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

Abstract

Mitochondrial deoxynucleoside triphosphates are formed and regulated by a network of anabolic and catabolic enzymes present both in mitochondria and the cytosol. Genetic deficiencies for enzymes of the network cause mitochondrial DNA depletion and disease. We investigate by isotope flow experiments the interrelation between mitochondrial and cytosolic deoxynucleotide pools as well as the contributions of the individual enzymes of the network to their maintenance. To study specifically the synthesis of dGTP used for the synthesis of mitochondrial and nuclear DNA, we labeled hamster CHO cells or human fibroblasts with [ 3 H]deoxyguanosine during growth and quiescence and after inhibition with aphidicolin or hydroxyurea. At time intervals we determined the labeling of deoxyguanosine nucleotides and DNA and the turnover of dGTP from its specific radioactivity in the separated mitochondrial and cytosolic pools. In both cycling and quiescent cells, the import of deoxynucleotides formed by cytosolic ribonucleotide reductase accounted for most of the synthesis of mitochondrial dGTP, with minor contributions by cytosolic deoxycytidine kinase and mitochondrial deoxyguanosine kinase. A dynamic isotopic equilibrium arose rapidly from the shuttling of deoxynucleotides between mitochondria and cytosol, incorporation of dGTP into DNA, and degradation of dGMP. Inhibition of DNA synthesis by aphidicolin marginally affected the equilibrium. Inhibition of DNA synthesis by blockage of ribonucleotide reduction with hydroxyurea instead disturbed the equilibrium and led to accumulation of labeled dGTP in the cytosol. The turnover of dGTP decreased, suggesting a close connection between ribonucleotide reduction and pool degradation. Mitochondrial (mt)2 deoxynucleotides are formed and regulated by a network of anabolic and catabolic enzymes located in both the cytosol and mitochondria. The enzymes are of considerable medical interest. Many drugs used in cancer and virus therapy are deoxynucleoside analogs whose activity requires phosphorylation by kinases (1) and whose efficacy and toxicity is affected by the interplay between the enzymes in the network. The enzymes may also be involved in oxidative damage by activating modified bases such as 8-oxoguanine for incorporation into RNA and DNA (2). Moreover, an increasing number of diseases with depletion of mt DNA arise from genetic deficiency of enzymes in the network (3). In some cases the genetic deficiency leads to depletion of mt deoxyribonucleoside triphosphate (dNTP) (4 -6), in other cases to overproduction (7-9). In both instances the disturbed normal balance between the four dNTPs results in disease. Early examples are the catabolic phosphorylases that degrade purine (7) or thymine nucleosides (9). Their loss causes overproduction of dGTP or dTTP, with the affected individuals suffering from immune deficiency (7,8) or mitochondrial neurogastrointestinal encephalomyopathy (9), respectively. Later discovered examples are the anabolic mt salvage enzymes deoxyguanosine kinase (dGK) (4...

Neurological Disorders of Purine and Pyrimidine Metabolism

“…A balanced dNTP pool is constantly required for mtDNA replication, which occurs throughout the whole cell cycle. Balance is maintained by either import of cytosolic dNDPs and dNTPs through dedicated transporters, or by dNTP synthesis achieved through specific salvage pathways, such as dGuo phosphorylation by dGK, since mitochondria are devoid of the de novo nucleotide synthesis [175]. Cytosolic de novo pathway is most active in the S-phase of the cell cycle and significantly decreases in quiescent or differentiated cells.…”

A Review Comparing Deoxyribonucleoside Triphosphate (dNTP) Concentrations in the Mitochondrial and Cytoplasmic Compartments of Normal and Transformed Cells

“…Thus, interpretation of the exact nature of that transport is complicated. Using isotope experiments, Bianchi and colleagues have established the importance of cytoplasmic enzymes towards the maintenance of mitochondrial dTTP and dGTP pools [33, 34]. By following the appearance and dilution of radioactivity, they observed two-way transport of thymidine and deoxyguanosine nucleotides between the cytoplasm and mitochondria and determined that while the cytoplasmic de novo pathway is the predominant source of mitochondrial dTTP in proliferating cells, even non-proliferating cells depend on ribonucleotide reduction in the cytoplasm.…”

SAMHD1: Recurring roles in cell cycle, viral restriction, cancer, and innate immunity

“…The opposing anabolic and catabolic pathways work in competing directions to maintain a dynamic equilibrium of intracellular dNTPs in a process referred to as substrate cycling (19). Isotope flow experiments of radiolabeled nucleotides have traced this phenomenon and elucidated its critical role as a regulatory mechanism for maintaining homeostatic dNTP levels (20–22). It is also important to note that cytosolic dNTP pools do not exist in isolation, but are directly related to mitochondrial dNTP pools.…”