Biochemical Analysis of Sister Chromatid Cohesion

描述（由申请人提供）：本研究项目的长期目标是了解染色体凝聚和分离的分子机制。这一过程的核心是进化保守的染色体ATP酶家族的成员，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，在每个臂的远端具有ATP结合的“头”结构域。在真核生物中，被称为粘附素的SMC蛋白复合物在有丝分裂和减数分裂中的姐妹染色单体粘附中起关键作用。在这项提案中，将采取多学科的方法来了解SMC蛋白如何在体外机制水平上工作，以及它们的功能如何在体内调节。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蛋白的基本作用的模型系统。一套创新的功能测定，结合广泛的定点诱变，将用于解决如何SMC的机械循环可能耦合到其催化循环。这些生化研究将得到其他方法的补充，包括单DNA分子纳米操纵和蛋白质结晶。其次，子和全复合体的粘连蛋白将重建从其重组亚基，其活动将在纯化的系统和爪蟾卵细胞的无提取物的特点。将构建复合物的突变体形式，以确定粘附素的机械化学循环如何参与姐妹染色单体的粘附及其释放。最后，将在非洲爪蟾卵提取物和哺乳动物组织培养细胞中研究粘附素的加载和稳定的调节。一个主要的重点将在3个不同类别的监管机构（Scc 2，Pds 5和Sgo）在这些过程中的作用。还将确定Scc 2和Pds 5对粘附素的ATP酶循环的影响。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，与肿瘤的发展和出生缺陷密切相关。

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.