Structure and function of Mis12 (Minichromosome Instability) centromere complex in mitosis

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Accurate chromosome segregation is essential for the genomic stability of all eukaryotes. The mitotic checkpoint is a failsafe mechanism that ensures accurate chromosome segregation. The kinetochore (kT) is the structural platform where mitotic checkpoint proteins dock and therefore, is the foundation of where mitotic checkpoint regulation occurs. Mis12 (minichromosome instability) is one of the key centromere proteins that specify kT and mitotic checkpoint protein assembly. The centromere protein Mis12 exists as a protein complex and specifies a CENP-A independent centromere assembly pathway. We hypothesize that the Mis12 complex is important for centromere structural integrity as well as mitotic checkpoint signaling. To understand the mechanism of Mis12 function, we propose to study the structure and function of Mis12. We will map the centromere localization domain of hMis12 by screening a random linker scanning as well as deletion and truncation mutant collection of hMis12. To understand the interaction between subunits in the Mis12 complex, we will screen the hMis12 mutant collection for protein-protein interaction domain(s), by yeast 2-hybrid assays, co-immunoprecipitation and GST pull-down experiments. Fluorescence Resonance Energy Transfer technique will be used to analysis protein interactions amongst the Mis12 complex in situ. The regulation of cell-cycle specific centromere dynamics of hMis12 and the hMis12 complex will be analyzed by the Fluorescent Recovery After Photobleaching technique. Mis12 mutants that are defective in specific protein-protein interaction will be analyzed for the functional consequence in vivo (effects on the mitotic checkpoint, the stability of kinetochore-MT interaction, and assembly of the kinetochore structure).