Kinetochore Function and Cell Cycle Progression Revision

描述(由申请者提供)：运动中枢功能所需基因的基础研究和纺锤体检查点的监测与癌症研究直接相关。我们的目标是鉴定和鉴定真核细胞有丝分裂染色体分离所需的蛋白质。着丝粒由着丝粒DNA和相关蛋白组成，在有丝分裂和减数分裂过程中对维持和分离染色体至关重要。在这些研究中，我们将使用萌芽酵母酿酒酵母，因为它的有丝分裂过程与多细胞真核生物相似。在目标1中，我们将描述纺锤体检查点靶向的DTS蛋白的功能。我们使用MAD2缺失突变体对一组酵母缺失突变进行的合成致死性筛选确定了32个基因，包括编码先前特征的动粒蛋白、微管结合蛋白、染色质结合蛋白和凝聚蛋白的基因。我们将这4个以前未鉴定的基因命名为DTS-1至DTS-4。Dts3突变体表现出典型的动粒突变体的表型，并且dts3与动粒蛋白相互作用。我们将通过执行一系列遗传和生化分析来表征Dts1、2和4的功能。在目标2中，我们将研究Bub1(纺锤体检查点激酶)如何控制Sgo1(Shugoshin，动粒张力传感器)。Sgo1保护着丝粒粘附素(Scc1/RAD21)。Bub1调节SGO1在人类细胞中的稳定性和着丝粒定位。尽管芽殖酵母Sgo1不调节有丝分裂中的凝聚力，但它是动粒的张力感受器。最近的研究表明，Bub1激酶结构域和Sgo1共同作用，以确保染色体的有效双向定位；因此，两者似乎都是张力检查点所必需的。然而，Bub1调控Sgo1功能的机制尚不清楚。我们发现Bub1与Scc1相互作用，并使Scc1磷酸化。因此，我们假设Bub1通过Scc1磷酸化来调节Sgo1的功能，并将进行一系列实验来验证这一假说。最后，在目标3中，我们将确定在细胞周期中调节纺锤体检查点的分子机制。磁盘轴检查点处于停用状态，不应在后期激活。后期促进复合体(APC)和纺锤体检查点的重要组成部分Mps1之间的相互抑制导致纺锤体检查点的持续失活。然而，APC是如何重新启动的仍不清楚。我们最近发现Bub1是酵母中APC的靶标，我们的初步数据表明，磷酸化的Bub1是APC后期的首选靶标，而在G1中不是。这些结果表明，中期磷酸化Bub1的积累是在长时间有丝分裂停止后启动纺锤体检查点活动沉默的信号。我们将进一步表征纺锤形检查点的“适应”机制。与公共卫生相关：在细胞分裂过程中，如果染色体没有准确分离，染色体可能会丢失或获得。染色体数目异常被称为非整倍体，这种情况会导致癌症的发展。因此，我们对染色体分离机制的研究将有助于理解癌症的发展。

DESCRIPTION (provided by applicant): Basic studies of genes required for the function of kinetochores and the surveillance of the spindle checkpoint are directly relevant to cancer research. Our goal is to identify and characterize proteins required for mitotic chromosome segregation in eukaryotes. The kinetochore, which consists of centromere DNA and associated proteins, is crucial for maintaining and segregating chromosomes during mitosis and meiosis. For these studies we will use the budding yeast Saccharomyces cerevisiae, as its process of mitotic division is comparable with that of multicellular eukaryotes. In Aim 1, we will characterize the functions of Dts proteins targeted by the spindle checkpoint. Our synthetic-lethality screen using a mad2-deletion mutant against a set of yeast deletion mutations identified 32 genes, including those encoding previously characterized kinetochore proteins, microtubule-binding proteins, chromatin-binding proteins, and cohesion proteins. We named the 4 previously uncharacterized genes DTS-1 through DTS-4. The dts3 mutants show a phenotype typical of kinetochore mutants, and Dts3 interacts with kinetochore proteins. We will characterize the function of Dts1, 2, and 4 by performing a series of genetic and biochemical assays. In Aim 2, we will investigate how Bub1 (a spindle checkpoint kinase) controls Sgo1 (Shugoshin, a tension sensor at the kinetochore). Sgo1 protects centromeric cohesin (Scc1/RAD21). BUB1 regulates the stability and centromeric localization of SGO1 in human cells. Although budding yeast Sgo1 does not regulate cohesion in mitosis, it is a tension sensor at kinetochores. It has recently been shown that the Bub1 kinase domain and Sgo1 act together to ensure the efficient bi-orientation of chromosomes; thus, both appear to be required for the tension checkpoint. However, the mechanism by which Bub1 controls Sgo1 function is unknown. We found that Bub1 interacts with Scc1 and phosphorylates Scc1. Thus, we hypothesize that Bub1 regulates Sgo1 function through Scc1 phosphorylation, and we will perform a series of experiments to test the hypothesis. Finally, in Aim 3 we will determine the molecular mechanism that regulates the spindle checkpoint during the cell cycle. The spindle checkpoint is deactivated and should not be activated during anaphase. Mutual inhibition between the anaphase-promoting complex (APC) and Mps1, an essential component of the spindle checkpoint, leads to sustained inactivation of the spindle checkpoint. However, how the APC is reactivated remains unclear. We have recently found that Bub1 is a target of the APC in yeast, and our preliminary data suggest that phosphorylated Bub1 is the preferred target of the APC during anaphase but not in G1. These results imply that the accumulation of phosphorylated Bub1 during metaphase is the signal that initiates the silencing of spindle checkpoint activity after a prolonged mitotic arrest. We will further characterize the mechanism of "adaptation" of the spindle checkpoint. PUBLIC HEALTH RELEVANCE: During cell division, chromosomes can be lost or gained when they do not segregate accurately. Having an abnormal number of chromosomes is called aneuploidy, and this situation can cause cancer to develop. Our studies on the mechanism of chromosome segregation will therefore contribute to the understanding of cancer development.