Biochemical Analysis of Sister Chromatid Cohesion

描述(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。这个过程的中心是一个进化保守的染色体ATPase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，它采用独特的双臂结构，在每条手臂的末端都有一个与ATP结合的“头”结构域。在真核生物中，一种被称为粘附素的SMC蛋白复合体在有丝分裂和减数分裂的姐妹染色单体凝聚中起着关键作用。在这项提案中，将采取多学科的方法来了解SMC蛋白在体外是如何在机制水平上发挥作用的，以及它们的功能在体内是如何调节的。首先，枯草芽孢杆菌的细菌SMC蛋白复合体将被用作理解SMC蛋白的基本作用的模型系统。一套创新的功能分析，结合广泛的定点突变，将被用于解决SMC的机械循环如何与其催化循环相耦合的问题。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，粘附素的亚复合体和全复合体将由它的重组亚基重组而来，它们的活性将在纯化系统和非洲爪哇卵无细胞提取物中进行表征。将构建复合体的突变形式，以确定粘附素的机械力化学循环如何参与姐妹染色单体的聚集及其释放。最后，将在非洲爪哇卵提取液和哺乳动物组织培养细胞中研究粘附素的负载和稳定性的调节。重点将重点放在3个不同级别的监管机构(SCC2、Pds5和SGO)在这些过程中的作用。Scc2和Pds5对粘附素ATPase周期的影响也将被确定。从这项工作中获得的信息最终将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.