The presence of rNTPs decreases the speed of mitochondrial DNA replication

In this time and age, it is delightful to see a publication which is not doing poorly controlled correlative stuff or omics for the sake of omics, but instead strong hypothesis driven biochemistry. The paper I am referring to comes from the lab of Sjoerd Wanrooij in Umea (Forslund et al. 2018).

Now, it has been known since the 70's that mitochondrial DNA (mtDNA) has ribonucleotides, but it has remained unclear whether they possess a function and how they affect mtDNA maintenance and gene expression. Until very recently, there has been very little attention given to these ribonucleotides, but it seems this subject is becoming trendy again. A recent paper from the Clausen lab showed for the first time the amount and distribution of ribonucleotides in whole mtDNA and also how the amount of ribonucleotides might be different in some patients with changes in nucleotide pools (Berglund et al. 2017). In this paper, Forslund et al. try to understand whether the mitochondrial DNA polymerase (POLG) is inhibited by ribonocleotides in the template strand or by changes in the relative ration of NTP to dNTP.

In the beginning of the paper Forslund et al. make a good point, which might be missed by a lot of people not working with POLG. Back in the 90's and early 00's, people performed all POLG in vitro experiments using the exonuclease-deficient mutant because it makes the experiments easier. However, it was later recognized that the exonuclease-deficient POLG might be doing some funky things such as increased strand-displacement (Farge et al. 2007). For this reason, one should interpret the old papers with some grain of salt.

I personally like these kind of experiments, where the models used are first established using previously published systems. Not only is this helpful in reproducing already published findings but it really makes you trust the experiment. I highly recommend reading the whole thing.

The weirdest finding of the paper is that only WT POLG but not the exonuclease-deficient POLG is sensitive to low NTP/high rNTP conditions. The authors suggest in the discussion that this could be caused idling of the WT POLG between polymerase/exonuclease modes. It is not obvious to be why the exonuclease-deficient POLG would not idle also. Could it be that the exo- mutation somehow alters the protein structure to have different nucleotide selection dynamics? Or perhaps the low NTP/high rNTP condition alters the efficiency of the WT POLG so that it will have increased removal of correct bases leading to decreased full length synthesis? I hope they will follow this up.

In the discussion the authors stated that their in vitro estimation of ribonucleotide content in mtDNA is somewhat lower than the values obtained from in vivo samples. One explanation for this could be that because the nucleotide pool sizes are so challenging to measure, it could be that the in vitro measurements are over-estimating the dNTP pools or under-estimating the NTP pools. To me, this is the most parsimonious answer. They also continue hypothesizing that some other polymerases, like Polβ or PrimPol, could introduce ribonucleotides in vivo. I'm yet to be convinced that either of these polymerases would be in mitochondrial matrix or have a mitochondrial function. For instance, depending on the study, small amount of PrimPol localizes to mitochondria or doesn't. Even when a fraction of PrimPol has been shown to localize within mitochondria, in subcellular fractination it seems to be degraded together with OPA1 (Torregrosa-Muñumer et al. 2017), suggesting PrimPol is in the inner membrane space. I put my money on over/under-estimated nucleotide pools.

In overall, once again strong biochemistry from Sjoerd's group.

References:

Berglund AK, Navarrete C, Engqvist MK, Hoberg E, Szilagyi Z, Taylor RW, Gustafsson CM, Falkenberg M, Clausen AR. Nucleotide pools dictate the identity and frequency of ribonucleotide incorporation in mitochondrial DNA. PLoS Genet. 2017. PMID: 28207748

Farge G, Pham XH, Holmlund T, Khorostov I, Falkenberg M. The accessory subunit B of DNA polymerase gamma is required for mitochondrial replisome function. Nucleic Acids Res. 2007. PMID: 17251196

Forslund JME, Pfeiffer A, Stojkovič G, Wanrooij PH, Wanrooij S. The presence of rNTPs decreases the speed of mitochondrial DNA replication. PLoS Genet. 2018 PMID: 29601571

Torregrosa-Muñumer R, Forslund JME, Goffart S, Pfeiffer A, Stojkovič G, Carvalho G, Al-Furoukh N, Blanco L, Wanrooij S, Pohjoismäki JLO. PrimPol is required for replication reinitiation after mtDNA damage. Proc Natl Acad Sci U S A. 2017. PMID:29073063