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

> **NIH NIH R35** · RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL · 2021 · $827,460

## Abstract

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.

## Key facts

- **NIH application ID:** 10196375
- **Project number:** 2R35GM118086-06
- **Recipient organization:** RBHS-ROBERT WOOD JOHNSON MEDICAL SCHOOL
- **Principal Investigator:** SMITA S PATEL
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $827,460
- **Award type:** 2
- **Project period:** 2016-05-01 → 2026-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10196375

## Citation

> US National Institutes of Health, RePORTER application 10196375, Mechanistic studies of nucleic acid enzymes involved in DNA replication, transcription, and innate immunity (2R35GM118086-06). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10196375. Licensed CC0.

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