Establishment of sister chromatid cohesion

 描述(申请人提供)：SMC(染色体的结构维护)复合体通过将染色体的不同区域连接在一起来调节更高阶的染色体结构。一种SMC复合体，粘附素，在几个DNA过程中发挥作用，包括那些需要在染色体之间系系的过程(姐妹染色单体凝聚和DNA修复)，以及那些需要在同一染色体的两个区域之间系系的过程(凝聚和转录调节)。粘附素的这些不同功能被认为可以调节干细胞中的基因表达，并防止导致癌症和出生缺陷的染色体传递错误。粘附素具有显着的环状结构，这是由四个ITS亚基Smc1、Smc3、Scc3和Mcd1(也称为Scc1/Rad21)相互作用产生的(图1)。粘附素还包含两个与SMC亚基相关的ATPase。粘附素的这些显著的生物学和生化特性激发了推动这一提议的两个问题：1)粘附素的结构和ATPase活性如何调节其与DNA双链的结合，并使其能够连接两个不同的DNA双链？2)如何调节粘附素，以确保它在非常不同的生物过程中正常发挥作用？目的1探讨SMC3ATPase和SMC1ATPase在体内调节粘附素与染色体结合和染色体拴系中的关键但尚未被探索的不对称作用(S)。目的2描述一种新发现的粘附素活性、其寡聚作用及其产生系链的潜力。这一目标的实验将：)测试粘附素寡聚在凝聚和缩合中的作用；2)询问寡聚化如何在细胞周期进程中变化，受DNA结合调控和粘附素调节剂控制；以及3)鉴定介导寡聚化的SMC亚单位内的氨基酸序列。通过通过SMC ATPase的新作用和粘附素齐聚来研究粘附素，AIMS 1和2将阐明体内粘附素功能的未知方面。在目标3中，将通过体外实验来研究粘附素在体内活性的分子基础，以测量粘附素在DNA上的结合和扩散，它的寡聚，它的拴系活性，以及与这些活性相关的任何结构变化。这些分析将为开发粘附素的DNA结合和拴系活动的特定分子模型提供框架。目的4将探索一组新发现的调节子如何在后期之前解析粘附素的功能，以确保姐妹染色单体凝聚力的正确维持和凝聚的建立。这些分析将提供对粘附素在染色体分离中的基本功能的洞察，并为如何调节SMC复合体的活动以执行不同的生物功能提供一个范例。这四个目标是开发捕获不同功能状态的粘附素的不寻常的遗传等位基因，用于粘附素齐聚的新的细胞生物学、遗传学和生化分析，以及用于在体外评估粘附素DNA结合和拴系的新群体和单分子分析。

 DESCRIPTION (provided by applicant): Smc (structural maintenance of chromosomes) complexes mediate higher-order chromosome structure by tethering together different regions of chromosomes. One Smc complex, cohesin, functions in several DNA processes, including those that require tethers between chromosomes (sister chromatid cohesion and DNA repair) and those that require tethers between two regions in the same chromosome (condensation and transcription regulation). These diverse functions of cohesin are thought to regulate gene expression in stem cells and to prevent errors in chromosome transmission that lead to cancer and birth defects. Cohesin has a remarkable ring architecture resulting from the interactions of four its subunits, Smc1, Smc3, Scc3, and Mcd1 (also known as Scc1/Rad21) (Fig. 1). Cohesin also contains two ATPases associated with the Smc subunits. These remarkable biological and biochemical properties of cohesin inspire the two questions that drive the aims in this proposal: 1) How do cohesin's architecture and ATPase activities mediate its binding to a DNA duplex and enable it to tether two different DNA duplexes? and 2) How is cohesin regulated to ensure that it functions properly in very diverse biological processes? Aim 1 explores the critical but unexplored asymmetric role(s) of the Smc3 and Smc1 ATPases in regulating cohesin binding to chromosomes and chromosome tethering in vivo. Aim 2 characterizes a newly-detected cohesin activity, its oligomerization and its potential to generate tethers. Experiments in this aim will: ) test the role of cohesin oligomerization in cohesion and condensation; 2) interrogate how oligomerization changes during cell cycle progression, is modulated by DNA binding and controlled by cohesin regulators; and 3) identify the amino acid sequences within the Smc subunits that mediate oligomerization. By studying cohesin through the novel roles of the Smc ATPases and cohesin oligomerization, Aims 1 and 2 will elucidate unexplored aspects of cohesin function in vivo. In Aim 3, the molecular basis for cohesin's in vivo activities will be interrogated by in vitro assays to measure cohesin's binding and diffusion along DNA, its oligomerization, its tethering activities, and any structural changes associated with these activities. These analyses will provide the framework to develop a specific molecular model for cohesin's DNA binding and tethering activities. Aim 4 will explore how a newly identified group of regulators parse cohesin function prior to anaphase to ensure the proper maintenance of sister chromatid cohesion and the establishment of condensation. These analyses will provide insights into cohesin's essential functions in chromosome segregation and provide a paradigm for how the activities of Smc complexes are regulated to carry out distinct biological functions. The four aims exploit unusual genetic alleles that trap distinct functional states of cohesin, new cell biological, genetic and biochemical assays for cohesin oligomerization and new population and single molecule assays for assessing cohesin DNA binding and tethering in vitro.