Mitochondrial deoxynucleotide pool sizes in mouse liver and evidence for a transport mechanism for thymidine monophosphate

Abstract

reported that mt pools of rat tissues instead are highly asymmetric, with the dGTP pool in some cases being several-hundred-fold larger than the dTTP pool, and suggested that the asymmetry contributes to increased mutagenesis during mt DNA replication. We have now investigated this discrepancy and determined the size of each dNTP pool in mouse liver mitochondria. We found large variations in pool sizes that closely followed variations in the ATP pool and depended on the length of time spent in the preparation of mitochondria. The proportion between dNTPs was in all cases without major asymmetries and similar to those found earlier in cultured resting cells. We also investigated the import and export of thymidine phosphates in mouse liver mitochondria and provide evidence for a rapid, highly selective, and saturable import of dTMP, not depending on a functional respiratory chain. At nM external dTMP the nucleotide is concentrated 100-fold inside the mt matrix. Export of thymidine phosphates was much slower and possibly occurred at the level of dTDP.mitochondrial diseases ͉ mitochondrial DNA ͉ regulation of mitochondrial dNTP pools M itochondria contain pools of the four canonical dNTPs for the synthesis of mitochondrial (mt) DNA, separated by the mt double membrane from the corresponding cytosolic dNTP pools for nuclear DNA synthesis (1). mt DNA synthesis occurs throughout the whole life of a cell, independent of nuclear DNA synthesis in S-phase, and mt dNTPs must therefore be available also in cells outside S-phase, when dNTP pools are not required for nuclear DNA synthesis. In cultured cells we found much larger dNTP pools in the cytosol than in mitochondria, but we found roughly equal proportions among the four dNTPs in these two compartments. This applied to both cycling (2) and resting (3) cells even though pool sizes were much smaller in the latter. Pulse-chase experiments with radioactively labeled thymidine demonstrated separate metabolic compartments for mt and cytosolic dTTP pools, in rapid communication with each other. mt dTTP was formed by two separate pathways: (i) de novo synthesis from ribonucleotides in the cytosol, followed by import into mitochondria; or (ii) phosphorylation of imported thymidine by a mt thymidine kinase. Cycling cells largely depended on the first pathway whereas resting cells used the second. These results may explain why genetic defects (4-6) involving enzymes of the second pathway mostly affect tissues with terminally differentiated cells.A recent article in PNAS by Song et al. (7) reported a large asymmetry among the four mt dNTP pools in various rat organs, with dGTP in some cases being several-hundred-fold more abundant than dTTP. In model experiments the asymmetries greatly affected the fidelity of DNA synthesis in vitro. They were suggested to lead to an increased mutability of the mt genome in vivo. The pool data appear to be at variance with the scenario for mt dNTP synthesis in cultured cells suggested by us.Earlier analyses of the size and composition of adenosine ...

The Role of the Mitochondrial Glycine Cleavage Complex in the Metabolism and Virulence of the Protozoan Parasite Leishmania major

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Metabolic Interrelations within Guanine Deoxynucleotide Pools for Mitochondrial and Nuclear DNA Maintenance

“…We studied by isotope-chase experiments with radioactive thymidine the flow of metabolites and found that in cycling cells the R1/R2 ribonucleotide reductase and, to a minor extent, salvage of thymidine by TK1 were the major sources of the nucleotide. dTMP enters mitochondria (22) and is phosphorylated to dTTP by intra-mt kinases. In quiescent cells dTMP is synthesized by the R1/p53R2 ribonucleotide reductase in the cytosol (14,15) and by salvage of thymidine by TK2 in mitochondria (23).…”