Linear mitochondrial DNA is rapidly degraded by components of the replication machinery

Viktoriya Peeva,Christian Becker,Pedro Rebelo-Guiomar,Alexei P Kudin,Michal Minczuk,Sarah Corsi,Janine Altmüller,Wolfram S Kunz,Maciej J Szukszto,Daniel Blei,Payam A Gammage,Genevieve Trombly,Gábor Zsurka

doi:10.1038/s41467-018-04131-w

Abstract

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Here, we show that, in cellular models of restriction endonuclease-induced mtDNA double-strand breaks, linear mtDNA is eliminated within hours by exonucleolytic activities. Inactivation of the mitochondrial 5′-3′exonuclease MGME1, elimination of the 3′-5′exonuclease activity of the mitochondrial DNA polymerase POLG by introducing the p.D274A mutation, or knockdown of the mitochondrial DNA helicase TWNK leads to severe impediment of mtDNA degradation. We do not observe similar effects when inactivating other known mitochondrial nucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, or RBBP8). Our data suggest that rapid degradation of linearized mtDNA is performed by the same machinery that is responsible for mtDNA replication, thus proposing novel roles for the participating enzymes POLG, TWNK, and MGME1.

Highlights

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown
In control mitoEagI-expressing cells, linear mtDNA species carrying an end that corresponded to the original cutting site were only abundant after 2 h of induction (Fig. 1a, c, mitoEagI)
We show here that rapid degradation of linearized DNA, which is unique for the mitochondrial genome and surpasses doublestrand break repair reactions, is executed by the same enzymes that are involved in mtDNA replication (Fig. 6a)

Summary

Introduction

Emerging gene therapy approaches that aim to eliminate pathogenic mutations of mitochondrial DNA (mtDNA) rely on efficient degradation of linearized mtDNA, but the enzymatic machinery performing this task is presently unknown. Damaged DNA molecules normally represent only a tiny fraction of total mtDNA of a cell, which can be discarded without severe consequences and replaced by replicating intact mtDNA. This idea of a 'disposable genome'[1, 2] plays a crucial role in emerging approaches in gene therapy of mitochondrial DNA diseases that aim to reduce the proportion of pathogenic mtDNA mutations by selectively cleaving and subsequently breaking down mutated mtDNA3–5. We demonstrate that the removal of linear mtDNA is an important aspect of proper maintenance of the mitochondrial genome

Methods

Results

Conclusion