Mechanisms of Kinetochore Assembly

描述(申请人提供)：为了确保细胞存活和保持基因组的完整性，在有丝分裂过程中，染色体必须均匀地分配给子细胞。动粒是染色体上的一个特殊区域，它绑定有丝分裂纺锤体的微管。所有真核生物在有丝分裂过程中都使用着丝点来分离染色体，但着丝点是如何在每个真核细胞的染色体上建立和维持的尚不清楚。着丝粒的染色质是独特的，因为组蛋白H3被组蛋白变体着丝粒蛋白A(CENP-A)取代。CENP-A在染色质中的掺入确定了着丝粒的染色体位置，对着丝粒的组装是必不可少的。这项提议的重点是了解细胞如何指定动粒的位置，以及动粒是如何在该位置组装的。我们提出了遗传学、生物化学和细胞生物学的方法来理解CENP-A是如何被特异性地结合到染色质中的。我们将首先确定和描述在运动中心建立和维持CENP-A的重要因素。我们的第一个特定目标是通过基于RNAi的果蝇基因组筛选来识别对CENP-A定位重要的基因。利用亲和生化方法，我们分离了一种能与CENP-A特异结合的染色质重塑酶。我们的第二个特定目标是研究该酶在着丝粒染色质形成中的作用。我们的第三个具体目标是使用无细胞非洲爪哇卵子提取系统作为一个体外系统来剖析动粒组装的机制。通过将调控动粒组装的蛋白质的发现与体外系统和细胞分析相结合来分析它们的功能，我们希望了解动粒是如何被指定和组装的。

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.