DYNAMICS OF DNA RECOGNITION BY DNA REPAIR POLYMERASE

DNA复制和修复是传播的基本过程 遗传信息从一代细胞传到另一代细胞 保护细胞免受DNA的破坏。在这些问题的核心 过程是DNA聚合酶催化合成DNA的过程。 哺乳动物聚合酶β提供了一个出色的模型系统来 研究其聚合酶作用的分子机制 简化的催化谱系及其在哺乳动物DNA修复中的作用。 POLβ是公认的四种DNA导向的聚合酶之一 真核细胞核。这种酶参与了缝隙填充合成， 在错配修复中，单功能加合物的修复，紫外线损伤的DNA， 和DNA中的基本损伤。 鉴于它在哺乳动物DNA修复中的关键作用，它具有基础性 理解POL-β分子机制的重要性 功能。了解聚合酶的机制细节 机制对于我们理解DNA修复过程是必不可少的 在人类细胞中，以及细胞自我防御的机制 对抗疾病，例如癌症。研究不同的步骤 分子水平应该提供必要的知识，如何 控制这些过程。反过来，这些知识将是非常有用的 在设计有效的疾病治疗方法方面。 DNA修复聚合酶被设计用来对有缝隙的DNA进行DNA合成 随着间隙大小的消失，这表明酶的模式 在DNA的过程中，与核酸的相互作用正在发生变化 综合。聚合酶β缺乏纠错活性，这是典型的 对于复制聚合酶，这表明DNA和dNTP 控制DNA合成保真度的识别过程领先于 化学步骤。因此，阐明了原子核的能量学和动力学 Polbeta的底物识别过程，包括转换 不同绑定模式之间的关系，是理解 酶作用的分子机制，特别是，保真度 DNA合成。 本项目的主要目标是建立一种新的分子模型。 Polbeta对dna和dNTP底物的识别过程。这个目标 将通过定量热力学、动力学和 溶液中其与DNA和dNTPs络合物的结构研究 定量荧光滴定，分析离心法， 荧光停止流动、温度跳跃、快速熄灭流动， 荧光能量转移和定点突变技术。

DNA replication and repair are fundamental processes for transmission of genetic information from one cell generation to the other and for defending the cell against damages in its DNA. At the heart of these processes is the synthesis of DNA catalyzed by the DNA polymerases. The mammalian polymerase beta provides an outstanding model system to study the molecular mechanism of the polymerase action due to its simplified catalytic repertoire and its role in mammalian DNA repair. Pol beta is one of the four recognized DNA-directed polymerases of the eucaryotic nucleus. The enzyme is involved in gap filling synthesis, in mismatch repair, repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA. In light of its key role in mammalian DNA repair, it is of fundamental importance to understand the molecular mechanism by which pol beta functions. Knowledge of the mechanistic details of the polymerase mechanism is essential to our understanding of the DNA repair process in a human cell and the mechanism by which the cell defends itself against diseases, e.g., cancer. Studying different steps on the molecular level should provide the necessary knowledge about how to control these processes. In turn, this knowledge will be very useful in designing efficient therapies for diseases. DNA repair polymerase is designed to perform DNA synthesis on gapped DNA with vanishing gap size which suggests that the mode of enzyme interactions with nucleic acids is changing, in the course of DNA synthesis. Polymerase beta lacks error correcting activities, typical for replicative polymerases, which indicates that DNA and dNTP recognition processes, which control fidelity of DNA synthesis, precede the chemical step. Thus, elucidation of the energetics and dynamics of the substrate recognition process by pol beta, including the transitions between different binding modes, is a prerequisite for understanding the molecular mechanism of the enzyme action, particularly, the fidelity of the DNA synthesis. The main goal of this project is to establish a molecular model of the recognition process of DNA and dNTP substrates by pol beta. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of its complexes with DNA and dNTPs in solution using quantitative fluorescence titrations, analytical centrifugation methods, fluorescence stopped-flow, temperature-jump, rapid-quench-flow, fluorescence energy transfer, and site-directed mutagenesis techniques.