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.

Public Health Relevance

This work will make major contributions to understanding how helicases separate and polymerases synthesize DNA and RNA. These insights can provide the basis for understanding and treatment of diseases caused by dysregulation or malfunction of these enzymes, for example, mutations in mitochondrial DNA polymerase and helicase affect energy production and result in a wide variety of neuromuscular diseases, such as Alpers disease. The RIG-I family of helicases are the cytoplasmic detectors of RNA viral infections, such as Dengue fever, West Nile, influenza, and hepatitis C, and our research will address key gaps in understanding the essential role of RIG-I helicases in initiating innate immunity by identifying crucial viral RN recognition features, how viruses evade detection, and mechanisms that activate RIG-I as the first line of defense.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Unknown (R35)
Project #
5R35GM118086-03
Application #
9470899
Study Section
Special Emphasis Panel (ZGM1)
Program Officer
Barski, Oleg
Project Start
2016-05-01
Project End
2021-04-30
Budget Start
2018-05-01
Budget End
2019-04-30
Support Year
3
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Rbhs-Robert Wood Johnson Medical School
Department
Biochemistry
Type
Schools of Medicine
DUNS #
078795875
City
Piscataway
State
NJ
Country
United States
Zip Code
Koh, Hye Ran; Roy, Rahul; Sorokina, Maria et al. (2018) Correlating Transcription Initiation and Conformational Changes by a Single-Subunit RNA Polymerase with Near Base-Pair Resolution. Mol Cell 70:695-706.e5
Sultana, Shemaila; Solotchi, Mihai; Ramachandran, Aparna et al. (2017) Transcriptional fidelities of human mitochondrial POLRMT, yeast mitochondrial Rpo41, and phage T7 single-subunit RNA polymerases. J Biol Chem 292:18145-18160
Ramachandran, Aparna; Basu, Urmimala; Sultana, Shemaila et al. (2017) Human mitochondrial transcription factors TFAM and TFB2M work synergistically in promoter melting during transcription initiation. Nucleic Acids Res 45:861-874
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Nandakumar, Divya; Patel, Smita S (2016) Methods to study the coupling between replicative helicase and leading-strand DNA polymerase at the replication fork. Methods 108:65-78
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Sen, Doyel; Patel, Gayatri; Patel, Smita S (2016) Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE. Nucleic Acids Res 44:4200-10