Cell cycle Assembly of Nucleoprotein Complexes

描述（由申请人提供）：该提案侧重于dna -蛋白质复合物的形成，触发染色体复制的发生，长期目标是了解控制细菌细胞生长的分子机制。在细菌中，染色体复制的起始速率决定了细胞复制的频率，并且需要一个复制前复合体（pre-RC）的组装，该复合体包括与复制起点oriC结合的保守启动dna的多个拷贝。我们的假设是，oriC序列包含所有必要的信息，以确保pre-RC在细胞周期中以所有生长速率在正确的时间组装。我们基于以下观察得出这一假设：1)大肠杆菌oriC含有高亲和力的dna结合位点，在整个细胞周期中占据，形成细菌起源识别复合物（ORC）；2) ORC位点的低亲和位点侧阵列仅在染色体复制开始时与dna相互作用形成pre-RC；因此，低亲和力位点的占用决定起始时间；3)低亲和力位点是通过调节dna结合调控起始的多种机制的靶点；4)在没有ORC的情况下，dna不能占据低亲和力位点。基于这些观察结果，我们的目标是确定大肠杆菌oriC中高亲和力和低亲和力dna结合位点的排列如何指导ORC向pre-RC过渡并调节细胞周期中的起始时间，并了解这种过渡如何通过生长速率依赖机制进行调节。具体目的是：1)验证低亲和力dna识别位点的加载和稳定占据依赖于近端ORC位点的假设；2)验证低亲和力dna识别位点的相控阵加载是pre-RC形成的后期，决定了细胞周期中dna - atp的需求并调节起始时间的假设；3)检验生长速度调节Fis和IHF与oriC结合的假设，并调节低亲和力位点占用所需的dna水平。完成这些目标将促进我们对细菌起源中的序列信息如何精确地指导定时的、细胞周期特定的复合物组装的理解。这一信息将提高我们对肠道和非肠道病原体生长调节的理解。这里描述的研究也将有助于确定新的目标，以指导设计针对细菌病原体的新型细胞生长抑制剂。

DESCRIPTION (provided by applicant): This proposal focuses on formation of DNA-protein complexes that trigger the onset of chromosome duplication, with the long term objective of understanding molecular mechanisms that control bacterial cell growth. In bacteria, the rate of initiation of chromosome replication determines the frequency of cell duplication and requires assembly of a pre-replication complex (pre-RC), comprising multiple copies of the conserved initiator DnaA bound to an origin of replication, oriC. Our hypothesis is that the oriC sequence contains all the information required to ensure that the pre-RC is assembled at the correct time in the cell cycle at all growth rates. We base this hypothesis on our observations that: 1) E. coli oriC contains high affinity DnaA binding sites that are occupied throughout the cell cycle, forming a bacterial Origin Recognition Complex (ORC); 2) The ORC sites flank arrays of lower affinity sites which interact with DnaA to form the pre-RC only at the time of initiation of chromosome replication; thus occupancy of low affinity sites determines initiation timing; 3) Low affinity sites are targets for multiple mechanisms that regulate initiation by modulating DnaA binding; and 4) DnaA cannot occupy low affinity sites in the absence of ORC. Based on these observations our goals for this proposal are to define how arrangement of high and low affinity DnaA binding sites in E. coli oriC directs ORC to pre-RC transition and regulates timing of initiation during the cell cycle, and to understand how this transition may be regulated by growth rate-dependent mechanisms. The Specific Aims are to: 1) Test the hypothesis that loading and stable occupation of low affinity DnaA recognition sites is dependent on proximal ORC sites; 2) Test the hypothesis that loading of phased arrays of low affinity DnaA recognition sites is a late stage of pre-RC formation that determines the requirement for DnaA-ATP and regulates initiation timing during the cell cycle; and 3) Test the hypothesis that growth rate regulated binding of Fis and IHF to oriC modulates the level of DnaA required for low affinity site occupation. Accomplishing these aims will advance our understanding of how sequence information in bacterial origins directs precisely timed, cell cycle specific complex assembly. This information will improve our understanding of growth regulation in both enteric and non-enteric pathogens. Studies described here will also aid in the identification of new targets appropriate to guide the design of novel cell growth inhibitors for bacterial pathogens. PUBLIC HEALTH RELEVANCE: The long term goal of the proposed research is to understand mechanisms that control cell growth. Understanding these mechanisms will be useful in identifying new targets to guide the design of cell growth inhibitors to treat cancer or diseases caused by pathogenic bacteria.