Structural studies on the P1 plasmid partition apparatus

描述（申请人提供）：任何物种的生存都需要遗传信息的忠实继承。这一过程的关键是染色体或质粒的定向运动和定位，以便它们在细胞分裂时准确地分布到子细胞中。虽然原核生物不经历与真核细胞相关的复杂的有丝分裂步骤，但原核生物的染色体在细胞周期中通过分离或par系统的作用动态排列。这一过程的协调需要由par系统进行精确的蛋白质-DNA和蛋白质-蛋白质相互作用。所有已知的质粒编码的par位点都有三个组成部分：一个顺式作用的着丝粒位点（parS）和两个蛋白质ParB和ParA。coli P1 par系统，该系统已经作为理解分配的范例，帕拉和ParB的基因形成操纵子，并且大约74 bp的分配位点parS位于parB的紧下游。ParB是一种与任何蛋白质都没有序列同源性的38 kDa蛋白质，是一种DNA结合蛋白质，并且与IMF一起沿着或单独结合到高度复杂的着丝粒样位点parS上，以形成分配复合物。帕拉是一种分子量为44 kDa的Walker型ATP酶，在与ParB相互作用后，利用ATP水解的能量驱动质粒分离。我们最近的结构测定的ParB和最小分区网站揭示了新的DNA结合特性的ParB解释其能力，结合，传播和配对质粒。因此，这些结构提供了前所未有的深入了解分区复合物的形成机制。然而，完全未知的是质粒分离的结构基础，这一步骤由ParA进行。因此，在本提案中，我们将建立在我们最近的进展，以充分阐明P1分配与以下具体目标：（1）充分阐明P1分配复合物形成的机制，通过对ParB-parS分配复合物的结构和生物化学研究。(2)通过对P1帕拉关键末端状态（apo帕拉、ParA-ADP和帕拉（K188 Q）-（AMP-PCP）-ParB（1-28）复合物）的结构和生化研究，阐明P1质粒分离的机制。这些结构将为理解分区系统如何发挥作用以驱动原核生物中的染色体分离提供基础，并可能提供针对致病细菌的治疗干预的潜在点，这些致病细菌也依赖于par系统进行分离，从而存活。

DESCRIPTION (provided by applicant): The survival of any species demands the faithful inheritance of genetic information. Essential to this process are the directed movements and positioning of chromosomes or plasmids such that they are accurately distributed to the daughter cells at cell division. Although prokaryotes do not undergo the complex mitotic steps associated with eukaryotic cells, prokaryotic chromosomes are nevertheless dynamically arranged during the cell cycle via the action of segregation, or par systems. Coordination of this process requires precise protein-DNA and protein-protein interactions carried out by par systems. All known plasmid encoded par loci specify three components: a cis-acting centromere site (parS), and two proteins, ParB and ParA. In the E. coli P1 par system, which has served as a paradigm for understanding partition, the genes for ParA and ParB form an operon, and the approximately 74 bp partition site, parS, is located immediately downstream of parB . ParB, a 38 kDa protein with no sequence homology to any protein, is a DNA-binding protein and, along with IMF or alone, binds to a highly complex centromere-like site, parS, to form the partition complex. ParA, a 44 kDa Walker-type ATPase, utilizes the energy of ATP hydrolysis to drive plasmid separation after interacting with ParB in the partition complex. Our recent structure determinations of ParB and the minimal partition site have revealed novel DNA-binding characteristics of ParB that explain its ability to bind, spread and pair plasmids. Thus, these structures have provided unprecedented insight into the mechanism of partition complex formation. Completely unknown, however, are structural bases for plasmid separation, the step carried out by ParA. Thus, in this proposal we will build on our recent progress towards a full elucidation of P1 partition with the following Specific Aims: (1) fully elucidate the mechanism of P1 partition complex formation through structural and biochemical studies on the ParB-parS partition complex. (2) Clarify the mechanism of P1 plasmid separation via structural and biochemical studies on the key end states of P1 ParA (apoParA, ParA-ADP and the ParA(K188Q)-(AMP-PCP)-ParB(1-28) complex). These structures will provide the foundation for understanding how partition systems function to drive chromosome segregation in prokaryotes and may provide potential points of therapeutic intervention against pathogenic bacteria, which also depend on par systems for segregation and thus, survival.