Mechanistic studies of nucleic acid enzymes involved in DNA replication, transcription, and innate immunity

参与DNA复制、转录和先天免疫的核酸酶的机制研究

• 批准号: 9266427
• 负责人: SMITA S PATEL
• 金额: $ 64.2万
• 依托单位: RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9266427
• 关键词: ATP phosphohydrolase; Address; Affect; Alpers' Syndrome; Antiviral Agents; Bacteriophage T7; Biochemical; Biochemical Reaction; Complex; Computer-Assisted Image Analysis; Crystallography; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Dengue; Detection; Development; Disease; Enzymes; Family; Genetic Recombination; Genetic Transcription; Goals; Hepatitis C; Human Development; Influenza C Virus; Kinetics; Malignant Neoplasms; Mitochondria; Mitochondrial DNA; Modeling; Molecular Motors; Mutation; Natural Immunity; Neuromuscular Diseases; Nucleic Acids; Polymerase; Process; Production; RNA; RNA Helicase; RNA Virus Infections; Reaction; Research; Resolution; Role; Structure; Viral; Virus; West Nile virus; Work; antimicrobial drug; biophysical techniques; biophysical tools; detector; helicase; insight; pathogen; public health relevance; single molecule; therapy development; transcription factor; viral RNA

 DESCRIPTION (provided by applicant): The overarching goal of our research is to understand the mechanisms of helicases and polymerases in processes such as DNA replication, transcription, and role of RIG-I helicase in innate immunity. Our research has made major contributions to understanding how these molecular motors move on nucleic acids to catalyze DNA and RNA strand separation and synthesis. These insights can provide the basis for understanding and treatment of diseases caused by dysregulation or malfunction of these enzymes. The unifying approach is quantitative characterization of the enzymatic reactions using rigorous biochemical and biophysical methods such as transient state kinetics, single molecule kinetics, computational kinetic modeling, and crystallography. Integration of structural and functional studies allows development of a complete mechanistic picture. The elegantly simple phage T7 enzymes allowed us to probe replication reactions with unprecedented temporal and spatial resolution, to develop new biophysical tools that correlate structure with function, and to propose new mechanisms that serve as a basis for studying more complex mitochondrial replication and transcription enzymes. Mitochondrial DNA deletions caused by defects in mitochondrial helicase and DNA polymerase affect energy production and result in a wide variety of neuromuscular diseases. Hence, in depth understanding of the enzymatic mechanisms of the mitochondrial DNA enzymes are critically needed. Our research on T7 and mitochondrial DNA replication will address key gaps in understanding the structure of the replisome, the proofreading mechanism of the DNA polymerase, and the DNA recombination activities of mitochondrial DNA helicase Twinkle. Our research on mitochondrial DNA transcription will provide mechanistic insights into the initiation mechanism, roles of the transcription factors, and address challenges in solving the structure of the initiation complex. Recently, we ventured into investigating the roles of RNA helicases in innate immunity by biochemically and structurally characterizing the RIG-I family of helicases. The RIG-I family of helicases are the cytoplasmic detectors of RNA viral infections, e.g. Dengue fever, West Nile, influenza, and hepatitis C. Our research will address key gaps in understanding the essential role of RIG-I helicases in initiating innate immunity by identifying crucial viral RNA recognition features, how viruses evade detection, and mechanisms that activate RIG-I. We will also address challenges in understanding the role of ATPase in RIG-I activation. This research will provide the mechanistic framework to quantitatively model the reactions of replication, transcription, and pathogen recognition that will guide in the development of therapies for human diseases including cancer, antiviral, and antimicrobial agents.

 描述（由适用提供）：我们的研究的总体目标是了解在DNA复制，转录和RIG-I解旋酶在先天免疫中的过程中的解旋酶和聚合酶的机制。我们的研究为理解这些分子电动机如何在核酸上移动以催化DNA和RNA链分离和合成做出了重大贡献。这些见解可以为理解和治疗这些酶失调或故障引起的疾病提供基础。统一的方法是使用严格的生化和生物物理方法（例如瞬时状态动力学，单分子动力学，计算动力学建模和晶体学）等严格的生化和生物物理方法对酶促反应进行定量表征。结构和功能研究的整合允许开发完整的机械图。优雅的简单噬菌体T7酶使我们能够用前所未有的临时和空间分辨率探测复制反应，以开发将结构与功能相关的新生物物理工具，并提出了新机制，这些机制是研究更复杂的线粒体复制和转录酶的基础。线粒体DNA缺失是由线粒体解旋酶和DNA聚合酶缺陷引起的，会影响能量产生，并导致多种神经肌肉疾病。因此，非常需要深入了解线粒体DNA酶的酶促机制。我们对T7和线粒体DNA复制的研究将解决理解复制体结构，DNA聚合酶的校对机理以及线粒体DNA Helicase Twinkle的DNA重组活性的关键差距。我们对线粒体DNA转录的研究将提供有关主动机制，转录因子的作用的机械见解，并解决解决倡议络合物结构方面的挑战。最近，我们冒险通过生化和结构表征RIG-1的解旋酶家族来研究RNA解旋酶在先天免疫中的作用。 RIG-1的解旋酶家族是RNA病毒感染的细胞质检测器，例如登革热，西尼罗河，影响力和乙型肝炎。我们的研究将解决RIG-I解放酶在识别重要的病毒RNA识别特征，病毒逃避检测以及激活RIG-I的机制方面来理解RIG-I解放酶在启动先天免疫中的重要作用方面的关键差距。我们还将解决了解ATPase在RIG-I激活中的作用方面的挑战。这项研究将提供机械框架，以定量地对复制，转录和病原体识别的反应进行定量建模，该反应将指导在包括癌症，抗病毒和抗菌药物在内的人类疾病疗法开发中。