THERMODYNAMICS OF PROCARYOTIC DNA REPLICATION

对蛋白质与蛋白质相互作用的机制的极大兴趣 核酸的结果来自这些相互作用的重要性， 这些重要的细胞过程，如DNA复制，重组， 修复转录和翻译 特别是复制 涉及一系列复杂的高度协调的反应 新的DNA链在每一条DNA链上被启动和延长， 亲本链 在大肠大肠杆菌dnaB蛋白起着重要的作用 在这些过程中。 dnaB系统提供了一个很好的模型 来研究这些重要的蛋白质-核酸相互作用。 阐明这些基本的机械细节， 相互作用对于理解为什么这些过程 在各种病理状况下的功能障碍，例如，癌症和 遗传病 在分子水平上研究不同的步骤 应提供关于如何管理和 控制他们。 这些知识反过来有助于设计 有效治疗疾病。 此外，参与 病毒DNA复制中的dnaB蛋白提供了机会， 研究病毒如何破坏正常的调节机制。 我们 总体目标是获得定量的、分子的理解， 大肠杆菌dnaB蛋白如何作为“移动的复制”发挥作用 启动子”和解旋酶通过细菌的复制 染色体，以及在病毒和质粒DNA复制期间。 dnaB蛋白的解旋酶活性涉及DNA的解旋， 易位沿着DNA 这些都是至关重要的， 限制复制步骤。了解的生物合成 DNA在分子水平上，有必要阐明 蛋白质-DNA形成和稳定性的热力学 复杂的参与。 我们将应用稳态和寿命 荧光光谱，分析超离心，快速 化学（停流）动力学和各种其他生物化学和 分子生物学方法研究热力学、动力学和 dnaB蛋白与核酸相互作用的结构方面 acids. 在第一步中， 将确定相互作用，主要使用平衡 荧光滴定法 这些实验将 ATP酶和DNA解旋（解旋酶）活性研究。 接下来 不同复合物的拓扑结构将通过 荧光能量转移、消化保护实验和 与其观察到的功能活动相关。 在决赛中 步骤详细的机制方面的相互作用将是 使用荧光停流技术研究。

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.