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。已知染色体分离中的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。每个真核染色体都有一个称为着丝粒的位点，它是染色体分离所必需的。在有丝分裂过程中，着丝粒作为有丝分裂动粒的模板，将染色体附着在微管纺锤体上进行分离。我们的长期目标是了解着丝粒和动粒是如何组装产生一个微管结合位点，介导染色体分离。着丝粒是通过用H3变体着丝粒蛋白A（CENP-A）替换组蛋白H3来表观遗传地确定的。在没有CENP-A的情况下，着丝粒和动粒不能组装，染色体分离失败。这项建议有三个目标。首先，我们将使用基于人细胞和细胞提取物的测定，通过研究APC依赖性蛋白水解在将着丝粒组装因子靶向染色体以组装新的CENP-A核小体中的功能，来确定将着丝粒组装与细胞周期偶联的机制。其次，使用重建的生化系统，我们将研究如何CENP-A核小体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，使用细胞提取物中的体外复制和组装系统，我们将确定CENP-A核小体在复制期间如何分布以及新CENP-A组装的位点如何确定。我们的实验计划应该提供新的洞察力的问题，着丝粒染色质是如何组装，以及如何识别染色质组装着丝粒和动粒。 公共卫生相关性：细胞分裂期间准确的染色体分离对所有生物体的生长和发育至关重要。染色体分离错误导致遗传疾病，并促进人类癌症的进展。我们的建议集中在了解确保准确染色体分离的分子机制，以告知染色体错误分离如何产生并导致人类疾病。

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.