This thesis investigates the structure and function of the kinetochore centromere binding factor 3 (CBF3) complex. The kinetochore is a multi-protein complex, which controls the chromosome attachment to the mitotic or meiotic spindle and nucleates on the centromere. The latter is a specific chromosomal loci divided in two groups highly divergent in length and composition: regional and point centromeres. Regional centromeres are composed of long arrays of repetitive DNA supporting multiple microtubule nucleations. On the contrary, point centromeres are characterised by a short and conserved sequence supporting a single microtubule attachment. Despite this discrepancy, both point and regional centromere establishment start with the replacement of the histone H3 by the Cse4 histone variant. The point centromeres H3 replacement by Cse4 (loading) solely relies on the recognition of a conserved DNA sequence (licensing) by the CBF3 complex, a crucial centromere element composed by four essential proteins: Ndc10, Cep3, Ctf13 and Skp1. Ctf13 and Skp1 regulate the CBF3 assembly. Cep3, the licensing element, recognises the point centromere DNA sequence. Finally, Ndc10 acts as the loading factor of the CBF3 complex by recruiting Cse4. At the start of this work the structural basis underlying the mechanism of Cse4 loading by Ndc10 was speculative and the structure of the complex was unknown, mainly because of the inherent instability of Ctf13. In this study, I solved the X-ray structure of Ndc10 Nterminal domain (Ndc10NTD) at 1.9 Å and highlighted the unsuspected similarities of the Ndc10NTD fold with the tyrosine recombinase/λ-integrase family. Interestingly, Ndc10 lost the catalytic activity characteristic to the family but conserved a strong DNA binding, which I characterised by structural and mutagenesis studies. Furthermore, I described the Ndc10 C-terminal domain, which displays another DNA binding domain and supports the Ndc10 dimerisation. Ultimately, this work will lead to the structural characterisation of the Ndc10 dimer bound to DNA, of which preliminary crystallisation and data collection results are presented. Finally, I will introduce early results on the purification of the Ctf13 subdomains and the CBF3 reconstitution, a challenging step toward the comprehension of the point centromere establishment.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:647256 |
| Date | January 2015 |
| Creators | Perriches, T. R. A. |
| Publisher | University College London (University of London) |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://discovery.ucl.ac.uk/1463174/ |
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