RNR is essential for both nuclear and mtDNA replication. The enzyme is limiting for mtDNA copy number in yeast, but whether the same is true in mammals has not been studied earlier.
dNTP pool regulation has emerged as a potential tool to increase mtDNA levels, and thereby to slow down progression of mtDNA disease. In vitro
supplementation with two deoxyribonucleoside monophosphates (dNMPs) rescued mtDNA depletion in cultured patient myotubes with mutations in the mitochondrial deoxyribonucleoside salvage pathway enzyme deoxyguanosine kinase (dGK) (32
). However, mtDNA depletion due to patient mutations in the mtDNA polymerase gamma (Pol γ) was not restored through nucleotide supplementation in the same study. Likewise, nucleotide supplementation to healthy myotubes did not increase mtDNA levels significantly above normal (32
). These results suggested that although dNTP pool expansion may be beneficial in cases of dNTP deficiency, other factors than the size of the mitochondrial dNTP pool are limiting for mtDNA copy number in normal cells or in mtDNA replication defects.
RNR overexpression in yeast resulted in elevated mtDNA levels (12
), and complementation of the respiratory chain deficient phenotype of Pol γ disease mutations (13
). The results presented here suggest that RNR overexpression has partially opposite effects in mammals, which illustrates the marked differences in dNTP pool maintenance between organisms. However, we cannot exclude the possibility that increased RNR activity or dNTP availability in vivo
could be beneficial in cases with increased mtDNA turnover or mutagenesis. The increased carcinogenesis in RNRTg
) illustrates the hazards of altering dNTP pool maintenance and further reduces the potential of RNR as a therapeutic tool.
The mechanisms governing mtDNA copy number in tissues are starting to come into focus. In mice, overexpression of the histone-like packaging protein TFAM or of the mtDNA helicase Twinkle increase mtDNA copy number 2- to 3-fold (14
). The mRNA level of Twinkle and the protein level of TFAM correlate linearly with mtDNA content, suggesting that these may be limiting factors in determining the amount of mtDNA. Defects in mitochondrial dNTP pool maintenance proteins cause loss of mtDNA, indicating that these factors are essential for mtDNA maintenance (7
). However, their contribution to the physiological control of mtDNA copy number is not well characterized. Heart-specific overexpression of the salvage pathway enzyme thymidine kinase 2 (TK2) in mice resulted in a 300-fold increase in enzyme activity and produced a ~30% increase in mtDNA copy number (37
). Therefore, a very large increase in TK2 activity modestly influences mtDNA levels, potentially through increases in deoxycytidine and thymidine nucleotide pools. Our results show that although RNR is involved in the synthesis of all four dNTPs, very high levels of the enzyme do not increase mtDNA copy number and instead perturb mtDNA homeostasis. This finding is in line with Twinkle and TFAM being the main regulators of mtDNA level under normal circumstances.
A possible mechanism for nuclear control over mtDNA copy number would be to alter the transcription of mtDNA maintenance genes. In yeast, the first established signaling pathway that regulated mtDNA copy number is activated by the Mec1p/Rad53p kinases and leads to induction of RNR expression (12
). The related ATM (ataxia-telangiectasia mutated) kinase in humans was also found to influence RNR expression and mtDNA homeostasis (38
). We found the transcription of Rrm1
to be downregulated in two independent mouse models with increased mtDNA copy number, suggesting an intimate feedback mechanism between transcriptional regulation of RNR subunits and mtDNA levels. RNR could thus contribute to a regulatory mechanism for nuclear control of mtDNA copy number in vivo
. Such a signaling pathway could involve p53, a tumor suppressor and ATM target, which influences the expression level of p53R2 and is known to localize in small amounts to mitochondria (39
RNR is rate-limiting for de novo
dNTP synthesis, so any alteration to RNR activity is expected to induce changes in dNTP pools. These changes are transmitted into mitochondria, since the mitochondrial and cytosolic dNTP pools are in rapid communication (40
). Direct measurement of dNTP pools from animal tissues is complicated by dNTP dephosphorylation caused by the anaerobiosis that immediately follows the death of the animal (30
). Our extraction method was optimized to minimize nucleotide degradation during extraction, and the ATP, ADP and AMP levels were used as internal controls to ensure comparability across samples. We documented clear and reproducible differences in total dNTP pools between RNR overexpressors and wild-type mice.
First, there were significant increases in the levels of dATP and dCTP both in the Rrm2Tg
mice. Rrm2 induced higher dNTP increases than p53R2, which is consistent with Rrm2 being more active than p53R2 in vitro
). Rrm1 overexpression alone was able to induce a detectable, albeit not statistically significant, increase in dATP and dCTP levels, and overexpression of Rrm1 together with either small subunit led to a clear trend towards higher levels of dATP and dCTP. This apparent synergy suggested that co-overexpression of both subunits led to an increase in the abundance of the tetramer, with subsequent effects on nucleotide pools.
The allosteric regulation of RNR promotes a balanced production of all four dNTPs and therefore manipulation of RNR expression might be expected to influence the levels of all four dNTPs equally (1
). However, we found remarkable dNTP pool imbalances in the RNRTg
mice. The dGTP pools were unchanged whereas dTTP pools were actually decreased in p53R2Tg
as well as Rrm1Tg
mice. This finding illustrates the need for tight control of the relative activities of the large number of anabolic and catabolic enzymes that determine the final dNTP composition in vivo
). For instance, the synthesis of thymidine phosphates requires—as an additional step—reductive methylation of RNR-generated deoxyuridine monophosphate, which is catalyzed by thymidylate synthase. Hence, induction of RNR could cause a substrate overload for the endogenous thymidylate synthase, which in turn could explain why increased RNR does not increase the dTTP level. Furthermore, the specificity of RNR for GDP reduction is induced by the binding of dTTP to the specificity site of Rrm1 (1
). Thus, a relative lack of dTTP could explain why the dGTP pools did not increase in our mice. The dNTP imbalance was not due to depletion of any of the substrates of RNR, since the levels of ADP, CDP, UDP and GDP were similar in all genotypes.
Unbalanced dNTP pools are known to cause mutagenesis in both nuclear (43
) and mitochondrial (28
) genomes. Given the presence of altered dNTP pools in RNRTg
mice, the likely mechanism of progressive mtDNA depletion in bitransgenic mice is inefficient mtDNA replication caused by perturbed dNTP balance. Initiation of nuclear DNA replication is influenced by the dNTP pool (44
), and an imbalanced pool could lead to reduced frequency of mtDNA replication initiation. mtDNA depletion was specifically restricted to bitransgenic mice, which was consistent with them displaying the largest dNTP alterations. The fact that Rrm2Tg
mice also had altered dNTP pools but no mtDNA depletion, suggests a threshold effect for the relative dNTP levels above which mtDNA replication becomes inefficient. Nevertheless, we cannot exclude that co-overexpression of Rrm1 and the small subunit gave rise to additional, unidentified effects, which did not occur upon overexpression of one subunit. Moreover, mtDNA depletion was observed only in the skeletal muscle, although the transgenes were overexpressed in both the skeletal and cardiac muscles, suggesting tissue specific mechanisms in the regulation of nucleotide pools and/or mtDNA maintenance.
Altered dNTP pool balance is a hallmark of MNGIE disease, where deficiency of the catabolic enzyme thymidine phosphorylase (TP) leads to elevated dTTP pools, which cause mtDNA depletion, deletions and point mutations in humans (28
). Contrary to MNGIE, however, we found no increase in mtDNA point mutations or deletions in the RNRTg
mice. There are at least two possible explanations for the absence of mtDNA instability, other than depletion, in our mice. First, the dNTP changes were almost opposite to those in MNGIE, i.e. the relative dTTP level decreased instead of increasing. Further studies are needed to elucidate the exact effect of changes in the relative levels of each of the four dNTPs on mtDNA replication frequency and fidelity. Second, the life-span of a mouse may be too short to develop significant amounts of mtDNA deletions or point mutations in the setting of dNTP imbalance. In support of this, the MNGIE mouse model lacking TP and the related uridine phosphorylase (UP) displayed increased dTTP in brain, and, similar to RNRTg
mice, developed progressive mtDNA depletion, but no deletions or point mutations (27
). The authors argued that this was at least partly due to the short life-span of mice, emphasizing the differences in dNTP maintenance and disorders between species.
In conclusion, we have established that expression of recombinant RNR in mice leads to dNTP pool imbalance and progressive depletion of mtDNA. This is in contrast to previous findings in yeast, in which RNR is a positive regulator of mtDNA abundance. Furthermore, endogenous RNR expression is responsive to increased mtDNA copy number and a potential modifier of mtDNA homeostasis. The dNTP pool imbalance and mtDNA decrease caused by excess RNR suggests that balanced amounts of RNR are essential for mtDNA maintenance in vivo, and that RNR is unlikely to be an optimal target for therapeutic engineering of mtDNA levels in mammals.