Kinetochore Function and Cell Cycle Progression

描述(申请人提供)：当染色体分离出现错误时，癌细胞中就会发生非整倍体。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因。根据这一模型，动粒及其调控系统对基因组的稳定性至关重要，在防止癌症发展方面至关重要。该项目的目标是通过使用酿酒酵母作为实验有机体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。 SGT1和核心动粒蛋白Skp1通过激活Ctf13促进动粒核心复合体(CBF3)的组装。此外，Skp1和SGT1是SCF复合体(E3泛素连接酶)的一部分。具体目的1是研究SGT1的生物学功能。这将通过确定SGT1与CEN结合的时间，以及分析SGT1的S磷酸化状态来进行研究。为了验证SGT1和Skp1是动粒激活和泛素通过SCF复合体介导的降解之间潜在联系的关键成分的假设，将进行基因外抑制物筛选。具体目标2是分离和鉴定与SGT1相互作用的蛋白质。免疫沉淀质谱仪将用于分离SGT1的相互作用。分离蛋白的功能(S)将使用我们将构建的突变株进行遗传学和生化表征。 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，但人们对此知之甚少。主要研究人员已经证明纺锤体检查点蛋白Bub1与Skp1结合，Bub1通过Skp1与CEN DNA结合。因此，特定的目标3是描述动点和有丝分裂纺锤体检查点之间的分子相互作用，特别是Bub1-Skp1相互作用。将分析Bub1-Skp1复合体在有丝分裂缺陷引起的G2/M延迟中的作用，Bub1‘S Skp1结合结构域和CEN结合结构域，以及额外的Bub1相互作用元件。 这项工作将揭示Skp1和SGT1的动粒功能以及新的动粒或细胞周期调节因子，它们可以作为进一步分析染色体传递和细胞周期进程中的动粒的切入点，实现这些目标将进一步阐明癌症的发生机制。

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.