mechanisms of Mad2 conversion from the open
to closed state by NMR.
Keywords
Bub1, Mad1, Mitotic Checkpoint Complex, Cdc20, Spindle Assembly Checkpoint, Mps1
Sponsorship
Elyse Fischer was funded by a Gates Cambridge Scholarship. This work was funded by MRC grant (MC_UP_1201/6) and CRUK grant (C576/A14109) to David Barford.
Identifiers
This record's DOI: https://doi.org/10.17863/CAM.80226
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