| Summary: | If the kinetochore is incorrectly or not attached to the spindle, the mitotic checkpoint arrests the cell cycle by inhibiting the ubiquitin ligase activity of the anaphase promoting complex/cyclosome. BubR1 (Budding uninhibited by benomyl), a multi-domain kinase essential for the mitotic checkpoint, localises at incorrectly or not attached kinetochores and directly inhibits the anaphase promoting complex/cyclosome. A critical BubR1 region is an N-terminal α-helical segment conserved also in fellow mitotic checkpoint protein Bub1. In higher eukaryotes, this region interacts with kinetochore protein Blinkin and contains a conserved KEN box (consecutive lysine, glutamate and asparagines residues) essential for binding anaphase promoting complex/cyclosome co-activator Cdc20 (Cell division cycle). Obtaining a molecular view of this critical BubR1-N-terminal region has previously been hindered by an inability to produce stable protein for structural studies. this thesis reports the establishment of a method for purifying stable BubR1- and yeast orthologue Mad3 (Mitotic arrest deficient)-N-terminal proteins, as well as biochemical and biophysical investigations of their behaviour, shape and structure. Crystallographic analysis at both 1.8 and 3.5 Å resolutions shows that the core of the BubR1-N-terminal region adopts an all α-helical fold reminiscent of a tetratricopeptide repeat domain. Subsequent interpretation and mutagenesis studies provide insight into the binding of this tetratricopeptide repeat-like domain to mitotic checkpoint-relevant protein ligands. The work presented in this thesis is thus a significant step in describing the molecular detail of BubR1 mitotic checkpoint function.
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