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

项目摘要 我们研究的主要目标是了解解旋酶和聚合酶的作用机制。 病毒RNA识别、DNA转录和复制等过程。统一的方法是 用严格的生化和生物物理方法对酶反应进行定量表征 例如暂态动力学、单分子动力学、计算动力学建模和冷冻电子 显微镜。结构和功能研究的整合允许开发一个完整的 机械化的画面。在项目1中，我们正在研究RIG-I样受体对病毒RNA的识别 解旋酶是病毒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 产生抗病毒状态的免疫反应。我们的研究重点是了解RNA的作用机制 利用生化、结构和细胞功能识别RIG-I样受体及其ATPase/Helicase功能 信号分析。我们正在阐明RIG-I中实现自我与非我的内在机制 认识并制定新的战略，以了解它们是如何被激活和监管的。在项目2中， 我们正在研究rna催化线粒体dna转录的机制和调控。 类似噬菌体T7但受转录因子调控的聚合酶。转录起始和 过渡到伸长是受转录因子调控的关键阶段。我们使用的是低温电子 显微镜和系综/单分子动力学来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复制DNA的机制。我们学习 解旋酶和聚合酶如何共同作用催化链置换DNA合成，尤其是如何 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、闪烁和线粒体单链结合蛋白的作用。一个In- 深入了解酶的作用机制对于了解线粒体DNA是至关重要的。 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 为了对复制、转录和病原体识别的反应进行定量建模，这些反应将指导 开发针对病毒感染、癌症、线粒体疾病的治疗方法。

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.