Kinetochore Function and Cell Cycle Progression Revision

动粒功能和细胞周期进程修订

• 批准号: 7730160
• 负责人: KATSUMI KITAGAWA
• 金额: $ 32.76万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7730160
• 关键词: Anaphase; Aneuploidy; BUB1 gene; Binding Proteins; Biochemical; Biological Assay; Cancer Etiology; Cell Cycle; Cell Cycle Checkpoint; Cell Cycle Progression; Cell division; Cells; Centromere; Chromatin; Chromosomal Instability; Chromosomal Stability; Chromosome Segregation; Chromosomes; DNA; Data; Defect; Deletion Mutation; Detection; Development; Ensure; Eukaryota; Genes; Genetic; Goals; Human; Kinetochores; Laboratory Organism; Light; MXI1 gene; Malignant Neoplasms; Meiosis; Metaphase; Microtubule Proteins; Microtubules; Mitosis; Mitotic; Mitotic Chromosome; Molecular; Monitor; Mutation; Names; Pathway interactions; Phenotype; Phosphorylation; Phosphotransferases; Process; Proteins; Saccharomyces cerevisiae; Saccharomycetales; Series; Signal Transduction; Testing; Yeasts; anaphase-promoting complex; anticancer research; cohesin; cohesion; mutant; novel; protein function; public health relevance; research study; sensor; tumor; tumorigenesis

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.

描述（由申请人提供）：动物学功能所需的基因基础研究和主轴检查点的监视与癌症研究直接相关。我们的目标是识别和表征真核生物中有丝分裂染色体隔离所需的蛋白质。由丝粒DNA和相关蛋白组成的动力学对于在有丝分裂和减数分裂过程中维持和隔离染色体至关重要。在这些研究中，我们将使用酿酒酵母的萌芽酵母菌，因为其有丝分裂的过程与多细胞真核生物相当。在AIM 1中，我们将表征由主轴检查点靶向的DTS蛋白的功能。我们使用MAD2-局部突变体针对一组酵母缺失突变的合成杀菌性筛选确定了32个基因，包括编码先前表征的动力学蛋白，微管结合蛋白，染色质结合蛋白和内线蛋白的基因。我们命名了4个以前未表征的基因DTS-1至DTS-4。 DTS3突变体显示了典型的动力学突变体的表型，而DTS3与动力学蛋白相互作用。我们将通过执行一系列遗传和生化测定法来表征DTS1、2和4的功能。在AIM 2中，我们将研究BUB1（主轴检查点激酶）如何控制SGO1（Shugoshin，Kinetochore的张力传感器）。 SGO1保护着丝粒粘着素（SCC1/RAD21）。 BUB1调节了人类细胞中SGO1的稳定性和丝状定位。尽管萌芽的酵母菌SGO1不调节有丝分裂的内聚力，但它是动力学上的张力传感器。最近已经显示，Bub1激酶结构域和SGO1共同起作用，以确保染色体的有效双向化。因此，两者似乎都是张力检查点所需的。但是，BUB1控制SGO1函数的机制尚不清楚。我们发现BUB1与SCC1相互作用并磷酸化SCC1。因此，我们假设BUB1通过SCC1磷酸化调节SGO1功能，我们将执行一系列实验以检验该假设。最后，在AIM 3中，我们将确定在细胞周期期间调节主轴检查点的分子机制。主轴检查点被停用，不应在后期激活。主轴检查点的基本组成部分，促成后期的复合物（APC）（APC）（APC）（APC）之间的相互抑制作用导致纺锤体检查点的持续失活。但是，如何重新激活APC尚不清楚。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是后期期间APC的首选目标，而不是G1中的APC。这些结果表明，中期过程中磷酸化的BUB1的积累是在延长有丝分裂停滞后引发纺锤体检查点活性沉默的信号。我们将进一步表征主轴检查点“适应”的机制。公共卫生相关性：在细胞分裂期间，染色体在不准确隔离时会​​丢失或获得。具有异常数量的染色体称为非整倍性，这种情况可能导致癌症发展。因此，我们对染色体分离机理的研究将有助于理解癌症的发展。