The Role of the DnaX Complex in Assembling Pol III Initation Complexes

DnaX 复合物在组装 Pol III 引发复合物中的作用

• 批准号: 7782784
• 负责人: Christopher Dale Downey
• 金额: $ 5.22万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-04-28 至 2011-04-27
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7782784
• 关键词: ATP phosphohydrolase; Affect; Bacteria; Binding; Biological Assay; Biological Models; Cell Division Process; Cells; Complex; DNA; DNA Polymerase III; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Development; Devices; Disease; Escherichia coli; Fluorescence Resonance Energy Transfer; Genes; Genetic; Genome; Goals; Holoenzymes; Human; In Vitro; Infection; Kinetics; Laboratories; Lead; Length; Malignant Neoplasms; Measures; Modeling; Molecular; Molecular Chaperones; Play; Polymerase; Process; Property; Protein Subunits; Proteins; Reaction; Recruitment Activity; Research; Role; Site; Slide; System; Techniques; Testing; Time; Work; base; effective therapy; fluorophore; in vivo; public health relevance; reconstitution; research study; stopped-flow fluorescence

DESCRIPTION (provided by applicant): All cells must rapidly and accurately replicate their DNA during the process of cell division. DNA replication is carried out by complex multi-protein assemblies, which must be recruited to and loaded onto the template DNA in a highly coordinated fashion. The DNA polymerase III (Pol III) holoenzyme from the bacterium E. coli is a model system for studies of DNA replication. A key component of the Pol III holoenzyme is the 5 subunit DnaX complex, which has known functions in loading the "sliding clamp" processivity factor onto the DNA and in coordinating leading and lagging strand synthesis. One of the DnaX subunits, the product of the DnaX gene, occurs in vivo as either a full length or truncated version. While both forms support processive DNA synthesis, there are important functional differences between the two components. The goal of this proposal is to ascertain how the two forms of the DnaX protein affect the mechanism for initiating DNA replication and to thereby better understand the role of the DnaX complex. Rapid kinetic assays will be developed to measure the rate for assembling the Pol III holoenzyme on a DNA template. Both a KinTek rapid mix/quench device and fluorescence resonance energy transfer techniques will be utilized. The kinetic advantage of the full length DnaX protein over the truncated version will be established experimentally. The mechanism by which holoenzyme/DNA initiation complexes are formed will be elaborated by examining the kinetic effects as the concentration of each holoenzyme component is varied. These experiments will test the hypothesis that the core Pol III DNA polymerase is "chaperoned" to the initiation complex by DnaX complexes containing the full length DnaX protein. PUBLIC HEALTH RELEVANCE The fast and accurate replication of genetic information is a fundamental process of cell division. Thus, research that deepens our understanding of this process is crucial for developing more effective treatments for cancer or pathogenic infection. The results of this proposal will establish the role of a key component in the DNA replication machinery and will lead to a greater understanding of how DNA replication is initiated at the molecular level.

描述（由申请人提供）：所有细胞在细胞分裂过程中都必须快速、准确地复制其 DNA。 DNA 复制是通过复杂的多蛋白组装体进行的，必须以高度协调的方式将其招募并加载到模板 DNA 上。来自大肠杆菌的 DNA 聚合酶 III (Pol III) 全酶是研究 DNA 复制的模型系统。 Pol III 全酶的一个关键成分是 5 亚基 DnaX 复合物，已知其具有将“滑动钳”持续合成因子加载到 DNA 上以及协调前导链和滞后链合成的功能。 DnaX 亚基之一是 DnaX 基因的产物，在体内以全长或截短形式存在。虽然这两种形式都支持持续 DNA 合成，但两种成分之间存在重要的功能差异。该提案的目的是确定 DnaX 蛋白的两种形式如何影响启动 DNA 复制的机制，从而更好地了解 DnaX 复合物的作用。将开发快速动力学测定法来测量 Pol III 全酶在 DNA 模板上的组装速率。将使用 KinTek 快速混合/猝灭装置和荧光共振能量转移技术。全长 DnaX 蛋白相对于截短版本的动力学优势将通过实验确定。通过检查随着每种全酶成分浓度变化的动力学效应，将详细阐述全酶/DNA起始复合物形成的机制。这些实验将检验以下假设：核心 Pol III DNA 聚合酶由含有全长 DnaX 蛋白的 DnaX 复合物“陪伴”至起始复合物。公共卫生相关性 遗传信息的快速、准确复制是细胞分裂的基本过程。因此，加深我们对这一过程的理解的研究对于开发更有效的癌症或病原体感染治疗方法至关重要。该提案的结果将确定 DNA 复制机制中关键组件的作用，并将导致人们更好地了解 DNA 复制如何在分子水平上启动。