# Structure, Mechanism and Interactions of Type IA Topoisomerases

> **NIH NIH R35** · FLORIDA INTERNATIONAL UNIVERSITY · 2021 · $209,165

## Abstract

Project Summary/Abstract
Type IA topoisomerases are ubiquitous in the three kingdoms of life, and play critically important roles in
maintaining proper DNA topology during the vital cellular processes of replication, transcription, recombination,
and repair. The PI’s research activities have provided seminal biochemical and structural findings for this class
of essential genome regulator, and continue to address key questions on the catalytic mechanism of type IA
topoisomerases and provide new insights into their functional and regulatory interactions. This information is
needed to utilize type IA topoisomerases present in every bacterial pathogen as a novel therapeutic target for
finding new antibiotics to help face our serious global health challenge of antibiotic resistance. Type IA
topoisomerases catalyze the relaxation of negatively supercoiled DNA by cleaving a single DNA strand in the
underwound duplex DNA and passing the complementary DNA single strand through the break before
religation of the cleaved strand to change the DNA topology. The molecular mechanism of the large enzyme
conformational changes that are required for the coordinated movement of the passing DNA in and out of the
DNA gate is the critical barrier for elucidating how bacterial TOP1 can relax negatively supercoiled DNA with
high efficiency to prevent hypernegative DNA supercoiling and R-loop stabilization that can arise during
transcription. This important function of bacterial TOP1 is facilitated by the direct TOP1 interaction with RNA
polymerase that we have characterized and found to be targeted by endogenous toxin in mycobacteria. For
future studies, we will create new TOP1 mutants perturbed in interdomain interactions at a distance from the
active site and investigate the effect on the in vivo relaxation activity and in vitro interactions with DNA
substrate. Mutants with reduced catalytic efficiency will be further studied to determine if the mutations affected
the gate opening-closing dynamics and DNA strand passage. We will capture new structural conformations of
the TOP1-DNA complex that may represent different stages of the catalytic cycle with X-ray crystallography
and measure the gate opening-closing dynamics with single molecule assays. Structural studies will also
incorporate other ligands including RNA. Type IA topoisomerases have evolved to include TOP1 and TOP3
enzymes in all three kingdoms of life that possess dual activities on both DNA and RNA substrates. The RNA
topoisomerase activity of human TOP3B has been shown to be required for neurodevelopment and the
enzyme is also involved in R-loop suppression and genome stability. We are modeling the RNA interaction of
type IA topoisomerases with molecular dynamics simulations to determine how the DNA and RNA substrate
may be accommodated differentially by change in enzyme conformation and interacting residues. We have
initiated studies to identify a separation of function mutation or small molecule probe t...

## Key facts

- **NIH application ID:** 10093404
- **Project number:** 1R35GM139817-01
- **Recipient organization:** FLORIDA INTERNATIONAL UNIVERSITY
- **Principal Investigator:** Yuk-Ching Tse-Dinh
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $209,165
- **Award type:** 1
- **Project period:** 2021-02-01 → 2026-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10093404

## Citation

> US National Institutes of Health, RePORTER application 10093404, Structure, Mechanism and Interactions of Type IA Topoisomerases (1R35GM139817-01). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10093404. Licensed CC0.

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