Functional Dynamics of Mammalian and Viral DNA Repair Polymerases

描述(申请人提供)：DNA复制和修复是将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损伤或病毒感染的基本过程。这些过程的核心是DNA聚合酶催化的DNA合成。哺乳动物聚合酶3(Polp)和非洲猪瘟病毒聚合酶X(PolX)由于其结构简单和催化体系简单，为研究DNA修复聚合酶作用的分子机制提供了优秀的模型系统。因为PolP和PolX在人类DNA修复和病毒防御宿主对感染的反应中起着基础性的作用，所以它们是具有极其重要的生物医学意义的酶系统。Polp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、结直肠癌和沃纳综合征。PolX的突变使病毒容易受到细胞的DNA修饰装置的攻击，从而削弱了病毒感染的进程。鉴于polP(3)在人类DNA修复中的关键作用以及polX在哺乳动物细胞病毒感染的有效性中的重要作用，了解polp和polX在执行其活动中的分子机制是至关重要的。了解这些机制的机制细节对于我们理解人类细胞中的DNA修复过程、细胞防御疾病的机制以及细胞对抗病毒感染的机制是至关重要的。在分子水平上研究不同的步骤将为如何控制它们提供必要的知识。反过来，这些知识对于设计合理有效的遗传、癌症和病毒疾病治疗方法是非常宝贵的。复制聚合酶和修复聚合酶之间的深刻而根本的区别在于，DNA修复酶必须在催化之前识别损伤DNA的特定结构，在压倒性的dsDNA构象的背景下。这表明，控制DNA合成保真度的DNA和dNTP识别必须先于催化。因此，阐明Polp-DNA和ASFV polX-DNA复合体的能级、动力学和结构是理解这两种酶的分子机制，特别是催化效率和保真度的前提。本项目的主要目的是阐明PolP和PolX识别特定DNA结构的分子机制及其在DNA合成中的作用。这一目标将通过定量荧光滴定、分析离心、荧光停流、快速猝灭流动、荧光能量转移和定点突变技术对其与DNA底物和dNTPs在溶液中的络合物进行定量的热力学、动力学和结构研究来实现。

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 or against viral infections. At the heart of these processes is the synthesis of the DNA catalyzed by DNA polymerases. Mammalian Polymerase (3 (pol p) and African Swine Fever Virus Polymerase X (pol X) provide outstanding model systems to study the molecular mechanism of the DNA repair polymerase action due to its simplified structures and catalytic repertoires. Because of the fundamental role in human DNA repair and the virus defense against the host reaction to the infection, pol P and pol X are enzymatic systems of a paramount biomedical importance. Mutations and deletions in pol p have been implicated in several human cancers and genetic diseases including breast, prostate, kidney, lung, colorectal cancers, and Werner syndrome. Mutations in pol X render the virus vulnerable to the DNA- modifying apparatus of the cell, which weakens the progress of the virus infection. In light of the pol (3 key role in human DNA repair and the pol X essential role in the effectiveness of viral infection of the mammalian cell, it is of fundamental importance to understand the molecular mechanism by which pol p and pol X function in performing their activities. Knowledge of mechanistic details of the mechanisms is essential to our understanding of the DNA repair processes in a human cell, the mechanism by which the cell defends itself against diseases, and the mechanism by which the cell fights against viral infections. Studying different steps at the molecular level will provide the necessary knowledge about how to control them. In turn, this knowledge is invaluable for designing rational and efficient therapies for genetic, cancer and viral diseases. The profound and fundamental difference between the replicative and repair polymerases 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. This indicates that DNA and dNTP recognition, which controls fidelity of DNA synthesis, must precede the catalysis. Thus, elucidation of the energetics, dynamics and structure of pol p - DNA and ASFV pol X - DNA complexes is a prerequisite for understanding the molecular mechanisms of the enzymes, particularly, the efficiency and fidelity of catalysis. The main goal of this project is to elucidate the molecular mechanisms of the recognition of specific DNA structures by pol p and pol X and their role in DNA synthesis. This goal will be achieved through quantitative thermodynamic, kinetic, and structural studies of their complexes with DNA substrates and dNTPs in solution using quantitative fluorescence titrations, analytical centrifugation, fluorescence stopped-flow, rapid-quench-flow, fluorescence energy transfer and site-directed mutagenesis techniques.