Mechanisms of Kinetochore Assembly

描述（由申请人提供）：为了确保细胞存活和保持基因组完整性，染色体必须在有丝分裂期间均匀分布到子细胞。着丝点是染色体上结合有丝分裂纺锤体微管的特殊区域。在有丝分裂过程中，所有真核生物都使用着丝点分离染色体，但着丝点是如何在每条真核生物染色体上建立和维持的尚不清楚。着丝点的染色质是独特的，因为组蛋白H3被组蛋白变体着丝点蛋白A （Cenp-A）所取代。Cenp-A与染色质的结合确定了着丝点的染色体位置，对着丝点的组装至关重要。这个建议的重点是了解细胞如何指定着丝点的位置以及着丝点如何在该位置组装。我们提出遗传学、生物化学和细胞生物学方法来了解Cenp-A是如何特异性地结合到染色质中的。我们将首先确定和描述在着丝点建立和维持Cenp-A的重要因素。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。利用亲和生物化学方法，我们分离出了一种染色质重塑酶，它与Cenp-A特异性结合。我们的第二个具体目标是表征这种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系统作为体外系统来解剖着丝点组装机制。通过将调节着丝点组装的蛋白质的发现与体外系统和细胞分析相结合，我们希望了解着丝点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.