The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions.

The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions.

I posted recently how two labs have published data showing that the mitochondrial DNA  (mtDNA) polymerase gamma (POLGA) would degrade linear mtDNA fragments (Peeva et al. 2018) or mtDNA under starvation (Medeiros et al. 2018). Now a third group published similar results and they come from the lab of Carlos Moraes so you know are going to enjoy it (Nissanka et al. 2018).

In this study they used both mouse embryonic fibroblasts (MEFs) and actual mice. First, they expressed mitochondrially targeted restriction enzymes to produce linear mtDNA fragments and followed how quickly these are degraded in WT MEFs and mtDNA mutator MEFs. Clearly, MEFs expressing the exonuclease-deficient POLGA were not efficient in removing the linear mtDNA fragment which is a similar result to the Peeva et al. study.

Previously, Medeiros et al. and Peeva et al studied the degradation of mtDNA in yeast and cell culture, respectively, but it was unclear whether this would also take place in vivo. Moraes lab is rather experienced in introducing enzymes (restriction enzymes and mitoTALENs) into mitochondria in mice and as could be expected they introduced these mitochondrially targeted restriction enzymes into mice using adenovirus to study mtDNA degradation. As a result, it seems that POLGA is participating to the degradation of linear mtDNA fragments also in vivo.

Nissanka et al. also assessed whether the presence of linear mtDNA leads to mtDNA rearrangements such as circular mtDNA molecules with deletions. This seems to indeed be the case and it would be interesting to know whether these rearrangements would also take place in patients carrying pathogenic mutations in the replication machinery proteins. These results also suggest that mitochondrial zinc fingers (mtZFN) and mitoTALENs (Gammage et al. 2017) might have some unintended consequences. Both of these approaches are based on cutting the mtDNA molecules carrying a pathogenic mutation leading to the degradation of the molecule. In the ideal case, the loss of these pathogenic molecules would be replaced by the replication of the WT mtDNA molecules. Based on the results of Nissanka et al. in the non-ideal case the presence of these linear molecules could increase the amount of mtDNA rearrangements.

References:

Medeiros TC, Thomas RL, Ghillebert R, Graef M. Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation. J Cell Biol. 2018. PMID: 29519802

Nissanka N, Bacman SR, Plastini MJ, Moraes CT. The mitochondrial DNA polymerase gamma degrades linear DNA fragments precluding the formation of deletions. Nat Commun. 2018. PMID: 29950568

Gammage PA, Moraes CT, Minczuk M. Mitochondrial Genome Engineering: The Revolution May Not Be CRISPR-Ized. Trends Genet. 2017. PMID: 29179920

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun. 2018. PMID: 29712893

Replication machinery degrading linear mitochondrial DNA

Linear mitochondrial DNA (mtDNA) molecules with deletions are a curious case. To my knowledge, they are only present at high levels in a few genetic models, including the mtDNA mutator mouse(Trifunovic et al. 2004, Bailey et al. 2009), mtDNA mutator flies (Bratic et al. 2015), patients carrying mutations in mitochondrial genome maintenance exonuclease 1 (MGME1)(Nicholls et al. 2014) and MGME1 knockout mouse (Matic et al. 2018).

In the case of mtDNA mutator mouse, the level of this linear mtDNA with deletion remains stable with time (Kukat et al. 2009). Additionally, it has been shown using mitochondrially targeted restriction enzymes that linear mtDNA is rapidly degraded (Bayona-Bafaluy et al. 2005). Therefore the question remains, who is degrading these mtDNA fragments?

Moretton et al. tried to answer this question by linearizing mtDNA with mitochondrial targeted restriction enzyme followed by systematic knockdown of exonucleases known/suggested to be in mitochondria (Moretton et al. 2017). These exonucleases included ExoG, EndoG, MGME1, DNA2 and FEN1. EXD2 was not tested, but that was later shown to be localized on the mitochondrial outer membrane (Hensen et al. 2018). Perhaps surprisingly, none of the exonucleases studied by Moretton et al. seemed to participate in degradation of linear mtDNA fragments.

There is still at least one exonuclease that Moretton et al. did not study, which is the exonuclease activity of mitochondrial DNA polymerase (POLG). Indeed, in the absence of nucleotides POLG can engage in so called exonuclease mode where it starts degrading the primer in 3’-5’ direction (Bratic et al. 2015). A recent study in fruit flies tried to answer the question what enzyme degrades mtDNA in fly sperm (Yu et al. 2017). Yu et al. concluded that DmPOLG is involved in this degradation but it takes place independent of its exonuclease activity. I was not convinced by this paper because they saw mtDNA replication in punctae without DmPOLG-GFP and more importantly all the models were based on knocking down various proteins of the minimal mitochondrial replisome with variable efficiencies. Also, when I read this paper I was aware of some in-house work taking place in the group of Martin Graef.

Medeiros et al. showed in yeast that under starvation there is a strong mtDNA depletion if autophagy is inhibited and that this depletion can be rescued by increasing nucleoside pools through supplementation or genetically (Medeiros et al. 2018). Therefore, the authors hypothesized that under starvation the autophagy-deficient cells might have changes in nucleotide pools causing MIP1 (yeast POLG) to enter the above mentioned exonuclease mode and degrade mtDNA. Using an exonuclease-deficient POLG the authors showed that indeed it seems to be POLG which is degrading mtDNA.

The most recent paper on the subject comes from Peeva et al. (Peeva et al. 2018). Peeva et al. tested multiple exonucleases (EXOG, APEX2, ENDOG, FEN1, DNA2, MRE11, RBBP8, MGME1 and POLG) to see whether the knockout or knockdown of these enzymes would affect the degradation of linear mtDNA molecules. Somewhat similar to Medeiros et al., Peeva et al. also suggest that POLG together with MGME1 and TWINKLE would be degrading linear mtDNA.

So there it is. Both in yeast and human cells mtDNA seems to be degraded by the exonuclease activity of POLG. It would be interesting to see how the yeast mtDNA actually looks like at the molecular level when degraded as yeasts do not possess MGME1. One could therefore predict that these degraded molecules would not have blunt ends similarly to the results from humans where POLG is suggested to degrade mtDNA to the 3’-5’ direction and MGME1 to the 5’-3’ direction. It is still open question what controls this degradation, if it is controlled to begin with. Both Medeiros et al. and Peeva et al. hypothesize that low nucleotide pools might be a controlling factor. Although this hypothesis would make sense in the case of yeast under starvation and inhibited autophagy, I don’t see how this would work with these human cells under normal culture conditions. Clearly just linearizing mtDNA is sufficient to promote mtDNA degradation without manipulation of the dNTP pools.

Some open questions for the future:

• Is this kind of degradation present also in more in vivo conditions or whether it is only present in somewhat artificial conditions where most of mtDNA is linearized by restriction enzymes.
• What, if any, is controlling the balance between mtDNA synthesis and degradation?
• Is there a specific endonuclease cleaving the yeast mtDNA before POLG can start degrading mtDNA?
• How is mtDNA degraded in organisms without MGME1?

Perhaps this is a stretch but in Peeva et al. study the authors showed how in WT cells linear mtDNA is degraded piece by piece. Where mitophagy selective for damaged mtDNA to exist, which I think it does not (Kauppila et al. 2017), one would not expect to see these slowly degrading molecules but instead a bulk disappearance of linear mtDNA fragments.

References:

Bailey LJ, Cluett TJ, Reyes A, Prolla TA, Poulton J, Leeuwenburgh C, Holt IJ. Mice expressing an error-prone DNA polymerase in mitochondria display elevated replication pausing and chromosomal breakage at fragile sites of mitochondrial DNA. Nucleic Acids Res. 2009. PMID: 19244310

Bayona-Bafaluy MP, Blits B, Battersby BJ, Shoubridge EA, Moraes CT. Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci U S A. 2005. PMID: 16179392

Bratic A, Kauppila TE, Macao B, Grönke S, Siibak T, Stewart JB, Baggio F, Dols J, Partridge L, Falkenberg M, Wredenberg A, Larsson NG. Complementation between polymerase- and exonuclease-deficient mitochondrial DNA polymerase mutants in genomically engineered flies. Nat Commun. 2015. PMID: 26554610

Hensen F, Moretton A, van Esveld S, Farge G, Spelbrink JN. The mitochondrial outer-membrane location of the EXD2 exonuclease contradicts its direct role in nuclear DNA repair. Sci Rep. 2018. PMID: 29599527

Kauppila TES, Kauppila JHK, Larsson NG. Mammalian Mitochondria and Aging: An Update. Cell Metab. 2017. PMID: 28094012

Kukat A, Trifunovic A. Somatic mtDNA mutations and aging--facts and fancies. Exp Gerontol. 2009. PMID: 18585880

Matic S, Jiang M, Nicholls TJ, Uhler JP, Dirksen-Schwanenland C, Polosa PL, Simard ML, Li X, Atanassov I, Rackham O, Filipovska A, Stewart JB, Falkenberg M, Larsson NG, Milenkovic D. Mice lacking the mitochondrial exonuclease MGME1 accumulate mtDNA deletions without developing progeria. Nat Commun. 2018. PMID: 29572490

Medeiros TC, Thomas RL, Ghillebert R, Graef M. Autophagy balances mtDNA synthesis and degradation by DNA polymerase POLG during starvation. J Cell Biol. 2018. PMID: 29519802

Moretton A, Morel F, Macao B, Lachaume P, Ishak L, Lefebvre M, Garreau-Balandier I, Vernet P, Falkenberg M, Farge G. Selective mitochondrial DNA degradation following double-strand breaks. PLoS One. 2017. PMID: 28453550

Nicholls TJ, Zsurka G, Peeva V, Schöler S, Szczesny RJ, Cysewski D, Reyes A, Kornblum C, Sciacco M, Moggio M, Dziembowski A, Kunz WS, Minczuk M. Linear mtDNA fragments and unusual mtDNA rearrangements associated with pathological deficiency of MGME1 exonuclease. Hum Mol Genet. 2014. PMID: 24986917

Peeva V, Blei D, Trombly G, Corsi S, Szukszto MJ, Rebelo-Guiomar P, Gammage PA, Kudin AP, Becker C, Altmüller J, Minczuk M, Zsurka G, Kunz WS. Linear mitochondrial DNA is rapidly degraded by components of the replication machinery. Nat Commun. 2018. PMID: 29712893

Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly-Y M, Gidlöf S, Oldfors A, Wibom R, Törnell J, Jacobs HT, Larsson NG. Premature ageing in mice expressing defective mitochondrial DNA polymerase. Nature. 2004. PMID: 15164064

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

Accurate estimation of 5-methylcytosine in mammalian mitochondrial DNA

[Updated 26.06.2018]

I have always tried to avoid touching the field of nuclear DNA methylation or any epigenetic stuff. So many claims in the field seem so implausible that there is probably plentiful of BS out there but not being a specialist in the field makes it difficult to critically assess the literature. I do, however, have an interest in mitochondrial DNA (mtDNA) methylation. It is an interesting niche because there are almost 40 years of data showing that it is ineligible/non-existent but still some researchers consider it a subject worthy of research.

The short story, as shown recently by Matsuda et al (Matsuda et al. 2018), is that once you control you experiments properly, there is no mtDNA methylation.

Now to the longer version of the story. DNA methylation can be detected in various ways, including restriction enzymes, antibodies, mass spectrometry, and bisulfite sequencing (van der Wijst & Rots, 2015). These methods have different caveats as explained in the previous reference. For instance, bisulfite sequencing is known to be sensitive to mtDNA topology changes and the other methods can actually detect methylation of nuclear mitochondrial sequences (NUMTs). Pawar and Eide had recently a nice publication trying to assess the signal-to-noise ratio of various methylation detection methods (Pawar & Eide, 2017). They showed how the choice of buffer for restriction enzyme digestions can have a large effect on the results, how restriction enzyme based methods have 3-5% background noise level, how linearization of mtDNA is essential in bisulfite based methods and how antibody-based methods should use proper negative controls.

I tried to assemble a (probably non-exhaustive) timeline of both negative and positive mtDNA methylation studies. Unfortunately, most publications reporting positive findings do not take into consideration the methodological recommendations coming from several methods papers (Matsuda et al. 2018, Owa et al. 2018, Mechta et al. 2017, Pawar et al. 2017, Liu et al. 2016).

Figure 1: The history of mtDNA methylation research. Multiple publications have reported either the absence (upper part) or presence (lower part) of mtDNA methylation. Most studies claiming the presence of mtDNA methylation have questionable methods.

Paper	Method	Shortcoming
Shmookler Reis et al. 1983	Restriction enzyme	Below 5% detection threshold
Shock et al. 2011	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Infantino et al. 2011	Mass spectrometry	mdC levels below 0.25% could come from nuclear DNA
Chestnut et al. 2011	Immunofluorescence microscopy	Only some mdC punctae in the cytoplasm co-localize with mitochondrial SOD2
Chen et al. 2012	ELISA kit	No controls for mtDNA purity
Dzitoyeva et al. 2012	ELISA kit	No controls for mtDNA purity
Kelly et al. 2012	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Sun et al. 2013	Aba-seq	E. coli instead of PCR-amplified mtDNA as negative control
Bellizzi et al. 2013	Bisulphite	mtDNA was not linearized
Pirola et al. 2013	Bisulphite	mtDNA was not linearized
Wong et al. 2013	Bisulphite	mtDNA was not linearized
Ghosh et al. 2014	Bisulphite	Unclear whether mtDNA was linearized
Jia et al. 2016	Bisulphite	mtDNA was not linearized
Mawlood et al. 2016	Bisulphite	mtDNA was not linearized
Janssen et al. 2017	Bisulphite	mtDNA was not linearized
Saini et al. 2017	Restriction enzyme	Below 5% detection threshold
Ren et al. 2017	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Dewall et al. 2017	Immunoprecipitation	No PCR-amplified mtDNA as a negative control
Yu et al. 2017	Bisulphite	mtDNA was not linearized
Kim et al. 2018	Bisulphite	mtDNA was not linearized
Sanyal et al. 2018	Bisulphite	mtDNA was not linearized

So in the end only one of the papers detecting mtDNA methylation seemed valid. It is interesting to note however, that the only valid paper (Infantino et al. 2011) quantified mtDNA methylation using mass spectrometry and their results suggested the levels of mdC were <25%. Another paper also using mass spectrometry saw much lower mdC levels in mtDNA (0.3-0.5%) and concluded mtDNA methylation to be absent (Matsuda et al. 2018). The problem is that in order to measure mtDNA methylation levels by mass spectrometry, one has to first obtain highly pure mtDNA. This is extremely difficult. Even when one uses one of the best protocols out there to obtain pure mtDNA (Kennedy et al. 2014) there will always be nuclear DNA contaminations detectable by Illumina sequencing and that contamination will increase the detected amount of mdC.

So the take-home message method-wise is.

1) Restriction enzyme methods

The background noise level of this method is 3-5% so if you measure mtDNA methylation below this level, you are working with noise instead of signal.

            Optimize the buffer conditions for digestion.

2) Antibody methods

There is a reason why western blot results are not reproducible and the reason is crappy antibodies. Similarly, antibodies supposed to detect only methylated dC bind all kinds of sequences and as a negative control you should always have the same DNA without methylation. In case on mitochondria, one should have PCR amplified mtDNA as the negative control.

3) Bisulfite methods

MtDNA has a topology which will affect bisulfite conversion efficiency. Therefore, one has to linearize mtDNA before this chemical modification or you will be just detecting artefacts.

4) Mass spectrometry approaches

You will always have nuclear DNA contamination in your purified mtDNA. I would like to see a study where one would actually quantify the amount of this background using Illumina sequencing instead of trying to amplify a single nuclear gene by PCR.

In the end I think we can stop researching mtDNA methylation. It is just not there.

PS: Please everybody stop referencing the Rebelo et al (Rebelo et al. 2009) paper as proof of mtDNA methylation. Obviously you either did not read the paper or did not understand it.

References:

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Chestnut BA, Chang Q, Price A, Lesuisse C, Wong M, Martin LJ. Epigenetic regulation of motor neuron cell death through DNA methylation. J Neurosci. 2011. PMID: 22090490

Dzitoyeva S, Chen H, Manev H. Effect of aging on 5-hydroxymethylcytosine in brain mitochondria. Neurobiol Aging. 2012. PMID: 22445327

Chen H, Dzitoyeva S, Manev H. Effect of valproic acid on mitochondrial epigenetics. Eur J Pharmacol. 2012. PMID: 22728245

Ghosh S, Sengupta S, Scaria V. Comparative analysis of human mitochondrial methylomes shows distinct patterns of epigenetic regulation in mitochondria. Mitochondrion. 2014. PMID: 25058022

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Hong EE, Okitsu CY, Smith AD, Hsieh CL. Regionally specific and genome-wide analyses conclusively demonstrate the absence of CpG methylation in human mitochondrial DNA. Mol Cell Biol. 2013. PMID: 23671186

Infantino V, Castegna A, Iacobazzi F, Spera I, Scala I, Andria G, Iacobazzi V. Impairment of methyl cycle affects mitochondrial methyl availability and glutathione level in Down's syndrome. Mol Genet Metab. 2011. PMID: 21195648

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Jia L, Li J, He B, Jia Y, Niu Y, Wang C, Zhao R. Abnormally activated one-carbon metabolic pathway is associated with mtDNA hypermethylation and mitochondrial malfunction in the oocytes of polycystic gilt ovaries. Sci Rep. 2016. PMID: 26758245

Kelly RD, Mahmud A, McKenzie M, Trounce IA, St John JC. Mitochondrial DNA copy number is regulated in a tissue specific manner by DNA methylation of the nuclear-encoded DNA polymerase gamma A. Nucleic Acids Res. 2012. PMID: 22941637

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