Endonuclease G promotes mitochondrial genome cleavage and replication

I have always had in mind the textbook model where EndoG is localized in the mitochondrial intermembrane space (IMS) from where it is released upon apoptosis to degrade nuclear DNA. Every now and then though, there are papers suggesting it would have a mitochondrial function such as regulation of mitochondrial DNA (mtDNA) removal in fruit fly sperm (DeLuca et al. 2012, Yu et al. 2017).

In this paper from Wiehe et al. (Wiehe et al. 2018) the authors suggest EndoG has something to do with mtDNA replication. In the introduction the authors cite a classical paper showing the IMS localization of EndoG (Ohsato et al. 2002) but also another paper suggesting EndoG is either directly or indirectly associated with the mitochondrial inner membrane (Uren et al. 2005). It is important to point out that Uren et al. never showed that EndoG would be in the mitochondrial matrix.

There is data both for and against the function of EndoG in mtDNA maintenance although the former is rather weak. The EndoG knockout mice have normal mtDNA copy number (Irvine et al. 2005, David et al. 2006) providing the strongest evidence for the lack of mitochondrial function. There is one study, however, shoving that EndoG knockout mice have decreased mtDNA copy number (McDermott-Roe et al. 2011). Wiehe et al. refer to this same McDermott-Roe et al. study suggesting that EndoG would directly interact with mtDNA. This evidence seems spurious at best, because the authors performed chromatin immunoprecipitation (ChIP) using purified mitochondria (McDermott-Roe et al. 2011). It is expected that if you carry out a pulldown with a DNA binding protein (EndoG) and the only DNA around comes from mitochondria, one will enrich mtDNA. This does not show that EndoG would interact with mtDNA in vivo. It seems there is no strong evidence showing that EndoG would be localized in the mitochondrial matrix.

To visualize mitochondrial DNA replication and transcription in cells, the Wiehe et al. use so called mitochondrial Transcription and Replication Imaging Protocol (mTRIP). This method is based on fluorescence probes binding mtDNA (mREP) or mtRNA (mTRANS). The mtDNA binding probes is located upstream of the replication origin of the heavy strand, between the light- and heavy-strand promoters (LSP and HSP) (Chatre & Ricchetti 2013). It has been suggested that this probe can access mtDNA only during the initiation of mtDNA replication and 7S DNA synthesis. It is unclear to me, why it would not access this region during the initiation of mtDNA transcription from the LSP and how sensitive this probe is to changing mtDNA topology.

Wiehe et al. use this mREP mtDNA binding probe as evidence for initiation for mtDNA replication but it seems to me these probes can measure several indistinguishable things (initiation of transcription, initiation of mtDNA replication, initiation of 7S DNA synthesis, differences in mtDNA topology). The authors also used qPCR to quantify the levels of 7S DNA and mtDNA and it seems that the amount of 7S DNA was decreased as was the mtDNA copy number when measured proximal, but not distal, to the replication origin (Fig. 2). It would have been nice to see a Southern blot of mtDNA to actually see whether there is some paused/abortive replication going on.

Next, Wiehe et al over-expressed WT and catalytically inactive EndoG but it is not shown how strong this over-expression was (Fig. 3). Also, the over-expressed proteins were tagged (Myc-DDK) and it is not addressed whether the tagged proteins are functional. The authors compared the effects of these over-expressions on mREP signal and saw a statistically significant but to me a biologically meaningless increase in mREP signal, which, as explained above, can come from many sources.

Next in figure 4 the authors amplified mtDNA in two large overlapping fragments using long-range PCR and saw that upon EndoG knockdown the amount of amplified product decreases in only one of the fragments, which included the whole minor arc. This long-range PCR could be affected by various factors such as replication stalling, DNA base damage and nicking of DNA so it is unclear what the authors are observing here. Weirdly enough, this effect on long-range PCR amplification was not reproduced in a later experiment (Fig. 5D).

All in all the paper assumes without any evidence that EndoG is in the mitochondrial matrix. The results are largely based on fluorescence probes and long-range PCR, both of which are sensitive to various effectors.

Just to be pedantic I have to correct the first sentence of this manuscript. MtDNA encodes 13 proteins, 11 of which are part of the electron transport system and all 13 are part of the oxidative phosphorylation system.

Stats:

The authors used statistical tests which do not assume normal distribution. Therefore it would have been nice to see etc. box plots instead of bar plots to see the sample distribution. Also, the authors do not seem to correct any statistical methods for multiple comparisons.

References:

Chatre L, Ricchetti M. Prevalent coordination of mitochondrial DNA transcription and initiation of replication with the cell cycle. Nucleic Acids Res. 2013. PMID: 23345615

David KK, Sasaki M, Yu SW, Dawson TM, Dawson VL. EndoG is dispensable in embryogenesis and apoptosis. Cell Death Differ. 2006. PMID: 16239930

DeLuca SZ, O'Farrell PH. Barriers to male transmission of mitochondrial DNA in sperm development. Dev Cell. 2012. PMID: 22421049

Irvine RA, Adachi N, Shibata DK, Cassell GD, Yu K, Karanjawala ZE, Hsieh CL, Lieber MR. Generation and characterization of endonuclease G null mice. Mol Cell Biol. 2005. PMID: 15601850

McDermott-Roe C, Ye J, Ahmed R, Sun XM, Serafín A, Ware J, Bottolo L, Muckett P, Cañas X, Zhang J, Rowe GC, Buchan R, Lu H, Braithwaite A, Mancini M, Hauton D, Martí R, García-Arumí E, Hubner N, Jacob H, Serikawa T, Zidek V, Papousek F, Kolar F, Cardona M, Ruiz-Meana M, García-Dorado D, Comella JX, Felkin LE, Barton PJ, Arany Z, Pravenec M, Petretto E, Sanchis D, Cook SA. Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function. Nature. 2011. PMID: 21979051

Ohsato T, Ishihara N, Muta T, Umeda S, Ikeda S, Mihara K, Hamasaki N, Kang D. Mammalian mitochondrial endonuclease G. Digestion of R-loops and localization in intermembrane space. Eur J Biochem. 2002. PMID: 12444964

Uren RT, Dewson G, Bonzon C, Lithgow T, Newmeyer DD, Kluck RM. Mitochondrial release of pro-apoptotic proteins: electrostatic interactions can hold cytochrome c but not Smac/DIABLO to mitochondrial membranes. J Biol Chem. 2005. PMID: 15537572

Wiehe RS, Gole B, Chatre L, Walther P, Calzia E, Ricchetti M, Wiesmüller L. Endonuclease G promotes mitochondrial genome cleavage and replication. Oncotarget. 2018. PMID: 29719607

Yu Z, O'Farrell PH, Yakubovich N, DeLuca SZ. The Mitochondrial DNA Polymerase Promotes Elimination of Paternal Mitochondrial Genomes. Curr Biol. 2017. PMID: 28318978