BIOCHEMICAL ANALYSIS OF MITOTIC CHROMOSOME CONDENSATION

染色体凝聚是保证有丝分裂和减数分裂过程中遗传信息忠实分离的重要细胞过程。该项目的长期目标是了解这种动态和高度协调的dna -蛋白质组装过程是如何实现的，以及它是如何在真核细胞周期中精确调节的。重点将放在13S凝缩蛋白的结构和功能表征上，这是一种最近发现的在这一过程中起关键作用的蛋白质复合物。13S凝聚蛋白是一种dna刺激的atp酶，由两个SMC（染色体结构维持）亚基和三个非SMC亚基组成。该复合体具有通过以atp依赖的方式引入全局正扭曲来重新配置DNA的能力。在这个建议中，(1)五个亚基复合物将被生化剖析，以确定单个亚基和亚复合物如何促进凝缩蛋白的功能。(2)在体外重建细胞周期依赖的13S凝聚蛋白与染色质之间的相互作用，确定有丝分裂特异性凝聚的分子基础。(3)将结合结构、细胞生物学和生物物理学的方法来了解何时、何地以及如何在高阶染色体结构的背景下凝聚蛋白复合体起作用。(4)与13S凝缩蛋白合作构建有丝分裂染色体的新结构组分将被鉴定和表征。从这项工作中获得的信息将最终有助于更好地了解人类健康，因为染色体异常，如非整倍体和易位，与肿瘤发展或出生缺陷密切相关。

Chromosome condensation is an essential cellular process that ensures the faithful segregation of genetic information during mitosis and meiosis. The long-term goal of this project is to understand how this dynamic and highly orchestrated process of DNA-protein assembly is achieved, and how it is regulated precisely during the eukaryotic cell cycle. A major emphasis will be made on the structural and functional characterization of 13S condensin, a recently identified protein complex that plays a key role in this process. 13S condensin is a DNA-stimulated ATPase that consists of two SMC (structural maintenance of chromosomes) subunits and three non-SMC subunits. The complex has an ability to reconfigure DNA by introducing global positive writhe in an ATP-dependent manner. In this proposal, (1) the five-subunit complex will be dissected biochemically to determine how individual subunits and subcomplexes contribute to condensin functions. (2) A cell cycle-dependent interaction between 13S condensin and chromatin will be reconstituted in vitro and the molecular basis of mitosis-specific condensation will be determined. (3) Structural, cell biological and biophysical approaches will be combined to understand when, where and how the condensin complex works in the context of higher-order chromosome structure. (4) New structural components that cooperate with 13S condensin to build up mitotic chromosomes will be identified and characterized. The information obtained from this work will ultimately contribute to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocations, are tightly associated with tumor development or birth defects.