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：
2012
资助国家：
加拿大
起止时间：
2012-01-01 至 2013-12-31
项目状态：
已结题

来源：
https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=498122
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于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故障安全机制，可确保准确的染色体分离。动力学（KT）是有丝分裂检查点蛋白对接的结构平台，因此是有丝分裂检查点调节的基础。 MIS12（微小浓度小体不稳定性）是指定KT和有丝分裂检查点蛋白质组装的关键中心粒蛋白之一。 Centromere蛋白MIS12作为蛋白质复合物存在，并指定了CENP-A独立的中心粒总成途径。我们假设MIS12复合物对于中心粒结构完整性以及有丝分裂检查点信号很重要。为了了解MIS12功能的机制，我们建议研究MIS12的结构和功能。我们将通过筛选随机的链接器扫描以及HMIS12的删除和截断突变体收集来绘制HMIS12的CentRomere定位域。为了了解MIS12复合物中亚基之间的相互作用，我们将通过酵母2-杂化测定，共免疫沉淀和GST下拉实验来筛选蛋白质 - 蛋白质相互作用结构域的HMIS12突变体收集。荧光共振能量转移技术将用于分析MIS12复合物之间的蛋白质相互作用。 HMIS12和HMIS12复合物的细胞周期特异性中心动力学的调节将通过光漂白技术后的荧光回收率进行分析。将分析在特定蛋白质蛋白质相互作用中有缺陷的MIS12突变体，以分析体内功能后果（对有丝分裂检查点的影响，Kinetochore-MT相互作用的稳定性以及动力学结构的组装）。