# The Mechanism of Elimination of the Mitochondrial DNA Replisome

> **NIH NIH R15** · AUBURN UNIVERSITY AT MONTGOMERY · 2021 · $209,896

## Abstract

Abstract
 Defects in the human mitochondrial DNA (mtDNA) replication process result in the accumulation of
mutations that give rise to a broad spectrum of degenerative disorders involving multiple organs and systems of
the human body. In this project, we propose to investigate a putative mechanism that prevents the formation of
large-scale deletions in mtDNA, which are the most common (de novo) defects of the mitochondrial genome.
The mechanism of deletion formation is unknown, but studies reported to date indicate that they originate from
stalling of the mitochondrial DNA replication machinery (the replisome), which leads to the breaking of DNA
strands. Notably, prominent mtDNA replication stalling sites correspond with binding sites of the major
mitochondrial protease, Lon, which suggests that it might be involved in the elimination of halted replisomes. In
addition, the components of human mitochondrial Hsp70/40 chaperone system have been found to co-precipitate
with the catalytic subunit of the mitochondrial replicative polymerase, which suggests that these may be involved
in the assembly or disassembly of mitochondrial replisomes. In fact, in model organisms, Hsp70/40 systems have
been found to assist Lon protease by unfolding and delivering protein substrates. On the other hand, Hsp70/40
systems have also been found to cooperate with another protease, ClpXP, which deficiency results in
mitochondrial genome destabilization.
 This proposal aims to evaluate the hypothesis that stalled mtDNA replisomes are eliminated by two
alternative mechanisms that engage either Lon or ClpXP protease. In addition, the Hsp70/40 chaperone system
may serve to disassemble the replisome and deliver its components to the client protease. Confirmation and
characterization of a direct relationship between the capacity of a cell to remove defective mitochondrial
replisomes and the accumulation of damage in the mitochondrial genome, would bring a novel and exciting
perspective on the development of mtDNA deletions-linked human disorders, potentially identifying targets for
prospective therapeutic strategies. To this end, we will apply a comprehensive approach combining the cutting-
edge technique of biolayer interferometry for the analysis of molecular affinities and kinetic parameters, a
methodical biochemical analysis that entails specialized enzymatic assays, and testing the putative correlation
between cellular levels of Lon, Hsp70/40 and ClpX and the development of mtDNA deletions in vivo, using
Saccharomyces cerevisiae as the model organism. To date, we have demonstrated that Lon protease hydrolases
the catalytic subunit of the mitochondrial replicative polymerase, but only in the absence of the remaining
polymerase subunits. This finding confirms our initial hypothesis and warrants the investigation of the role of
Hsp70/40 and/or ClpX in the disassembly of the replisome complex to enable degradation of the catalytic core
by Lon protease.

## Key facts

- **NIH application ID:** 10291978
- **Project number:** 1R15GM139104-01A1
- **Recipient organization:** AUBURN UNIVERSITY AT MONTGOMERY
- **Principal Investigator:** Grzegorz Leszek Ciesielski
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $209,896
- **Award type:** 1
- **Project period:** 2021-09-01 → 2023-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10291978

## Citation

> US National Institutes of Health, RePORTER application 10291978, The Mechanism of Elimination of the Mitochondrial DNA Replisome (1R15GM139104-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10291978. Licensed CC0.

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