Structural studies on kinetochore linker complexes.
Doctoral thesis, UCL (University College London).
The MIND/Mis12 complex is an essential kinetochore component that is conserved throughout eukaryotes. This study has characterised both structurally and biochemically the recombinant MIND multiprotein complex from Saccharomyces cerevisiae. Hydrodynamic analysis identified that this complex is elongated in structure and comprises Dsn1p, Mtw1p, Nsl1p and Nnf1p in a stoichiometry of 1:1:1:1. Low-resolution structures obtained from electron microscopy (EM) and reconstructions from small angle X-ray scattering (SAXS) experiments are in agreement that the MIND/Mis12 complex in budding yeast exists as a 22 nm long structure, divided into ‘head’ and ‘tail’ regions. The head region is 8 nm in diameter and contains domains of all four subunits and is seen as a hook shape with a large cavity. The tail region appears as an elongated structure that is resistant to protease treatment. A model is proposed in this thesis for the overall structure and subunit orientation of the MIND complex. The MIND/Mis12 complex along with the Ndc80 complex and KNL-1/Spc105p are components of the KMN network that directly attaches the kinetochore to the mitotic spindle. This study has shown that the KMN network in Saccharomyces cerevisiae is conserved and formed by high affinity hydrophobic interactions between MIND and the C-terminal of Spc105p and MIND and the globular Spc24p/Spc25p domain of the Ndc80 complex. Spc105p and the Ndc80 complex bind MIND with comparable affinity, indicating tight associations of the KMN network with a 1:1:1 ratio that is also observed in higher eukaryotes The interaction between the MIND and Ndc80 complexes is proposed to point the tail of the MIND complex towards the centromere. The role of the Ipl1/Aurora B kinase was investigated for the MIND complex, with a single phosphorylation site found at serine 250 of Dsn1p. Phosphorylation did not affect either interactions between subunits of the MIND complex, nor binding between components of the KMN network, implying another role for this phosphorylation in kinetochore regulation.
|Title:||Structural studies on kinetochore linker complexes|
|Open access status:||An open access version is available from UCL Discovery|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of) > Structural and Molecular Biology|
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