Mechanisms of Kinetochore Assembly

描述（由申请方提供）：为确保细胞存活并保持基因组完整性，有丝分裂期间染色体必须均匀分布到子细胞。动粒是染色体的一个特殊区域，它与有丝分裂纺锤体的微管结合。所有的真核生物在有丝分裂过程中都使用动粒来分离染色体，但是动粒是如何在每个真核生物染色体上建立和维持的还不清楚。动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。将Cenp-A掺入染色质中鉴定动粒的染色体位置，并且对于动粒组装是必需的。这项建议的重点是了解细胞如何指定动粒的位置，以及动粒如何在该网站组装。我们提出了遗传，生物化学和细胞生物学方法来了解Cenp-A是如何特异性地掺入染色质的。我们将首先识别和描述对于在动粒中建立和维持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。使用亲和生物化学，我们已经分离出一种染色质重塑酶，特异性结合Cenp-A。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的非洲爪蟾卵提取物系统作为一个体外系统来剖析动粒组装的机制。通过结合蛋白质的发现，调节动粒装配与体外系统和细胞分析，以分析它们的功能，我们希望了解动粒是如何指定和组装。

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.