THERMODYNAMICS OF PROCARYOTIC DNA REPLICATION

原核 DNA 复制的热力学

• 批准号: 3306118
• 负责人: W. M. BUJALOWSKI
• 金额: $ 11.87万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1996-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3306118
• 关键词: DNA binding protein; DNA footprinting; DNA replication; DNA replication origin; adenosine triphosphate; adenosinetriphosphatase; analytical ultracentrifugation; bacterial genetics; chemical binding; chemical kinetics; chemical stability; circular dichroism; conformation; enzyme mechanism; fluorescence; fluorescence spectrometry; fluorescent dye /probe; helicase; molecular site; polynucleotides; stoichiometry; stop flow technique; synthetic nucleotide; thermodynamics

A great interest in the mechanism by which proteins interact with nucleic acids results from the importance of these interactions for such vital cellular processes as DNA replication, recombination, repair, transcription, and translation. In particular replication of DNA involves a complex, highly coordinated series of reactions in which new DNA chains are initiated and elongated on each parental strand. In E. coli dnaB protein plays an essential role in these processes. The dnaB system provides an excellent model to study these vital protein-nucleic acid interactions. Elucidation of the fundamental mechanistic details of these interactions is essential to understand why such processes dysfunction in various pathological conditions, e.g., cancer and genetic diseases. Studying different steps at the molecular level should provide necessary knowledge about how to regulate and control them. This knowledge should in turn help to design efficient therapy for the diseases. Moreover, the involvement of dnaB protein in the viral DNA replication gives an opportunity to study how viruses may subvert normal regulatory mechanisms. Our overall goals are to obtain a quantitative, molecular understanding of how the E coli dnaB protein functions as a "mobile replication promoter" and helicase through replication of the bacterial chromosome, as well as during viral and plasmid DNA replication. The helicase activity of dnaB protein involves unwinding of and translocation. along DNA. These are crucial and possibly rate limiting steps for replication . To understand the biosynthesis of DNA on the molecular level it is necessary to elucidate the thermodynamics of the formation and stability of the protein-DNA complexes involved. We will apply steady-state and life-time fluorescence spectroscopy, analytical ultracentrifugation, fast chemical (stop-flow) kinetic and various other biochemical and molecular biological methods to study thermodynamic, kinetic and structural aspects of the dnaB protein's interactions with nucleic acids. In the first step thermodynamic properties of the interactions will be determined, mainly using equilibrium fluorescence titrations. Following these experiments will be ATPase and DNA unwinding (helicase) activity studies. Next, the topology of the different complexes will be determined through fluorescence energy transfer, digestion protection experiments and correlated with their observed functional activities. In the final step detailed mechanistic aspects of the interactions will be studied using fluorescence stop-flow technique.

对蛋白质相互作用的机制非常感兴趣 核酸的产生源于这些相互作用的重要性 DNA复制、重组等重要的细胞过程， 修复、转录和翻译。 特别是复制 DNA 的合成涉及一系列复杂、高度协调的反应 其中新的 DNA 链在每个 父母链。 在大肠杆菌中 dnaB 蛋白起着至关重要的作用 在这些过程中。 dnaB系统提供了一个优秀的模型 研究这些重要的蛋白质-核酸相互作用。 阐明这些基本机制细节 相互作用对于理解为什么这样的过程至关重要 各种病理状况下的功能障碍，例如癌症和 遗传疾病。 在分子水平上研究不同的步骤 应提供有关如何监管和 控制他们。 这些知识反过来应该有助于设计 对疾病进行有效的治疗。 此外，参与 dnaB 蛋白为病毒 DNA 复制提供了机会 研究病毒如何破坏正常的调节机制。 我们的 总体目标是获得定量的分子理解 大肠杆菌 dnaB 蛋白如何发挥“移动复制 启动子”和解旋酶通过细菌的复制 染色体，以及病毒和质粒 DNA 复制过程中。 dnaB 蛋白的解旋酶活性涉及解旋 和 易位。沿着DNA。 这些都是至关重要的并且可能是重要的 限制复制步骤。了解生物合成 DNA在分子水平上有必要阐明 蛋白质-DNA 的形成和稳定性的热力学 涉及的复合体。 我们将应用稳态和寿命 荧光光谱、分析超速离心、快速 化学（停流）动力学和各种其他生化和 分子生物学方法来研究热力学、动力学和 dnaB 蛋白与核酸相互作用的结构方面 酸。 第一步热力学性质 相互作用将被确定，主要使用平衡 荧光滴定。 这些实验之后将 ATP 酶和 DNA 解旋（解旋酶）活性研究。 接下来， 不同复合体的拓扑将通过以下方式确定 荧光能量转移、消化保护实验和 与他们观察到的功能活动相关。 在决赛中 步骤详细的相互作用的机制方面将是 使用荧光停流技术进行研究。