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

SMC（染色体结构维持）蛋白在生命的所有三个领域的各种染色体动力学中起着核心作用。SMC蛋白是atp酶，是真核生物染色体内聚和压实以及细菌染色体分离所必需的几种蛋白质复合物的核心。SMC或其复杂伙伴（细菌中的ScpA和ScpB）的缺失导致无法进行有丝分裂，并导致原核细胞中染色体的压实和分离存在严重缺陷。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复合体作为细菌SMC复合体的模型，我们最近已经证明它在细胞中形成了几个不同的部分：不与ScpA和ScpB结合的SMC分子（形成紧密的亚复合体）在整个染色体中移动，而20%的SMC分子与ScpAB结合并静态定位在染色体上的不同位置（称为冷凝中心），在细胞周期的大部分时间里，每个细胞一半内一个。这些特定的组合对于染色体分离是必不可少的，并且在任何SMC复合体亚基耗尽时都会丢失。SMC和ScpA从缩聚中心交换的半时间不同，SMC的交换速度比ScpA快，大多数ScpA分子（86%）静态地定位在缩聚中心内，而剩余的分子则在细胞中自由扩散。因此，SMC显示出一种新的与DNA相互作用的模式：静态scab结合模式在体外不能与DNA从头结合，而动态自由模式可以与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化技术来分析SMC凝结中心的形成模式，SMC与DNA的结合以及SMC所经历的atp酶循环的功能。我们将利用单分子显微镜（SMM）分析枯草芽孢杆菌细胞中ATP结合、ATP酶和更多SMC突变体的运动，并分析在各种突变背景下ScpA聚集到凝聚中心的要求。所有的实验将辅以分析动力学的复杂的形成和DNA结合在体外。我们还将利用CHIP实验确定冷凝中心的静态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细菌染色体的首选排列和折叠很重要。我们将在体外使用ITC和热泳术来确定SMC、突变版本及其复杂伙伴的结合亲和力，以了解体内不寻常的交换率。在活细胞的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。

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.