Study of the mechanism of bacterial chromosome partitioning systems

DNA复制后，细菌染色体和低拷贝数质粒的子拷贝必须分离到两个子细胞中，以确保遗传。因此，在细胞分裂发生之前，系统已经进化到主动地将基因组的复制副本分割成细胞的两半。然而，原核生物没有真核生物的有丝分裂机制。一类常见的细菌系统包括三个组成部分：分离染色体上的一个特定DNA序列，其功能相当于细菌着丝粒；以及两个蛋白质因子，一个与着丝粒结合，另一个具有ATP依赖的非特异DNA结合活性的ATPase。大肠杆菌的P1-质粒和F-质粒都配备了这样的系统。这些系统的着丝粒通常称为PARB着丝粒结合蛋白，PARB着丝粒结合蛋白与PARB着丝粒结合蛋白结合，ParA是分区ATPase。涉及同源蛋白的密切相关系统不仅参与了多种细菌物种的染色体DNA分离，而且还参与了细菌中大型蛋白质细胞器的分离，如蓝藻中的羧基体。 对其中一些系统的体内成像研究表明，在DNA复制之前，ATPase蛋白形成了振荡焦点，并伴随着DNA在细胞内的振荡。在一些系统中，在细胞分裂之前，PARB结合的PARS基因座的一个复制副本停留在细胞的一端附近，另一个副本向另一端移动。然而，这些生物分子转运系统的详细分子机制仍然知之甚少。这个项目旨在通过结合各种技术来研究这些反应系统动态方面的生化和生物物理机制，包括我们建立的重组的无细胞反应系统，它概括了体内系统动力学的方面。 使用灵敏的荧光显微镜系统研究这些动态反应体系的技术和仪器已经被开发出来。利用荧光标记的ParA和PARB蛋白，在不同的反应条件下，监测了这些蛋白与密集固定在模拟细菌核仁的载玻片表面(DNA地毯)上的DNA分子的结合/解离动力学。通过使用P1和F质粒Para/B/S作为模型系统，我们了解到在三磷酸腺苷存在的情况下，ParA与非特异性DNA结合，具有快速的开关速率。此外，还研究了对位ATPase反应的稳态前动力学分析和ATP诱导的对位构象变化。已经观察到DNA结合所需的准构象变化发生在ATP结合之后的一段时间延迟，导致了一个质粒DNA运动的机制模型。我们已经成功地重组了无细胞系统，以观察在DNA地毯覆盖的流动细胞中，在ParA和PARB蛋白存在的情况下，携带PARS的荧光标记DNA的动态行为。这一研究导致我们提出了一类新的生物货物运输系统的力学模型，我们称之为扩散-棘轮机制。这个模型包括一个产生局部蛋白质分布梯度的反应-扩散过程和一个通过机械耦合机制将DNA地毯上的蛋白质分布梯度或体内细菌类核转化为驱动力的化学电泳法原理。结合生化、生物物理和数学方法，目前正在研究ATP驱动的质粒DNA动力学的进一步机制细节。我们目前的工作集中在表征在非特异性DNA和部分DNA存在下形成的对位PARB复合体。 该项目的最新进展包括我们对F-质粒分配复合体组装机制的了解进展。二十多年来，人们已经知道F-质粒及其同源物的PARB蛋白不仅与其同源部分DNA序列结合，而且DNA结合扩散到附近的非特异性DNA区域，形成含有大量PARB分子的浓缩分配蛋白-DNA复合体。然而，这些复合体是如何组装的仍然是一个谜。最近，该家族的PARB蛋白被发现与CTP结合并具有CTPase活性。我们发现，F-质粒的PARB蛋白与PARB蛋白相互作用，并通过PARB蛋白诱导ATP水解，将其从非特异(染色体)DNA中解离出来的方式是由CTP和PARS DNA协调控制的，以调节系统，通过我们提出的扩散-棘轮机制产生有效的动力。 我们还将该项目扩展到包括化脓性链球菌质粒pSM19035分区系统，该分区系统属于同一ParABS家族分区系统中的一个单独类别。该系统所属的一组ParABS系统具有PARB着丝粒结合蛋白，在结构上与F-和P1质粒系统以及染色体PAR系统无关。最值得注意的是，它们不具有CTPase活性，也缺乏PARB传播能力。我们发现pSM19035的PAR系统通过配位复杂的结构和对ATPase的激活机制发挥作用，与那些涉及PARB-CTPase蛋白的系统非常不同。 这项研究在一定程度上是为了提高我们的一般知识，即一组蛋白质分子如何协调细胞事件的空间控制，这些事件发生的长度规模比涉及的单个蛋白质分子大得多，而不需要组装跨越距离的聚合蛋白质细丝。

After DNA replication, daughter copies of the bacterial chromosome and low copy number plasmids must be segregated into two daughter cells to ensure genetic inheritance. Therefore, systems have evolved to actively partition the replicated copies of the genome to two halves of the cell before cell division takes place. However, prokaryotes do not have the mitotic machinery of eukaryotes. One commonly found class of bacterial systems involve three components; a specific DNA sequence on the segregating chromosome that functions as the bacterial equivalent of a centromere, and two protein factors, one binds to the centromere and the other an ATPase with ATP-dependent non-specific DNA binding activity. E. coli P1-plasmid and F-plasmid are both equipped with such systems. The centromeres of these systems are generally called parS, to which ParB centromere-binding protein binds, and ParA is the partition ATPase. Closely related systems involving homologous proteins have been found to be involved not only in the chromosomal DNA segregation in a variety of bacterial species, but also in the segregation of large proteinous organelles in bacteria, such as carboxysomes in cyanobacteria. In vivo imaging studies on some of these systems have demonstrated oscillating focus formation of the ATPase protein and accompanied oscillation of the plasmid DNA within the cell prior to DNA replication. In some of the systems, one replicated copy of the ParB-bound parS locus stays near one end of the cell and the other copy moves toward the other end prior to cell division. However, the detailed molecular mechanism of these bio-molecular transport systems is still poorly understood. This project aims to investigate the biochemical and biophysical mechanism of the dynamic aspects of these reaction systems by combining a variety of techniques, including a reconstituted cell-free reaction systems we have established that recapitulates aspects of the in vivo system dynamics. Techniques and instruments have been developed to study these dynamic reaction systems by using a sensitive fluorescence microscope system. By using fluorescence-labeled ParA and ParB proteins, association/dissociation dynamics of these proteins with DNA molecules densely immobilized on a slide glass surface (DNA-carpet) mimicking bacterial nucleoid were monitored under a variety of reaction conditions. By using P1- and F-plasmid ParA/B/S as model systems, we learned that ParA, in the presence of ATP, associates with non-specific DNA with rapid on- and off-rates. A pre-steady state kinetic analysis of the ParA ATPase reaction and the ATP-induced conformational change of ParA have also been studied. The ParA conformational change necessary for DNA binding has been observed to take place with a time delay following ATP binding, leading to a mechanistic model of plasmid DNA motion. We have successfully reconstituted cell-free systems to observe ATP-driven dynamic behaviors of the fluorescence-labeled plasmid DNA carrying parS in the presence of ParA and ParB proteins, within a flow cell coated by DNA-carpet. This study led us to propose a new class of mechanistic model for bio-cargo transport systems we call diffusion-ratchet mechanism. This model comprises a reaction-diffusion process that generates a local protein distribution gradient and a chemophoretic principle of motive force generation that converts the protein distribution gradient on the DNA-carpet, or bacterial nucleoid in vivo, to the motive force via a mechano-coupling mechanism. Further mechanistic details of the ATP-driven plasmid DNA dynamics are currently studied combining biochemical, biophysical and mathematical approaches. Our efforts are currently focused on the characterization of ParA-ParB complexes that form in the presence of both non-specific and parS DNA. Latest advances in this project include progress in our understanding of the mechanism of the F-plasmid partition complex assembly. For more than two decades, it has been known that ParB protein of the F-plasmid and its homologues not only bind to their cognate parS DNA sequence, DNA-binding spread to nearby non-specific DNA areas, forming condensed partition protein-DNA complexes containing large numbers of ParB molecules. However, how such complexes are assembled remained a mystery. Recently, ParB proteins of this family have been found to bind CTP and have CTPase activity. We found that the way ParB protein of the F-plasmid interacts with ParA protein and induce ATP hydrolysis by ParA to dissociate it from non-specific (chromosomal) DNA is coordinately controlled by CTP and parS DNA to tune the system for efficient motive force generation by the diffusion-ratchet mechanism we proposed. We have also expanded this project to include Streptococcus pyogenes plasmid pSM19035 partition system, which belongs to a separate class within the same ParABS family of partition systems. The group of ParABS systems to which this system belongs have ParB centromere-binding proteins structurally unrelated to those of F- and P1 plasmid systems and chromosomal Par systems. Most notably, they do not have the CTPase activity and lack the ParB spreading capability. We are finding the pSM19035 Par system functions via partition complex architecture and ParA ATPase activation mechanism quite distinct from those systems involving ParB-CTPase proteins. This study is in part aimed at advancement of our general knowledge on how a set of protein molecules could orchestrate a spatial control of cellular events that occur with a much larger length-scale than the individual protein molecules involved, without assembling polymeric protein filaments that spans the distance.