Mechanisms of Kinetochore Assembly

描述（由申请人提供）：准确的基因组分离对所有生物的生存和发展至关重要。染色体分离错误导致细胞非整倍体，从而导致唐氏综合症等人类遗传疾病，并成为大多数人类癌症类型的特征。细胞生物学的一个主要问题是什么是细胞机制，以确保高保真染色体分离，以避免遗传不稳定和由此产生的非整倍体。我们的研究重点是人类着丝粒和着丝点的形成和功能。着丝点的功能是结合微管，通过有丝分裂检查点监测染色体的正常附着，并在后期分离染色体。这些过程中的任何缺陷都会导致染色体分离错误。着丝粒是染色体上有丝分裂着丝点的唯一组装位点。着丝粒功能由一种称为着丝粒蛋白a （CENP-A）的特殊组蛋白变体决定，CENP-A的突变或丢失会导致着丝粒和着丝点功能障碍。我们的第一个目标是确定将CENP-A组装成染色质的机制。我们建议通过确定CENP-A组装所需的两个关键蛋白HJURP和M18BP1如何在终末期和G1期间靶向着丝粒组装CENP-A核小体来实现这一点。其次，我们提出表征基本着丝粒蛋白CENP-C与着丝粒染色质阵列相互作用的机制。利用生化实验的见解，我们将测试人类细胞中组织和加强着丝粒和着丝粒结构的CENP-C功能模型。第三，我们建议使用一种新的体外着丝粒和着丝粒组装系统来了解染色质结构在促进着丝粒和着丝粒功能中的作用。总之，我们的目标应该为脊椎动物着丝粒的组装和功能以及它们的活动如何确保忠实的染色体分离提供新的见解。

DESCRIPTION (provided by applicant): Accurate genome segregation is essential for the survival and development of all organisms. Mistakes in chromosome segregation result in cellular aneuploidies that give rise to human genetic diseases such as Down syndrome and that characterize most human cancer types. A major question in cell biology is what are the cellular mechanisms that ensure high fidelity chromosome segregation to avoid genetic instability and the resulting aneuploidy. Our studies focus on the formation and function of human centromeres and kinetochores. The functions of the kinetochore are to bind microtubules, to monitor proper chromosome attachment via the mitotic checkpoint and to segregate chromosomes in anaphase. Defects in any of these processes result in chromosome segregation errors. The centromere is sole the assembly site for the mitotic kinetochore on the chromosome. Centromere function is determined by a specialized histone variant called centromere protein A (CENP-A) and mutation or loss of CENP-A causes centromere and kinetochore dysfunction. Our first objective in this proposal is to identify the mechanisms that assemble CENP-A into chromatin. We propose to do this by identifying how two of the key proteins required for CENP-A assembly, HJURP and M18BP1, are targeted to centromeres to assemble CENP-A nucleosomes during telophase and G1. Second, we propose to characterize the mechanisms by which the essential centromere protein CENP-C interacts with arrays of centromeric chromatin. Using insights from biochemical experiments, we will test models for CENP-C function in human cells in organizing and reinforcing centromere and kinetochore structure. Third, we propose to use a novel in vitro centromere and kinetochore assembly system to understand the role of chromatin structure in promoting centromere and kinetochore function. Together our aims should provide new insight into the assembly and function of vertebrate centromeres and how their activities ensure faithful chromosome segregation.