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

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

doi:10.1038/s41467-018-04131-w

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

Nat Commun. 2018 Apr 30;9(1):1727. doi: 10.1038/s41467-018-04131-w.

Authors

Viktoriya Peeva¹, Daniel Blei¹, Genevieve Trombly¹, Sarah Corsi^{1

2}, Maciej J Szukszto³, Pedro Rebelo-Guiomar^{3

4}, Payam A Gammage³, Alexei P Kudin¹, Christian Becker⁵, Janine Altmüller^{5

6}, Michal Minczuk⁷, Gábor Zsurka^{8

9}, Wolfram S Kunz^{10

11}

Affiliations

¹ Institute of Experimental Epileptology and Cognition Research, University of Bonn, Sigmund-Freud-Str. 25, D-53105, Bonn, Germany.
² Human Nutrition Research Centre, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
³ MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, CB2 0XY, UK.
⁴ Graduate Program in Areas of Basic and Applied Biology (GABBA), University of Porto, Porto, 4200-135, Portugal.
⁵ Cologne Center for Genomics, Center for Molecular Medicine Cologne (CMMC), University of Cologne, Weyertal 115b, Cologne, D-50931, Germany.
⁶ Institute of Human Genetics, University of Cologne, Kerpener Str. 34, Cologne, D-50931, Germany.
⁷ MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, CB2 0XY, UK. [email protected].
⁸ Institute of Experimental Epileptology and Cognition Research, University of Bonn, Sigmund-Freud-Str. 25, D-53105, Bonn, Germany. [email protected].
⁹ Department of Epileptology and Life & Brain Center, University of Bonn, Sigmund-Freud-Str. 25, Bonn, D-53105, Germany. [email protected].
¹⁰ Institute of Experimental Epileptology and Cognition Research, University of Bonn, Sigmund-Freud-Str. 25, D-53105, Bonn, Germany. [email protected].
¹¹ Department of Epileptology and Life & Brain Center, University of Bonn, Sigmund-Freud-Str. 25, Bonn, D-53105, Germany. [email protected].

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence
CRISPR-Cas Systems
DNA Breaks, Double-Stranded
DNA Cleavage*
DNA Helicases / genetics
DNA Helicases / metabolism
DNA Polymerase gamma / genetics
DNA Polymerase gamma / metabolism
DNA Replication*
DNA, Mitochondrial / genetics*
DNA, Mitochondrial / metabolism
Deoxyribonucleases, Type II Site-Specific / genetics
Deoxyribonucleases, Type II Site-Specific / metabolism
Electron Transport Complex IV / genetics
Electron Transport Complex IV / metabolism
Exodeoxyribonucleases / genetics
Exodeoxyribonucleases / metabolism
Gene Editing / methods*
Genetic Therapy
HEK293 Cells
Humans
Mitochondria / genetics*
Mitochondria / metabolism
Mitochondria / pathology
Recombinant Fusion Proteins / genetics
Recombinant Fusion Proteins / metabolism

Substances

DNA, Mitochondrial
Recombinant Fusion Proteins
COX8C protein, human
Electron Transport Complex IV
DNA Polymerase gamma
POLG protein, human
Exodeoxyribonucleases
MGME1 protein, human
endodeoxyribonuclease XmaIII
CTGCAG-specific type II deoxyribonucleases
Deoxyribonucleases, Type II Site-Specific
DNA Helicases

Abstract

Publication types

MeSH terms

Substances

Grants and funding