Mechanisms of Kinetochore Assembly

着丝粒组装机制

• 批准号: 7678371
• 负责人: Aaron F Straight
• 金额: $ 25.34万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7678371
• 关键词: Affinity; Affinity Chromatography; Binding; Biochemical; Biochemistry; Biological; Biological Assay; Cell Survival; Cells; Cellular Assay; Centromere; Chromatin; Chromosome Segregation; Chromosomes; Complex; Cultured Cells; DNA; Dissection; Drosophila genome; Ensure; Enzymes; Eukaryota; Genes; Genetic; Genomics; Goals; Histone Deacetylase; Histone H3; Histones; Homologous Gene; Human; In Vitro; Kinetochores; Methods; Microtubules; Mitosis; Mitotic Chromosome; Mitotic spindle; Molecular; Monitor; Multiprotein Complexes; Nucleosomes; Phenotype; Positioning Attribute; Process; Proteins; RNA Interference; Research; Research Personnel; Screening procedure; Site; Specific qualifier value; Structure; System; Tail; Variant; Xenopus; Xenopus laevis; base; centromere protein A; chromatin remodeling; daughter cell; egg; programs; research study; sperm cell

DESCRIPTION (provided by applicant): To ensure cell survival and maintain genomic integrity, chromosomes must be equally distributed to daughter cells during mitosis. The kinetochore is a specialized region of the chromosome that binds microtubules of the mitotic spindle. All eukaryotes use kinetochores to segregate chromosomes during mitosis but how the kinetochore is established and maintained on each eukaryotic chromosome is unknown. The chromatin of the kinetochore is unique in that histone H3 is replaced with the histone variant centromere protein A (Cenp-A). The incorporation of Cenp-A into chromatin identifies the chromosomal position of the kinetochore and is essential for kinetochore assembly. This proposal is focused on understanding how cells specify the position of the kinetochore and how kinetochores are assembled at that site. We propose genetic, biochemical and cell biological methods to understand how Cenp-A is specifically incorporated into chromatin. We will first identify and characterize factors that are important for establishing and maintaining Cenp-A at kinetochores. Our first specific aim is to identify genes that are important for Cenp-A localization by RNAi based screening of the Drosophila genome. Using affinity biochemistry we have isolated a chromatin-remodeling enzyme that binds specifically to Cenp-A. Our second specific aim is to characterize the function of this enzyme in centromeric chromatin formation. Our third specific aim is to use the cell free Xenopus egg extract system as an in vitro system to dissect the mechanisms of kinetochore assembly. By combining the discovery of proteins that regulate kinetochore assembly with in vitro systems and cellular assays to analyze their functions we hope to understand how kinetochores are specified and assembled.

描述（由申请人提供）：为了确保细胞存活并维持基因组完整性，染色体必须在有丝分裂期间均匀分配给子细胞。着丝粒是染色体的一个特殊区域，它结合有丝分裂纺锤体的微管。所有真核生物在有丝分裂期间都使用动粒来分离染色体，但动粒如何在每条真核染色体上建立和维持尚不清楚。着丝粒染色质的独特之处在于组蛋白 H3 被组蛋白变体着丝粒蛋白 A (Cenp-A) 取代。 Cenp-A 掺入染色质可识别着丝粒的染色体位置，并且对于着丝粒组装至关重要。该提案的重点是了解细胞如何指定着丝粒的位置以及着丝粒如何在该位点组装。我们提出了遗传、生化和细胞生物学方法来了解 Cenp-A 如何特异性整合到染色质中。我们将首先识别和表征对于在着丝粒建立和维持 Cenp-A 重要的因素。我们的第一个具体目标是通过基于 RNAi 的果蝇基因组筛选来鉴定对 Cenp-A 定位重要的基因。利用亲和生物化学，我们分离出了一种与 Cenp-A 特异性结合的染色质重塑酶。我们的第二个具体目标是表征该酶在着丝粒染色质形成中的功能。我们的第三个具体目标是使用无细胞爪蟾卵提取物系统作为体外系统来剖析着丝粒组装的机制。通过将调节着丝粒组装的蛋白质的发现与体外系统和细胞测定相结合来分析它们的功能，我们希望了解着丝粒是如何被指定和组装的。