Mechanisms of Kinetochore Assembly

着丝粒组装机制

• 批准号: 6956647
• 负责人: Aaron F Straight
• 金额: $ 28.77万
• 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/6956647
• 关键词: Drosophilidae; RNA interference; Xenopus oocyte; binding sites; cell cycle; centromere; chromosome movement; cytogenetics; enzyme activity; genetic regulation; molecular assembly /self assembly; protein binding; protein localization; protein structure function; regulatory gene

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过程中平均分配给子细胞。动力学是结合有丝分裂主轴的微管的染色体的专门区域。所有真核生物都使用动力学在有丝分裂过程中隔离染色体，但是如何在每个真核染色体上建立和维持动力学的染色体是未知的。动力学的染色质是独一无二的，因为组蛋白H3被组蛋白变体丝粒蛋白A（CENP-A）取代。将CENP-A掺入染色质中可以识别动力学的染色体位置，对于动力学组装至关重要。该建议的重点是了解细胞如何指定动力学的位置以及如何在该位点组装动力学。我们提出遗传，生化和细胞生物学方法，以了解CENP-A如何专门掺入染色质中。我们将首先确定和表征对于在动力学上建立和维持CENP-A很重要的因素。我们的第一个具体目的是鉴定果蝇基因组的基于RNAi的筛选对CENP-A定位很重要的基因。使用亲和力生物化学，我们分离了一种与CENP-A结合的染色质复制酶。我们的第二个具体目的是表征该酶在丝粒染色质形成中的功能。我们的第三个具体目的是将无细胞的Xenopus鸡蛋提取系统用作体外系统，以剖析动力学组装的机制。通过将调节动力学组装的蛋白质与体外系统和细胞测定法相结合，以分析其功能，我们希望了解如何指定和组装动力学。