Kinetochore Function and Cell Cycle Progression

描述（由申请人提供）：当染色体分离发生错误时，癌细胞中发生非整倍性。在某些癌症中导致染色体错误分离增加的突变可能是加速肿瘤发生的诱发因素。通过这个模型，动粒及其调控系统，这是基因组稳定性所必需的，是至关重要的，在防止癌症的发展。该项目的目标是通过使用酿酒酵母作为实验生物来鉴定和表征真核生物中有丝分裂染色体分离所需的蛋白质。 Sgt 1和核心动粒蛋白Skp 1通过激活Ctf 13促进动粒核心复合物（CBF 3）的组装。此外，Skp 1和Sgt 1是SCF复合物（E3泛素连接酶）的一部分。具体目的1是研究Sgt 1的生物学功能。这将通过确定Sgt 1与CEN结合的时间和分析Sgt 1的磷酸化状态来研究。为了检验Sgt 1和Skp 1是动粒激活和泛素介导的降解之间通过SCF复合物的潜在联系的关键组分的假设，将进行基因外抑制剂筛选。具体目标2是分离和表征与Sgt 1相互作用的蛋白质。将进行免疫沉淀质谱法以分离Sgt 1相互作用物。分离的蛋白质的功能将使用我们将构建的突变菌株在遗传学和生物化学中表征。 从有缺陷的动粒到有丝分裂检查点的信号被认为是发生的，但了解甚少。主要研究者已经表明，纺锤体检查点蛋白Bub 1与Skp 1结合，Bub 1通过Skp 1与CEN DNA结合。因此，具体目标3是表征动粒和有丝分裂纺锤体检查点之间的分子相互作用，特别是Bub 1-Skp 1相互作用。将分析Bub 1-Skp 1复合物对由有丝分裂缺陷、Bub 1的Skp 1结合和CEN相关结构域以及其他Bub 1相互作用物引起的G2/M延迟的贡献。 这项工作将揭示Skp 1和Sgt 1的动粒功能和新的动粒或细胞周期调节因子，可以作为进一步分析染色体传递和细胞周期进程中动粒的切入点。

DESCRIPTION (provided by applicant): Aneuploidy occurs in cancer cells when errors in chromosome segregation happen. Mutations leading to increased chromosome missegregation in certain cancers might be predisposing factors that accelerate tumorigenesis. By this model, the kinetochore and its regulatory system, which are essential for genome stability, are crucial in protecting against cancer development. The project's goal is to identify and characterize proteins required for mitotic chromosome segregation in eukaryotes, by using Saccharomyces cerevisiae as an experimental organism. Sgt1 and the core kinetochore protein, Skp1, promote assembly of the kinetochore core complex (CBF3) by activating Ctf13. Moreover, Skp1 and Sgt1 are part of the SCF complex (an E3 ubiquitin ligase). Specific Aim 1 is to investigate the biological function of Sgt1. This will be investigated by determining the time at which Sgt1 binds to CEN, and analyzing Sgt1's phosphorylation status. To test the hypothesis that Sgt1 and Skp1 are key components in the potential connection between kinetochore activation and ubiquitin-mediated degradation via the SCF complex, extragenic suppressor screens will be performed. Specific Aim 2 is to isolate and characterize proteins that interact with Sgt1. Immunoprecipitation mass spectrometry will be performed to isolate Sgt1 interactors. The function(s) of the isolated proteins will be characterized in genetics and biochemistry using the mutant strains that we will construct. Signaling from defective kinetochores to the mitotic checkpoint is thought to occur but is poorly understood. The principal investigator has shown that the spindle checkpoint protein Bub1 binds to Skp1 and that Bub1 associates with CEN DNA via Skp1. Therefore, Specific Aim 3 is to characterize the molecular interaction between kinetochores and the mitotic spindle checkpoint, especially the Bub1-Skp1 interaction. The contribution of the Bub1-Skp1 complex to the G2/M delays caused by mitotic defects, Bub1's Skp1 binding and CEN-associating domains, and additional Bub1 interactors will be analyzed. This work will reveal kinetochore functions of Skp1 and Sgt1 and novel kinetochore or cell-cycle regulators that can serve as entry points for further analysis of kinetochores in chromosome transmission and cell-cycle progression Achieving these aims will further elucidate mechanisms of cancer development.