Nucleoside triphosphate pool asymmetry in mammalian mitochondria

Our laboratory has reported that deoxyribonucleoside triphosphate (dNTP) pools in rat tissue mitochondria are highly asymmetric, with dGTP predominating, and that the imbalance probably contributes toward the high spontaneous mutation rate of the mitochondrial genome. Ferraro et al. (Ferraro, P., Nicolosi, L., Bernardi, P., Reichard, P., and Bianchi, V. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 18586-18591) have challenged these findings, based upon their studies of mouse liver mitochondria. Moreover, they have identified a potential artifact in the DNA polymerase-based assay for dNTPs, based upon overestimation of dGTP when GTP levels in extracts are much higher than dGTP levels. We measured ribonucleoside triphosphate (rNTP) pools in rat mitochondrial extracts and found that GTP pools exceed dGTP pools by 50-fold or less, not enough to interfere with the dGTP assay. Analysis of dNTP pools in state 3 mitochondria, after incubation with ADP and oxidizable substrates, gave similar results. We confirmed our earlier finding that rat mitochondrial dNTP pools are highly asymmetric. dNTP pools in cytosolic extracts are uniformly low, suggesting that the dNTP pool asymmetry arises within the mitochondrion. Moreover, we found rat tissue rNTP pools to be even more highly asymmetric, with ATP, for example, at least 2 orders of magnitude more abundant than CTP in liver extracts. This finding raises the possibility that transcription of the mitochondrial genome is more error-prone than transcription in the nucleus.