Structural and biochemical characterisation of the centromere binding factor 3

Miah, Shomon (2020) Structural and biochemical characterisation of the centromere binding factor 3. PhD thesis, Birkbeck, University of London.

Abstract

The kinetochore is a critical multi protein complex of the mitotic machinery, which binds both the centromere DNA of the chromosome and the microtubules from the mitotic spindle. In S. cerevisiae, the centromere DNA is short and conserved, and associates with a specialised non-canonical nucleosome, containing a variant of histoneH3 called Cse4. The centromere is specifically recognised by the kinetochore protein complex called CBF3.The CBF3 complex is composed of a homodimer of a zinc cluster protein, Cep3, a homodimer of a DNA binding protein, Ndc10 and a heterodimer of the F-box proteinCtf13 and F-box binding protein Skp1. Genetic studies have determined that CBF3 is responsible for the recruitment of the specialised nucleosome to the point centromere, but the molecular mechanism of the recruitment is unknown. This thesis will discuss new strategies to purify different forms of the CBF3 complex and present structural analysis conducted on a truncated CBF3 complex using single particle cryo-EM. Further work was conducted to analyse the regulation of Zn2Cys6cluster domain of Cep3 DNA binding activity and whether the DNA binding domain of Ndc10 has oligomerisation activity. The results are discussed in the context of the overall implications on the current understanding of CBF3 complex function and how this adds to the understanding of kinetochore and centromere biology in budding yeast.

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