A BIOCHEMICAL AND STRUCTURAL STUDY OF THE KINETOCHORE - CENTROMERE INTERFACE

Faithful chromosome segregation during mitosis requires the dynamic interaction between spindle microtubules and kinetochores, multiprotein complexes built on centromeres. A group of kinetochore proteins associates with centromeres throughout the cell cycle and is thus named constitutive centromere-associated network (CCAN). Biochemical and functional analyses indicate that CCAN proteins are organized in sub-complexes. However, the exact organization of these sub-complexes has not been fully elucidated to date. The aim of my project has been the biochemical reconstitution of CCAN sub-complexes and their structural and functional characterization. In particular, this dissertation dwells upon the results I have obtained regarding three different but intrinsically related topics. First, I present a biochemical and structural characterization of the CCAN protein CENP-M (centromere protein M), which displays the fold, but not the enzymatic activity of a G protein. In addition, I disclose its unprecedented role in the context of a quaternary complex with CENP-H, CENP-K and CENP-I and provide information about the spatial organization of this complex. The first steps towards an in vivo validation of these results are also described. Second, I report the discovery of a direct interaction of CENP-H / CENP-K complex with CENP-C. Third, I have been involved in establishing in the laboratory techniques for the in vitro reconstitution of recombinant nucleosomes. The production of material of good quality and quantity has recently been achieved, supporting the analysis of in vitro interactions between nucleosomes and kinetochore components. Specifically, I illustrate some preliminary observations concerning a direct interaction of Mis12 complex with nucleosomes.