Kinetochore Function and Cell Cycle Progression Revision

描述（由申请人提供）：对着丝点功能和纺锤体检查点监测所需基因的基础研究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。着丝点由着丝粒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，我们将使用出芽酵母酿酒酵母，因为它的有丝分裂过程与多细胞真核生物的过程相当。在Aim 1中，我们将描述纺锤体检查点靶向的Dts蛋白的功能。我们使用一个mad2缺失突变体对一组酵母缺失突变体进行了合成致死性筛选，鉴定出32个基因，包括那些编码先前已知的着丝点蛋白、微管结合蛋白、染色质结合蛋白和内聚蛋白的基因。我们将这4个先前未被鉴定的基因命名为DTS-1至DTS-4。dts3突变体表现出典型的着丝点突变体的表型，并且dts3与着丝点蛋白相互作用。我们将通过进行一系列的遗传和生化分析来表征Dts1、2和4的功能。在Aim 2中，我们将研究Bub1（纺锤体检查点激酶）如何控制Sgo1 （Shugoshin，着丝点上的张力传感器）。Sgo1保护着丝粒内聚蛋白（Scc1/RAD21）。BUB1调控SGO1在人细胞中的稳定性和着丝点定位。虽然出芽酵母Sgo1不调节有丝分裂中的内聚，但它是着丝点的张力传感器。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控制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磷酸化调控Sgo1功能，我们将进行一系列实验来验证这一假设。最后，在Aim 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。纺锤体检查点是不激活的，不应该在后期激活。后期促进复合体（APC）和Mps1（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的持续失活。然而，APC如何重新激活仍不清楚。我们最近发现，在酵母中，Bub1是APC的一个靶点，我们的初步数据表明，磷酸化的Bub1是APC在后期的首选靶点，而不是在G1期。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性沉默的信号。我们将进一步描述纺锤体检查点的“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，它们可能会丢失或获得。染色体数量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.