Kinetochore Function and Cell Cycle Progression Revision

描述（由申请人提供）：着丝粒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表征真核生物有丝分裂染色体分离所需的蛋白质。着丝粒由着丝粒DNA和相关蛋白质组成，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，我们将使用芽殖酵母酿酒酵母，因为它的有丝分裂过程与多细胞真核生物相当。在目标1中，我们将表征由纺锤体检查点靶向的Dts蛋白的功能。我们的合成致死性筛选使用一组酵母缺失突变的mad 2-缺失突变体确定了32个基因，包括那些编码先前表征的动粒蛋白，微管结合蛋白，染色质结合蛋白和凝聚蛋白。我们将这4个以前未表征的基因命名为p2p-1至p2p-4。dts 3突变体显示典型的动粒突变体的表型，并且Dts 3与动粒蛋白相互作用。我们将通过一系列的遗传和生化检测来表征Dts 1，2和4的功能。在目标2中，我们将研究Bub 1（一种纺锤体检查点激酶）如何控制Sgo 1（Shugoshin，动粒处的张力传感器）。Sgo 1保护着丝粒粘附素（Scc 1/RAD 21）。BUB 1调节SGO 1在人细胞中的稳定性和着丝粒定位。虽然芽殖酵母Sgo 1不调节有丝分裂中的凝聚力，但它是动粒处的张力传感器。最近的研究表明，Bub 1激酶结构域和Sgo 1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需的。然而，Bub 1控制Sgo 1功能的机制尚不清楚。我们发现Bub 1与Scc 1相互作用并磷酸化Scc 1。因此，我们假设Bub 1通过Scc 1磷酸化调节Sgo 1功能，我们将进行一系列实验来验证这一假设。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。纺锤体检查点已停用，不应在后期激活。后期促进复合物（APC）和Mps 1（纺锤体检查点的重要组成部分）之间的相互抑制导致纺锤体检查点持续失活。然而，APC如何重新启动仍不清楚。我们最近发现Bub 1是酵母中APC的靶点，我们的初步数据表明磷酸化的Bub 1是后期APC的首选靶点，但在G1期不是。这些结果表明，在中期磷酸化的Bub 1的积累是启动沉默的纺锤体检查点活动后，延长有丝分裂停滞的信号。我们将进一步表征纺锤体检查点的“适应”机制。公共卫生相关性：在细胞分裂过程中，当染色体不能准确分离时，染色体可能丢失或获得。染色体数目异常被称为非整倍体，这种情况可能导致癌症的发展。因此，我们对染色体分离机制的研究将有助于了解癌症的发展。

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.