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: 10396601
• 负责人: SMITA S PATEL
• 金额: $ 82.83万
• 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 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10396601
• 关键词: ATP phosphohydrolase; Antiviral Agents; Bacteriophage T7; Binding Proteins; Biochemical; Biochemical Reaction; Biological Assay; Coupled; Cryoelectron Microscopy; DNA; DNA biosynthesis; DNA-Directed DNA Polymerase; DNA-Directed RNA Polymerase; Defect; Development; Disease; Enzymes; Foundations; Genetic Transcription; Goals; Human; Immune; Immune response; Immunologic Receptors; In Vitro; Kinetics; Knowledge; Maintenance; Malignant Neoplasms; Mitochondria; Mitochondrial DNA; Mitochondrial Diseases; Mitochondrial RNA; Modeling; Molecular; Natural Immunity; Nucleic Acids; Pattern; Polymerase; Process; Production; Property; RNA; RNA Virus Infections; Reaction; Regulation; Research; Role; Signal Transduction; Structure; Transcription Initiation; Vaccine Adjuvant; Viral Genome; Virus Diseases; Virus Replication; Work; Yeasts; biophysical techniques; enzyme mechanism; first responder; helicase; kinetic model; pathogen; receptor; reconstitution; single molecule; therapy development; transcription factor; viral RNA

Project Summary The overarching goal of our research is to understand the mechanisms of helicases and polymerases in processes such as viral RNA recognition, DNA transcription, and replication. The unifying approach is the quantitative characterization of the enzymatic reactions using rigorous biochemical and biophysical methods such as transient state kinetics, single-molecule kinetics, computational kinetic modeling, and cryo-electron microscopy. The integration of structural and functional studies allows the development of a complete mechanistic picture. In project 1, we are studying viral RNA recognition by RIG-I like receptors which are helicases serving as the first responders of viral RNA infections. The RIG-I like receptors recognize pathogen- associated molecular patterns on viral genomes and replication intermediates and respond by triggering an immune response to create an antiviral state. Our research focuses on understanding the mechanisms of RNA recognition and ATPase/helicase functions of RIG-I like receptors using biochemical, structural, and cell- signaling assays. We are elucidating the intrinsic mechanisms in RIG-I that enable self versus non-self recognition and developing new strategies to understand how they are activated and regulated. In project 2, we are studying the mechanism and regulation of mitochondrial DNA transcription catalyzed by RNA polymerases that resemble phage T7 but regulated by transcription factors. Transcription initiation and transition into elongation are key stages that are regulated by transcription factors. We are using cryo-electron microscopy, and ensemble/single-molecule kinetics to elucidate the structure and dynamics at these stages of transcription using in vitro reconstituted yeast and human mitochondrial RNA polymerases. In project 3, we are studying the mechanism of DNA replication by phage T7 and human mitochondrial replisomes. We study how helicase and polymerase work together to catalyze strand-displacement DNA synthesis, in particular, how they are energetically coupled. We are studying the mechanism of DNA synthesis by mitochondrial DNA polymerase to understand the role of helicase, Twinkle, and mitochondrial single-strand binding protein. An in- depth understanding of the enzymatic mechanisms is critically necessary to understand mitochondrial DNA deletions caused by defects in helicase and polymerase. 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 viral infections, cancer, mitochondrial diseases.

项目摘要 我们研究的首要目标是了解解旋酶和聚合酶在细胞内的作用机制。 病毒RNA识别、DNA转录和复制等过程。统一的方法是 使用严格的生物化学和生物物理方法对酶反应进行定量表征 例如瞬态动力学、单分子动力学、计算动力学建模和低温电子 显微镜结构和功能研究的整合允许开发一个完整的 机械图在项目1中，我们正在研究RIG-I样受体对病毒RNA的识别， 解旋酶作为病毒RNA感染的第一反应者。RIG-I类受体识别病原体 病毒基因组和复制中间体上的相关分子模式，并通过触发 免疫反应来产生抗病毒状态。我们的研究重点是了解RNA的机制， 识别和ATP酶/解旋酶功能的RIG-I样受体使用生化，结构，和细胞- 信号传导测定。我们正在阐明RIG-I的内在机制， 识别和开发新的策略，以了解它们是如何被激活和调节的。在项目2中， 我们正在研究RNA催化线粒体DNA转录的机制和调节 类似于噬菌体T7但受转录因子调控的聚合酶。转录起始和 向伸长的过渡是受转录因子调控的关键阶段。我们用低温电子 显微镜和合奏/单分子动力学，以阐明在这些阶段的结构和动力学， 使用体外重组酵母和人线粒体RNA聚合酶进行转录。在项目3中，我们 正在研究噬菌体T7和人类线粒体复制体的DNA复制机制。我们研究 解旋酶和聚合酶如何协同作用催化链置换DNA合成，特别是， 它们是能量耦合的。我们正在研究线粒体DNA合成DNA的机制 聚合酶，以了解解旋酶，闪光，和线粒体单链结合蛋白的作用。一个在- 深入了解酶的机制是至关重要的理解线粒体DNA 由解旋酶和聚合酶缺陷引起的缺失。这项研究将提供一个机制框架 定量模拟复制，转录和病原体识别的反应，这将指导 病毒感染、癌症、线粒体疾病的治疗方法的开发。