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

有丝分裂中Mis12（微型染色体不稳定性）着丝粒复合体的结构和功能

基本信息

批准号：
355777-2008
负责人：
Chan, Gordon
金额：
$ 2.55万
依托单位：
University of Alberta
依托单位国家：
加拿大
项目类别：
Discovery Grants Program - Individual
财政年份：
2010
资助国家：
加拿大
起止时间：
2010-01-01 至 2011-12-31
项目状态：
已结题

来源：
https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=459557
关键词：
Structure function Mis12 Minichromosome Instability

项目摘要

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).

准确的染色体分离对所有真核生物的基因组稳定性至关重要。有丝分裂检查点是一种确保准确染色体分离的故障保险机制。着丝点（kT）是有丝分裂检查点蛋白停靠的结构平台，因此是有丝分裂检查点调控发生的基础。Mis12（小染色体不稳定性）是指定kT和有丝分裂检查点蛋白组装的关键着丝粒蛋白之一。着丝粒蛋白Mis12作为一种蛋白质复合物存在，并指定一个独立于CENP-A的着丝粒组装途径。我们假设Mis12复合体对着丝粒结构完整性和有丝分裂检查点信号传导很重要。为了了解Mis12的作用机制，我们建议对Mis12的结构和功能进行研究。我们将通过筛选hMis12的随机连接子扫描以及缺失和截断突变体收集来绘制hMis12的着丝粒定位域。为了了解Mis12复合体中亚基之间的相互作用，我们将通过酵母2杂交测定、共免疫沉淀和GST下拉实验筛选hMis12突变体收集的蛋白质-蛋白质相互作用域。荧光共振能量转移技术将用于原位分析Mis12复合物之间的蛋白质相互作用。hMis12和hMis12复合物对细胞周期特异性着丝粒动力学的调控将通过光漂白后荧光恢复技术进行分析。在特定蛋白质-蛋白质相互作用中存在缺陷的Mis12突变体将被分析其在体内的功能后果（对有丝分裂检查点的影响，着丝粒- mt相互作用的稳定性以及着丝粒结构的组装）。