Cell cycle Assembly of Nucleoprotein Complexes

描述(由申请人提供)：这项建议侧重于触发染色体复制开始的DNA-蛋白质复合体的形成，长期目标是了解控制细菌细胞生长的分子机制。在细菌中，染色体复制的启动速度决定了细胞复制的频率，并需要组装复制前复合体(Pre-RC)，该复合体由与复制起点ORIC结合的保守启动子Dna A的多个副本组成。我们的假设是，ORIC序列包含确保前RC在细胞周期的正确时间在所有生长速度下组装所需的所有信息。我们基于我们的观察，认为：1)E.coliORIC含有高亲和力的Dna A结合位点，这些结合位点在整个细胞周期中被占据，形成一个细菌起源识别复合体(ORC)；2)ORC位点位于低亲和力位点阵列的两侧，只有在启动染色体复制时才与Dna A相互作用形成前-RC；因此，低亲和力位点的占据决定了起始时间；3)低亲和力位点是通过调节Dna A结合来调节启动的多种机制的靶点；以及4)在没有ORC的情况下，Dna A不能占据低亲和力位点。基于这些观察，我们这项建议的目标是定义E.ColiORIC中高亲和力和低亲和力DNAA结合位点的排列如何引导ORC到前RC转变，并调控细胞周期中的启动时间，并了解这种转变可能如何受到生长速度依赖的机制的调控。其具体目的是：1)测试低亲和力DNAA识别位点的加载和稳定占据依赖于ORC近端位点的假说；2)测试低亲和力DNAA识别位点相控阵的加载是前RC形成的后期阶段的假说，它决定了对DNAA-ATP的需求并调节细胞周期中的起始时间；以及3)测试生长速度调节FIS和IHF与ORIC的结合调节低亲和力位点占据所需的DNAA水平的假说。实现这些目标将促进我们对细菌起源中的序列信息如何指导精确定时的细胞周期特异性复杂组装的理解。这些信息将提高我们对肠道和非肠道病原体生长调节的理解。这里描述的研究还将有助于识别新的靶点，以指导针对细菌病原体的新型细胞生长抑制剂的设计。 与公共卫生相关：拟议研究的长期目标是了解控制细胞生长的机制。了解这些机制将有助于确定新的靶点，以指导细胞生长抑制剂的设计，以治疗癌症或由致病菌引起的疾病。

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.