Investigation of the dynamics of the SMC chromosome condensation complex at the single molecule level

单分子水平上SMC染色体缩合复合体动力学研究

• 批准号: 274661393
• 负责人: Professor Dr. Peter Graumann
• 金额: --
• 依托单位: Zentrum für Synthetische Mikrobiologie (SYNMIKRO)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2015
• 资助国家: 德国
• 起止时间: 2014-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/274661393?language=en
• 关键词: Investigation; dynamics; SMC; chromosome; condensation

SMC (structural maintenance of chromosomes) proteins perform central roles in various chromosome dynamics in all three domains of life. SMC proteins are ATPases and form the core of several protein complexes essential for chromosome cohesion and compaction in eukaryotes, and for chromosome segregation in bacteria. Loss of SMC or of its complex partners (ScpA and ScpB in bacteria) leads to the inability to proceed through Mitosis, and to severe defects in the compaction and segregation of chromosomes in prokaryotic cells. Thus, SMC proteins are key components of the cell cycle. We are studying the Bacillus subtilis SMC complex as a model for the bacterial SMC complex, which we have recently shown to form several distinct fractions in cells: SMCs not bound to ScpA and ScpB (which form a tight subcomplex) move throughout the entire chromosome, while 20% of SMC molecules are bound to ScpAB and statically localize to distinct sites on the chromosomes (termed condensation centres), one within each cell half, during most of the cell cycle. These specific assemblies are essential for chromosome segregation and are lost upon depletion of any of the SMC complex subunits. Exchange of SMC and ScpA from the condensation centres is different in terms of half time, with SMC exchanging faster than ScpA, and most ScpA molecules (86%) are statically positioned within the centres, while the remaining molecules freely diffuse through the cells. Thus, SMC shows a novel mode of interaction with DNA: the a static ScpAB-bound mode can not bind to DNA de novo in vitro, and the dynamic free mode can interact with and bind to DNA. We will use the visualization and tracking of single SMC-YFP molecules in live cells and advanced biochemical techniques to analyse the mode of formation of condensation centres, binding of SMC to DNA and the function of the ATPase cycle that SMC undergoes. We will analyse the movement of ATP binding, ATPase and many more mutant versions of SMC using single molecule microscopy (SMM) in B. subtilis cells, and analyse the requirement of recruitment of ScpA to condensation centres in a variety of mutant backgrounds. All experiments will be complemented by the analysis of the kinetics of complex formation and DNA binding in vitro. We will also determine if the static ScpA molecules in the condensation centres are associated with any specific region on the chromosome, using CHIP experiments, or if the structures are dynamically interacting with many sites on the chromosome, which we speculate is important for the maintenance of the preferred arrangement and folding of the bacterial chromosome. We will determine binding affinities of SMC, and mutant versions, and its complex partners in vitro using ITC and thermophoresis, to understand the unusual exchange rates in vivo. Visualizing SMC at the single molecule level in live cells will also be informative on the general principle of the interaction with DNA for SMC proteins in eukaryotic cells.

SMC（染色体结构维持）蛋白在生命的所有三个领域中的各种染色体动力学中起着核心作用。SMC蛋白是ATP酶，并形成真核生物中染色体凝聚和压实以及细菌中染色体分离所必需的几种蛋白质复合物的核心。SMC或其复合伴侣（细菌中的ScpA和ScpB）的丧失导致不能进行有丝分裂，并导致原核细胞中染色体的致密和分离的严重缺陷。因此，SMC蛋白是细胞周期的关键组成部分。我们正在研究枯草芽孢杆菌SMC复合物作为细菌SMC复合物的模型，我们最近已经证明在细胞中形成几个不同的部分：不与ScpA和ScpB结合的SMC SMC分子（形成紧密的亚复合物）在整个染色体上移动，而20%的SMC分子与ScpAB结合并静态定位于染色体上的不同位点。在细胞周期的大部分时间里，每个细胞半体中有一个冷凝中心。这些特定的组件是必不可少的染色体分离，并失去了任何SMC复合体亚基耗尽。SMC和ScpA从冷凝中心的交换在半衰期方面是不同的，SMC交换比ScpA快，并且大多数ScpA分子（86%）静态地定位在中心内，而其余分子自由地扩散通过细胞。因此，SMC与DNA的相互作用呈现出一种新的模式：ScpAB的静态结合模式在体外不能与DNA从头结合，而动态游离模式可以与DNA相互作用并结合。我们将使用活细胞中单个SMC-YFP分子的可视化和跟踪以及先进的生物化学技术来分析凝聚中心的形成模式，SMC与DNA的结合以及SMC经历的ATP酶循环的功能。我们将在B中使用单分子显微镜（SMM）分析ATP结合、ATP酶和更多SMC突变形式的运动。枯草杆菌细胞，并分析在各种突变体背景下的ScpA的凝聚中心招聘的要求。所有实验将通过分析体外复合物形成和DNA结合的动力学来补充。我们还将使用CHIP实验确定缩合中心中的静态ScpA分子是否与染色体上的任何特定区域相关，或者这些结构是否与染色体上的许多位点动态相互作用，我们推测这对于维持染色体上的许多位点很重要。细菌染色体的排列和折叠。我们将确定SMC的结合亲和力，和突变版本，其复杂的合作伙伴在体外使用ITC和热泳，以了解不寻常的交换率在体内。在活细胞中在单分子水平上可视化SMC也将为真核细胞中SMC蛋白与DNA相互作用的一般原理提供信息。