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Structural and functional studies of the budding yeast kinetochore complex CBF3

Leber, Vera; (2018) Structural and functional studies of the budding yeast kinetochore complex CBF3. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

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.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Structural and functional studies of the budding yeast kinetochore complex CBF3
Event: UCL
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2018. Original content in this thesis is licensed under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) Licence (https://creativecommons.org/licenses/by/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10064549
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