Dynamics of DNA Recognition by DNA Repair Polymerase

描述(申请人提供)：DNA复制和修复是遗传信息从一代细胞传递到另一代细胞以及保护细胞免受DNA损伤的基本过程。这些过程的核心是DNA聚合酶催化的DNA合成。由于其简化的催化体系和在人类DNA修复中的作用，聚合酶13为研究聚合酶作用的分子机制提供了一个出色的模型系统。该酶参与缝隙填充合成、错配修复、单功能加合物的修复、紫外线损伤的DNA以及DNA的基本损伤。Pol13基因的突变和缺失与几种人类癌症和遗传性疾病有关，包括乳腺癌、前列腺癌、肾癌、肺癌、结直肠癌和韦默综合征。鉴于Pol13在人类DNA修复中的关键作用，了解Pol13在其活动中发挥作用的分子机制是至关重要的。了解酶机制的机制细节对于我们理解人类细胞中的DNA修复过程以及细胞抵御疾病的机制至关重要。在分子水平上研究不同的步骤将提供如何控制它们的知识。反过来，这些知识在设计有效的疾病治疗方法方面将是至关重要的。聚合酶13缺乏复制聚合酶的典型纠错活性。这表明，控制DNA合成保真度的DNA和dNTP识别必须先于催化。此外，复制聚合酶和修复聚合酶之间的一个深刻区别是，DNA修复酶必须在催化之前识别受损DNA的特定结构，在压倒性的dsDNA构象的背景下。因此，阐明Pol13-DNA识别过程的能量学和动力学以及形成的络合物的结构是理解该酶的分子机制，特别是DNA合成的效率和保真度的先决条件。本项目的主要目标是建立Pol13识别特定DNA结构的分子模型。这一目标将通过定量研究其与DNA底物和dNTPs在溶液中的络合物的热力学、动力学和结构，使用荧光滴定、分析离心法、荧光停流、温度跳跃、快速猝灭流、荧光能量转移和定点突变技术来实现。

DESCRIPTION (provided by applicant): 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. Polymerase 13 provides an outstanding model system to study the molecular mechanism of the polymerase action, due to its simplified catalytic repertoire and role in human DNA repair. The enzyme is involved in gap filling synthesis, mismatch repair, the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA. Mutations and deletions in pol 13 have been implicated in several human cancers and genetic diseases including breast, prostate, kidney, lung, colorectal cancers, and Wemer's syndrome. In light of its key role in human DNA repair, it is of fundamental importance to understand the molecular mechanism by which pol 13 functions in performing its activities. Knowledge of the mechanistic details of the enzyme 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. Studying different steps at the molecular level will provide the knowledge about how to control them. In turn, this knowledge will be of paramount importance in designing efficient therapies for diseases. Polymerase 13 lacks error-correcting activities typical for replicative polymerases. This indicates that DNA and dNTP recognition, which controls the fidelity of DNA syntheses, must precede the catalysis. Moreover, a profound difference between the replicative and repair polymerase is that the DNA repair enzyme must recognize a specific structure of the damaged DNA prior to the catalysis, in the context of overwhelmingly dsDNA conformation. Thus, elucidations of the energetics and dynamics of pol 13-DNA recognition processes and the structures of the formed complexes are prerequisites for understanding the molecular mechanism of the enzyme, particularly, the efficiency and fidelity of the DNA synthesis. The main goal of this project is to establish a molecular model of the recognition of specific DNA structures by pol 13. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of its complexes with DNA substrates and dNTPs in solution, using fluorescence titrations, analytical centrifugation, fluorescence stopped-flow, temperature-jump, rapid-quench-flow, fluorescence energy transfer, and site-directed mutagenesis techniques.