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

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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.