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

SMC(染色体的结构维持)蛋白在生命的所有三个领域的各种染色体动力学中发挥着核心作用。SMC蛋白是ATPase，是几种蛋白质复合体的核心，对真核生物中的染色体凝聚和紧凑以及细菌中的染色体分离是必不可少的。SMC或其复杂配对(细菌中的SCPA和SCPB)的丢失导致无法进行有丝分裂，并导致原核细胞染色体紧凑和分离的严重缺陷。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复合体作为细菌SMC复合体的模型，我们最近发现它在细胞中形成几个不同的组分：未与SCPA和SCPB结合的SMC在整个染色体上移动，而20%的SMC分子与ScpAB结合并静态地定位在染色体上的不同位置(称为凝聚中心)，在细胞周期的大部分时间里，每个细胞一半内一个SMC。这些特定的组装对染色体分离是必不可少的，一旦任何SMC复合体亚基耗尽，它们就会丢失。SMC和SCPA从凝聚中心交换的时间在一半时间上是不同的，SMC交换的速度比SCPA快，大多数SCPA分子(86%)静态地定位在凝聚中心，而其余的分子自由扩散到细胞中。因此，SMC呈现出一种新的与DNA相互作用的模式：静态的ScpAB结合模式不能在体外与DNA从头结合，而动态的自由模式可以与DNA相互作用和结合。我们将利用活细胞中单个SMC-YFP分子的可视化和跟踪以及先进的生化技术来分析SMC凝聚中心的形成方式、SMC与DNA的结合以及SMC经历的ATPase循环的功能。我们将利用单分子显微镜(SMM)分析枯草杆菌细胞中ATP结合、ATPase和更多SMC突变版本的运动，并分析在各种突变背景下SCPA向凝聚中心募集的需求。所有实验都将得到络合物形成和DNA体外结合动力学分析的补充。我们还将使用芯片实验来确定凝聚中心的静态SCPA分子是否与染色体上的任何特定区域有关，或者这些结构是否与染色体上的许多位置动态相互作用，我们推测这对于维持细菌染色体的首选排列和折叠是重要的。我们将在体外利用ITC和热电泳法测定SMC、突变型及其复杂伙伴的结合亲和力，以了解体内异常的交换率。在活细胞中在单分子水平上可视化SMC也将有助于了解真核细胞中SMC蛋白与DNA相互作用的一般原理。

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.