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Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Visualization of Mitochondrial DNA Replication in Individual Cells by EdU Signal Amplification

Published on: November 15, 2010

Mitochondrial DNA replication is regulated by endoplasmic reticulum-mitochondrial contact sites, the mitochondrial

Amaia Lopez de Arbina^1,2, Angelica Zamudio-Ochoa³, Mikel Muñoz-Oreja^1,2

¹Biogipuzkoa Health Research Institute, San Sebastián 20014, Spain.

Nucleic Acids Research

|April 25, 2026

Abstract:

Mitochondrial DNA replication occurs at contact sites between the endoplasmic reticulum (ER) and mitochondria (ERMCS). Beyond the known role of the tubular ER protein RTN4, the factors regulating this process are poorly defined. Here, we show that repressing the ER protein ERLIN2 in human fibroblasts depletes ER-mitochondrial contact sites and inhibits mitochondrial DNA replication, as does silencing RTN4 or the ER-mitochondrial tether GRP75. GRP75 or RTN4 scarcity also decreases the level of the mitochondrial calcium uniporter (MCU), whose inhibition blocks mitochondrial DNA synthesis. Because ERMCS depletion did not diminish mitochondrial calcium, and MCU complex can transport manganese, we tested whether manganese could bypass these defects. Manganese supplementation restored mitochondrial DNA replication in cells lacking ERMCS or with inhibited MCU, identifying manganese as a critical mediator. We then considered mitochondrial transcription as a potential manganese target, since it provides both transcripts for gene expression and primers for DNA replication. In vitro, manganese inhibits transcription re-start and stimulates RNA synthesis at the light-strand origin of replication. These findings support a model in which ER-mitochondrial contact sites, in conjunction with MCU, deliver manganese from the ER to mitochondria to promote DNA replication, potentially by modulating mitochondrial RNA polymerase activity.

Insights

Endoplasmic reticulum-mitochondrial contact sites regulate DNA replication by controlling manganese delivery. Manganese supplementation restores DNA replication, highlighting its crucial role in this process.

Area of Science:

Cell Biology
Mitochondrial Biology
Molecular Genetics

Background:

Mitochondrial DNA replication is essential for cellular energy production and occurs at endoplasmic reticulum-mitochondrial contact sites (ERMCS).
While RTN4 is known to regulate ERMCS, other factors controlling mitochondrial DNA replication remain largely undefined.

Purpose of the Study:

To investigate the role of ER proteins, specifically ERLIN2, in regulating ERMCS and mitochondrial DNA replication.
To elucidate the mechanism by which ERMCS influence mitochondrial DNA synthesis, focusing on the role of manganese.

Main Methods:

Human fibroblasts were used to study the effects of repressing ER proteins (ERLIN2, RTN4) and the ER-mitochondrial tether (GRP75) on ERMCS and mitochondrial DNA replication.
Mitochondrial calcium levels and manganese transport were assessed.
Manganese supplementation was used to test its ability to rescue replication defects.
In vitro transcription assays were performed to evaluate the effect of manganese on mitochondrial transcription.

Main Results:

Repressing ERLIN2, RTN4, or GRP75 depleted ERMCS and inhibited mitochondrial DNA replication.
Scarcity of GRP75 or RTN4 reduced the levels of the mitochondrial calcium uniporter (MCU), inhibiting DNA synthesis.
Manganese supplementation restored mitochondrial DNA replication in cells with depleted ERMCS or inhibited MCU.
In vitro, manganese affected mitochondrial transcription initiation and RNA synthesis at the replication origin.

Conclusions:

ERMCS, potentially involving ERLIN2, RTN4, and GRP75, are critical for mitochondrial DNA replication.
Manganese acts as a key mediator, delivered from the ER to mitochondria via ERMCS and MCU, to promote DNA replication.
Manganese may modulate mitochondrial RNA polymerase activity, influencing transcription and providing primers for replication.

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