Unleashing a novel function of Endonuclease G in mitochondrial genome instability
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Curated by eLife
Evaluation Summary:
This manuscript is of interest for researchers in the field of mitochondrial genome stability and mitochondrial genetic diseases. The authors show that endonuclease G preferentially binds to mitochondrial genome regions which have a potential for forming G4 tetraplexes, inducing DNA breaks that may lead to a common 9 bp deletion in the mitochondrial genome by microhomology-mediated endjoining.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
Having its genome makes the mitochondrion a unique and semiautonomous organelle within cells. Mammalian mitochondrial DNA (mtDNA) is a double-stranded closed circular molecule of about 16 kb coding for 37 genes. Mutations, including deletions in the mitochondrial genome, can culminate in different human diseases. Mapping the deletion junctions suggests that the breakpoints are generally seen at hotspots. ‘9 bp deletion’ (8271–8281), seen in the intergenic region of cytochrome c oxidase II/tRNA Lys , is the most common mitochondrial deletion. While it is associated with several diseases like myopathy, dystonia, and hepatocellular carcinoma, it has also been used as an evolutionary marker. However, the mechanism responsible for its fragility is unclear. In the current study, we show that Endonuclease G, a mitochondrial nuclease responsible for nonspecific cleavage of nuclear DNA during apoptosis, can induce breaks at sequences associated with ‘9 bp deletion’ when it is present on a plasmid or in the mitochondrial genome. Through a series of in vitro and intracellular studies, we show that Endonuclease G binds to G-quadruplex structures formed at the hotspot and induces DNA breaks. Therefore, we uncover a new role for Endonuclease G in generating mtDNA deletions, which depends on the formation of G4 DNA within the mitochondrial genome. In summary, we identify a novel property of Endonuclease G, besides its role in apoptosis and the recently described ‘elimination of paternal mitochondria during fertilisation.
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Evaluation Summary:
This manuscript is of interest for researchers in the field of mitochondrial genome stability and mitochondrial genetic diseases. The authors show that endonuclease G preferentially binds to mitochondrial genome regions which have a potential for forming G4 tetraplexes, inducing DNA breaks that may lead to a common 9 bp deletion in the mitochondrial genome by microhomology-mediated endjoining.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Joint Public review:
Endonuclease G (EndoG) is best known for its role in caspase-independent degradation of nuclear DNA during apoptosis, when the protein is released from mitochondria upon oxidative stress. This manuscript reveals a role of EndoG in generating a 9 bp deletion that is commonly found in the mitochondrial genome. The authors combine bioinformatics and experimental analysis to identify mitochondrial genome sequences that have the potential to form G4 tetraplexes. They focus the further analysis on the intergenic region of cytochrome c oxidase II/tRNALys that contains the most common mitochondrial deletion, a 9 bp deletion involving a repeated DNA sequence. Furthermore, using BG4, a presumptive anti-G4 DNA antibody, the authors use immunofluorescence and ChIP to provide evidence for the occurrence of G4 tetraplexes in the …
Joint Public review:
Endonuclease G (EndoG) is best known for its role in caspase-independent degradation of nuclear DNA during apoptosis, when the protein is released from mitochondria upon oxidative stress. This manuscript reveals a role of EndoG in generating a 9 bp deletion that is commonly found in the mitochondrial genome. The authors combine bioinformatics and experimental analysis to identify mitochondrial genome sequences that have the potential to form G4 tetraplexes. They focus the further analysis on the intergenic region of cytochrome c oxidase II/tRNALys that contains the most common mitochondrial deletion, a 9 bp deletion involving a repeated DNA sequence. Furthermore, using BG4, a presumptive anti-G4 DNA antibody, the authors use immunofluorescence and ChIP to provide evidence for the occurrence of G4 tetraplexes in the mitochondrial genome. Using purified Endo G and mitochondrial extracts, the authors show preferential binding of EndoG to mitochondrial genome sequences with the potential to form G4DNA and induction of DNA breaks at such sites using mutant oligonucleotide substrates that are predicted to break the G4 potential as controls. Moreover, the authors reconstitute in vitro the reaction to generate the 9 bp deletion using oligonucleotides and mitochondrial extracts, leading to the model that EndoG-mediated cleavage at G4 tetraplex forming sequences in the mitochondrial genome generates breaks that are repaired by MMEJ generating rearrangements including the common 9 bp deletion. Finally, the authors show that under oxidative stress induced by menadione, EndoG is released into the mitochondrial matrix, possibly making it more available to act on the mitochondrial genome. The manuscript spans a significant number of experiments ranging from bioinformatic analysis, protein biochemistry, cell genetics, and immunofluorescence. The data generally support the conclusions with some significant exceptions. The authors often inaccurately describe the results mixing in their interpretation, and the manuscript is difficult to read. Major problems are the specificity of the BG4 antibody, the resolution of the microscopy, and the lack of a critical control of separate incubation of the substrates for the reconstitution experiment. Conceptually, it is unclear what the physiological roles of EndoG release into the mitochondrial matrix and the generation of mitochondrial genome arrangements might be.
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