Structural studies on the P1 plasmid partition apparatus

描述（由申请人提供）：任何物种的生存都需要基因信息的忠实传承。这个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。虽然原核生物不经历与真核细胞相关的复杂有丝分裂步骤，但原核生物的染色体在细胞周期中通过分离或分离系统的作用动态排列。这个过程的协调需要精确的蛋白质- dna和蛋白质-蛋白质相互作用的par系统进行。所有已知的质粒编码par位点指定三个组成部分：一个顺式作用着丝粒位点（parS）和两个蛋白，ParB和ParA。在大肠杆菌P1 par系统中，ParA和ParB的基因形成一个操纵子，大约74 bp的分割位点parS位于ParB的下游。ParB是一种38 kDa的蛋白质，与任何蛋白质没有序列同源性，是一种dna结合蛋白，与IMF一起或单独与高度复杂的着丝粒样位点parS结合，形成分割复合物。ParA是一种44 kDa的walker型ATP酶，在与分割复合物中的ParB相互作用后，利用ATP水解的能量驱动质粒分离。我们最近对ParB和最小分割位点的结构测定揭示了ParB新的dna结合特征，这解释了它结合、传播和配对质粒的能力。因此，这些结构为分区复合体的形成机制提供了前所未有的见解。然而，完全未知的是质粒分离的结构基础，ParA进行的步骤。因此，在本提案中，我们将以我们最近的进展为基础，全面阐明P1的分配，具体目标如下：(1)通过对ParB-parS分配复合物的结构和生化研究，充分阐明P1分配复合物的形成机制。(2)通过对P1 ParA(apoParA、ParA- adp和ParA(K188Q)-（AMP-PCP)- parb（1-28）复合物）关键末端状态的结构和生化研究，阐明P1质粒分离的机制。这些结构将为理解分割系统如何驱动原核生物的染色体分离提供基础，并可能提供针对致病菌的潜在治疗干预点，致病菌也依赖于par系统进行分离并因此生存。

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.