Mechanisms of Kinetochore Assembly

描述(申请人提供)：在细胞分裂过程中，染色体必须被忠实地复制并分离到子细胞。众所周知，染色体分离的错误会导致人类遗传性疾病，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。每条真核染色体都有一个称为着丝粒的单一位置，这是染色体分离所必需的。在有丝分裂着丝粒模板期间，有丝分裂着丝粒的组装，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是如何组装起来的，以产生一个微管结合部位，从而调节染色体分离。着丝粒是由组蛋白H3被H3变异着丝粒蛋白A(CENP-A)所决定的表观遗传学。在没有CENP-A的情况下，着丝粒和着丝点不能组装，染色体分离失败。这项提案的目标有三个。首先，我们将使用基于人类细胞和细胞提取物的分析方法，通过研究APC依赖的蛋白分解在着丝粒组装因子靶向染色体以组装新的CENP-A核小体中的作用来确定着丝粒组装与细胞周期耦合的机制。其次，利用重组的生化系统，我们将研究CENP-A核小体如何被其他着丝粒蛋白识别来组装着丝粒，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，利用细胞提取物的体外复制和组装系统，我们将确定CENP-A核小体在复制过程中是如何分布的，以及如何确定新的CENP-A组装的位置。我们的实验计划应该为着丝粒染色质是如何组装的，以及该染色质是如何被识别来组装着丝粒和着丝粒的问题提供新的见解。 与公共卫生相关：细胞分裂过程中准确的染色体分离对所有生物体的生长和发育至关重要。染色体分离错误会导致遗传病，并促进人类癌症的进展。我们的建议侧重于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是如何发生并导致人类疾病的。

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.