Mechanisms of Kinetochore Assembly

描述（由申请人提供）：在细胞分裂期间，染色体必须忠实地复制并分离到子细胞。已知染色体分离错误会导致人类遗传疾病并促进癌症的进展。因此，了解控制染色体分离的机制将有助于深入了解人类疾病的病因学。每条真核生物染色体都有一个称为着丝粒的位点，它对染色体分离至关重要。在有丝分裂过程中，着丝粒模板将染色体连接到微管纺锤体上进行分离。我们的长期目标是了解着丝粒和着丝点如何组装以产生介导染色体分离的微管结合位点。着丝粒是由H3变异着丝粒蛋白A （CENP-A）取代组蛋白H3在表观遗传学上决定的。在缺乏CENP-A的情况下，着丝粒和着丝点不能组装，染色体分离失败。这项提议的目标有三个。首先，我们将使用基于人类细胞和细胞提取物的实验，通过研究APC依赖蛋白水解在着丝粒组装因子靶向到染色体上组装新的CENP-A核小体中的功能，来确定着丝粒组装与细胞周期耦合的机制。其次，利用重建的生化系统，我们将研究CENP-A核小体如何被其他着丝粒蛋白识别以组装着丝粒，以及其他着丝粒蛋白与CENP-A染色质的结合如何调节染色质结构。第三，利用细胞提取物中的体外复制和组装系统，我们将确定在复制过程中如何分布CENP-A核小体以及如何确定新CENP-A组装的位点。我们的实验计划将为着丝粒染色质如何组装以及如何识别染色质组装着丝粒和着丝粒的问题提供新的见解。

DESCRIPTION (provided by applicant): During cell division chromosomes must be faithfully replicated and segregated to daughter cells. Errors in chromosome segregation are known to result in human genetic diseases and promote the progression of cancer. Therefore, understanding the mechanisms that control chromosome segregation will provide insight into the etiology of human diseases. Each eukaryotic chromosome possesses a single site called the centromere that is essential for chromosome segregation. During mitosis centromeres template the assembly of the mitotic kinetochore that attaches chromosomes to the microtubule spindle for segregation. Our long-term goal is to understand how centromeres and kinetochores are assembled to generate a microtubule-binding site that mediates chromosome segregation. The centromere is epigenetically determined by the replacement of histone H3 with the H3 variant centromere protein A (CENP-A). In the absence of CENP-A, centromeres and kinetochores do not assemble and chromosome segregation fails. The goals of this proposal are threefold. First, we will use human cell and cell extract based assays to determine the mechanism that couples centromere assembly to the cell cycle by studying the function of APC dependent proteolysis in the targeting of centromere assembly factors to the chromosome to assemble new CENP-A nucleosomes. Second, using reconstituted biochemical systems we will study how CENP-A nucleosomes are recognized by other centromere proteins to assemble the centromere and how the binding of other centromere proteins to CENP-A chromatin regulates chromatin structure. Third, using in vitro replication and assembly systems in cell extracts we will determine how CENP-A nucleosomes are distributed during replication and how the sites of new CENP- A assembly are determined. Our experimental plan should provide new insight into the problems of how centromeric chromatin is assembled and how that chromatin is recognized to assemble the centromere and kinetochore. PUBLIC HEALTH RELEVANCE: Accurate chromosome segregation during cell division is essential for growth and development of all organisms. Chromosome segregation errors cause genetic disease and promote the progression of cancer in humans. Our proposal is focused on understanding the molecular mechanisms that ensure accurate chromosome segregation to inform how chromosome missegregation arises and contributes to human disease.