Cell cycle Assembly of Nucleoprotein Complexes

核蛋白复合物的细胞周期组装

• 批准号: 8006405
• 负责人: ALAN Carl LEONARD
• 金额: $ 24.81万
• 依托单位: FLORIDA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8006405
• 关键词: Affect; Affinity; Bacteria; Base Pairing; Base Sequence; Binding; Binding Sites; Biological Assay; Cell Cycle; Cells; Chromosomes; Complex; Coupled; DNA; DNA biosynthesis; Data; Disease; Ensure; Escherichia coli; Frequencies; Genome; Goals; Growth; Growth Inhibitors; In Vitro; Malignant Neoplasms; Measures; Modeling; Molecular; Mutate; Nucleoproteins; Nucleotides; Occupations; Pattern; Phase; Play; Positioning Attribute; Pre-Replication Complex; Process; Progress Reports; Proteins; Regulation; Replication Origin; Research; Role; Site; Staging; System; Testing; Time; Titrations; base; cell growth; chromosome replication; design; enteric pathogen; improved; in vivo; mutant; novel; origin recognition complex; pathogen; pathogenic bacteria; preference; protein complex; public health relevance

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.

描述（由申请人提供）：该提案侧重于触发染色体复制开始的DNA-蛋白质复合物的形成，长期目标是了解控制细菌细胞生长的分子机制。在细菌中，染色体复制的起始速率决定了细胞复制的频率，并且需要复制前复合物（pre-RC）的组装，所述复制前复合物包含结合至复制起点oriC的保守起始DNA A的多个拷贝。我们的假设是，oriC序列包含确保前RC在所有生长速率下在细胞周期中的正确时间组装所需的所有信息。我们的假设是基于我们的观察：1）E。coli oriC含有高亲和力的DnaA结合位点，这些位点在整个细胞周期中被占据，形成细菌起源识别复合物（ORC）2）ORC位点位于低亲和力位点阵列的两侧，这些低亲和力位点仅在染色体复制起始时与DnaA相互作用以形成前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DnaA结合来调节起始的多种机制的靶点;以及4）在不存在ORC的情况下，DnaA不能占据低亲和力位点。基于这些观察，我们的目标是确定如何安排高和低亲和力DnaA结合位点在大肠杆菌。大肠杆菌oriC指导ORC前RC转换，并在细胞周期中调节起始时间，并了解这种转换如何通过生长速率依赖性机制进行调节。具体目标是：2）测试以下假设：低亲和力DnaA识别位点的相控阵列的加载是前RC形成的晚期阶段，其决定对DnaA-ATP的需求并调节细胞周期期间的起始时间;和3）检验生长速率调节的Fis和IHF与oriC的结合调节低亲和力位点占据所需的DnaA水平的假设。实现这些目标将推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合物组装的理解。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。这里描述的研究也将有助于确定新的目标，以指导设计新的细胞生长抑制剂的细菌病原体。 公共卫生相关性：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，以治疗癌症或病原菌引起的疾病。