Biochemical Analysis of Sister Chromatid Cohesion

姐妹染色单体凝聚力的生化分析

• 批准号: 7081274
• 负责人: TATSUYA HIRANO
• 金额: $ 34.76万
• 依托单位: COLD SPRING HARBOR LABORATORY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-07-01 至 2007-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7081274
• 关键词: DNA binding protein; Xenopus oocyte; adenosinetriphosphatase; cell cycle; chromatin; chromosome movement; enzyme activity; eukaryote; genetic crossing over; human tissue; meiosis; molecular biology; molecular cloning; phosphorylation; protein purification; protein structure function; tissue /cell culture

DESCRIPTION (provided by applicant): The long-term goal of this research project is to understand the molecular mechanisms of chromosome cohesion and segregation. Central to this process are members of an evolutionary conserved family of chromosomal ATPase, known as the structural maintenance of chromosomes (SMC) family. SMC proteins form dimers, which adopt a unique two-armed structure with an ATP-binding "head" domain at the distal end of each arm. In eukaryotes, an SMC protein complex known as cohesin plays a key role in sister chromatid cohesion in mitosis and meiosis. In this proposal, multi-disciplinary approaches will be taken to understand how SMC proteins work at a mechanistic level in vitro and how their functions are regulated in vivo. First, the bacterial SMC protein complex from Bacillus subtilis will be used as a model system for understanding the basic: action of SMC proteins. An innovative set of functional assays, combined with extensive site-directed mutagenesis, will be employed to address how the mechanical cycle of SMC might be coupled to its catalytic cycle. These biochemical studies will be complemented by other approaches including single-DNA-molecule nanomanipulation and protein crystallization. Second, sub- and holo-complexes of cohesin will be reconstituted from its recombinant subunits, and their activities will be characterized in both purified systems and Xenopus egg cell-free extracts. Mutant forms of the complexes will be constructed to determine how the mechanochemical cycle of cohesin participates in sister chromatid cohesion and its release. Finally, regulation of cohesin's loading and stabilization will be studied in Xenopus egg extract:; and mammalian tissue culture cells. A major emphasis will be made on the role of 3 different classes of regulators (Scc2, Pds5 and Sgo) in these processes. The impacts of Scc2 and Pds5 on the ATPase cycle of cohesin will also be determined. The information obtained from this work will ultimately lead to a better understanding of human health because chromosome anomalies, such as aneuploidy and translocation, are tightly associated with tumor development and birth defects.

描述（由申请人提供）：本研究项目的长期目标是了解染色体内聚和分离的分子机制。这个过程的核心是染色体三磷酸腺苷酶进化保守家族的成员，被称为染色体结构维持（SMC）家族。SMC蛋白形成二聚体，其采用独特的双臂结构，在每条手臂的远端具有atp结合的“头”结构域。在真核生物中，一种称为内聚蛋白的SMC蛋白复合物在有丝分裂和减数分裂中姐妹染色单体内聚中起着关键作用。在本提案中，将采取多学科的方法来了解SMC蛋白在体外的机制水平上是如何工作的，以及它们的功能是如何在体内调节的。首先，来自枯草芽孢杆菌的细菌SMC蛋白复合物将被用作理解SMC蛋白基本作用的模型系统。一套创新的功能分析，结合广泛的定点诱变，将被用来解决SMC的机械循环如何与它的催化循环耦合。这些生化研究将由其他方法补充，包括单dna分子纳米操作和蛋白质结晶。其次，将其重组亚基重组为内聚蛋白的亚复合物和全复合物，并在纯化体系和爪蟾卵细胞提取物中对其活性进行表征。将构建这些复合物的突变形式，以确定内聚蛋白的机械化学循环如何参与姐妹染色单体的内聚及其释放。最后，对爪蟾卵提取物中黏结蛋白的加载和稳定规律进行了研究。哺乳动物组织培养细胞。重点将放在三种不同类型的监管机构（Scc2， Pds5和Sgo）在这些过程中的作用。Scc2和Pds5对内聚蛋白atp酶周期的影响也将被确定。从这项工作中获得的信息将最终导致更好地了解人类健康，因为染色体异常，如非整倍体和易位，与肿瘤发展和出生缺陷密切相关。