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Title: Structural and functional studies of the budding yeast kinetochore complex CBF3
Author: Leber, Vera
ISNI:       0000 0004 7660 0467
Awarding Body: UCL (University College London)
Current Institution: University College London (University of London)
Date of Award: 2018
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Kinetochores connect centromeres with spindle microtubules during mitosis and meiosis to ensure correct chromosome segregation. The initial step of kinetochore establishment in point centromere species, like budding yeast, is the sequence-specific recognition of the centromere by the essential CBF3 complex. CBF3 consists of four proteins, Ndc10, Cep3, Ctf13 and Skp1, and its binding to the centromere is required for the assembly of all other kinetochore proteins. Beside this, the complex is involved in the recruitment of the centromere-specific histone Cse4 through its interaction with the histone chaperone Scm3 and it also impacts the overall conformation of the centromere through DNA looping and bending. Besides its crucial role in kinetochore establishment, little structural data is available for the CBF3 complex, mainly due to difficulties with recombinantly expressing and purifying the full complex. This thesis describes a working co-expression and purification protocol for CBF3, which allowed structural as well as functional studies of the complex and led to the cryoEM structure of the core CBF3 complex, comprising the centromere-binding Cep3 and the regulatory subunits Ctf13 and Skp1. Besides the overall core architecture, this structure provides the first insights into the inherently unstable subunit Ctf13, as well as a potential new conformation of the Skp1/F-box interaction. Furthermore, it provides interesting insights into the unusual DNA-binding properties of the dimeric Cep3 to a single consensus site in the centromere, a matter of some debate in the field. Biochemical studies revealed a potential and previously undescribed regulatory mechanism of DNA-binding activity through phosphorylation of the Skp1 subunit. Additional work was undertaken to better understand how Ndc10 binds to the core complex and therefore how the full CBF3 assembles, as well as interaction studies with the centromere-specific nucleosome. As a side project, interaction studies between Ndc10 and Scm3 were undertaken, to elucidate the proposed Cse4-loading function of CBF3.
Supervisor: Singleton, M. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available