# Defining the mechanisms of kinetoplast DNA assembly by trypanosomal topoisomerase II for therapeutic target development

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2022 · $46,752

## Abstract

Project Summary/Abstract
Type IIA topoisomerases (topo II) are ubiquitous molecular machines that manage DNA superhelical structure
and decatenate DNA entanglements to support critical processes such as transcription, DNA replication, and
chromosome segregation. Topo II relies on ATP to capture and pass one DNA segment through a reversible,
topo-II mediated, double-strand break in a second DNA segment. With numerous clinically proven antibiotics
and anti-cancer drugs targeting the essential, yet risky, activities of these enzymes, the ATP-dependence of the
topo II strand passage reaction is well-established; however, how topo IIs use ATP and local DNA interactions
to favor unidirectional strand passage activity (ensuring genomic knots are removed, not formed) is unknown. In
sharp contrast to other topo IIs, the mitochondrial-specific type IIA topoisomerase from trypanosomes
(TxTopoIImt) is reported to possess an unexpected ATP-independent strand passage activity. In addition,
TxTopoIImt appears to switch between canonical topo II activities (e.g., decatenating DNA) and the antithetical
activity of catenating DNA molecules. Switching of strand passage directionality is believed to be how TxTopoIImt
perpetuates networks of mitochondrial DNA, known as kinetoplast or kDNA, which comprise thousands of DNA
circles interlocked into a giant structure akin to medieval chain mail. The unique kDNA structure is a hallmark of
trypanosomatids, recondite parasites that cause three neglected tropical diseases: African sleeping sickness,
Chagas disease, and leishmaniasis. The goal of this project is to develop TxTopoIImt as a therapeutic target by
understanding and exploiting the fundamental mechanisms underlying the enzyme’s deviant activities.
Strategies outlined in Aim 1 will mechanistically define the strange activities of TxTopoIImt in vitro, which are
now possible due to a recent breakthrough in producing soluble TxTopoIImt purified from recombinant sources.
Cofactor requirements for TxTopoIImt will be assessed (with in vitro topoisomerase decatenation and supercoil
relaxation assays) and the molecular determinants of DNA strand passage directionality will be explored (using
singly-catenated DNA substrates of various compositional topologies). The objectives of Aim 2 are to
characterize the mechanisms by which small-molecule inhibitors of purified TxTopoIImt (identified with in vitro
screens of both clinically known topo II inhibitors and the Johns Hopkins FDA-Approved Drug Library) affect
enzymatic activities, and then validate the anti-parasitic therapeutic potential of these inhibitors with killing assays
of bloodstream-form African trypanosome cultures. Together, these aims have the potential to offer novel
mechanistic insights into general topo II function, establish a molecular understanding of parasitic trypanosomes’
peculiar biology, and support future research efforts to develop novel treatments for trypanosome infections. As
such, this project dra...

## Key facts

- **NIH application ID:** 10386849
- **Project number:** 5F31GM142290-02
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Arman Alam Siddiqui
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $46,752
- **Award type:** 5
- **Project period:** 2021-04-01 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10386849

## Citation

> US National Institutes of Health, RePORTER application 10386849, Defining the mechanisms of kinetoplast DNA assembly by trypanosomal topoisomerase II for therapeutic target development (5F31GM142290-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10386849. Licensed CC0.

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