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

 描述(申请人提供)：我们研究的主要目标是了解解旋酶和聚合酶在DNA复制、转录等过程中的机制，以及Rig-I解旋酶在先天性免疫中的作用。我们的研究为理解这些分子马达如何在核酸上运动以催化DNA和RNA链的分离和合成做出了重大贡献。这些见解可以为理解和治疗由这些酶的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对酶反应进行定量表征，如瞬时动力学、单分子动力学、计算动力学模型和结晶学。结构和功能研究的结合可以形成一个完整的机械学图景。这种非常简单的噬菌体T7酶使我们能够以前所未有的时间和空间分辨率探索复制反应，开发将结构与功能相关联的新生物物理工具，并提出新的机制，作为研究更复杂的线粒体复制和转录酶的基础。线粒体解旋酶和DNA聚合酶缺陷引起的线粒体DNA缺失影响能量产生，导致多种神经肌肉疾病。因此，深入了解线粒体DNA酶的酶机制是非常必要的。我们对T7和线粒体DNA复制的研究将填补在了解复制体结构、DNA聚合酶的校对机制以及线粒体DNA解旋酶Twinkle的DNA重组活性方面的关键空白。我们对线粒体DNA转录的研究将提供对启动机制、转录因子的作用的机械性见解，并解决在解决启动复合体结构方面的挑战。最近，我们冒险通过对RIG-I解旋酶家族的生化和结构特征来研究RNA解旋酶在先天性免疫中的作用。RIG-I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、流感和丙型肝炎。我们的研究将通过识别关键的病毒RNA识别特征、病毒如何逃避检测以及激活RIG-I的机制来解决在理解RIG-I解旋酶在启动先天性免疫中的关键作用方面的关键空白。我们还将解决在理解ATPase在RIG-I激活中的作用方面的挑战。这项研究将提供定量建模复制、转录和病原体识别反应的机制框架，这将指导人类疾病治疗的发展，包括癌症、抗病毒和抗微生物药物。

 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.