Enzyme Kinetics of the Mitochondrial Deoxyribonucleoside Salvage Pathway Are Not Sufficient to Support Rapid mtDNA Replication

Abstract

Using a computational model, we simulated mitochondrial deoxynucleotide metabolism and mitochondrial DNA replication. Our results indicate that the output from the mitochondrial salvage enzymes alone is inadequate to support a mitochondrial DNA replication duration of as long as 10 hours. We find that an external source of deoxyribonucleoside diphosphates or triphosphates (dNTPs), in addition to those supplied by mitochondrial salvage, is essential for the replication of mitochondrial DNA to complete in the experimentally observed duration of approximately 1 to 2 hours. For meeting a relatively fast replication target of 2 hours, almost two-thirds of the dNTP requirements had to be externally supplied as either deoxyribonucleoside di- or triphosphates, at about equal rates for all four dNTPs. Added monophosphates did not suffice. However, for a replication target of 10 hours, mitochondrial salvage was able to provide for most, but not all, of the total substrate requirements. Still, additional dGTPs and dATPs had to be supplied. Our analysis of the enzyme kinetics also revealed that the majority of enzymes of this pathway prefer substrates that are not precursors (canonical deoxyribonucleosides and deoxyribonucleotides) for mitochondrial DNA replication, such as phosphorylated ribonucleotides, instead of the corresponding deoxyribonucleotides. The kinetic constants for reactions between mitochondrial salvage enzymes and deoxyribonucleotide substrates are physiologically unreasonable for achieving efficient catalysis with the expected in situ concentrations of deoxyribonucleotides.

The Human SLC25A33 and SLC25A36 Genes of Solute Carrier Family 25 Encode Two Mitochondrial Pyrimidine Nucleotide Transporters

“…Mitochondria contain the nucleotide salvage pathway that synthesizes nucleotides from the corresponding nucleosides that are transported into the organelles by the equilibrative nucleoside transporters hENT1 and hENT3 (40,41). As the contribution of the salvage pathway to mtDNA synthesis is insufficient in quiescent cells and even more in cycling cells (42)(43)(44), pyrimidine nucleotides must be transported from the cytosol to the mitochondrial matrix. In the past a partial purification from human mitochondria of a carrier that displayed an efficient transport activity for dCTP has been achieved (45).…”

“…As regards to SLC25A36, on the basis of our transport measurements the primary physiological function of this carrier (that transports cytosine and uracil (deoxy)nucleoside mono-, di-, and tri-phosphates) is probably to catalyze uptake of pyrimidine (d)NTPs into the mitochondrial matrix in exchange for internal pyrimidine (d)NMPs or, to a lesser extent, (d)NDPs. This exchange may play an important role in mitochondrial nucleic acid metabolism both in quiescent and cycling cells in which the contribution of the salvage pathway is significant and minimal, respectively (42)(43)(44). It should be noted that the SLC25A36-mediated import of pyrimidine Furthermore, SLC25A36 catalyzes a uniport transport mode besides exchange.…”

Defects in mitochondrial DNA replication and human disease

“…Mutations in the genes for enzymes involved in this salvage pathway cause several forms of mtDNA depletion syndromes. Computational models suggest that this salvage pathway is insufficient to support rapid mtDNA replication and the mitochondrial salvage pathway serves more as a backup role for the supply of nucleotide precursors for mtDNA replication (Gandhi and Samuels, 2011). This model helps to explain why defects in TK2 are restrictive to muscle and present as myopathic mtDNA depletion syndromes (Mancuso et al, 2002).…”

Human Mitochondrial DNA Polymerase γ Exhibits Potential for Bypass and Mutagenesis at UV-induced Cyclobutane Thymine Dimers

“…In response to damage to DNA, p53 is activated, which then increases mitochondrial nucleotide pools by the induction of the p53 inducible small subunit of ribonucleotide reductase, RRM2B (44,45). Nucleotide pools imported from the cytoplasm due to ribonucleotide reductase activity may serve as the main supply of mitochondrial nucleotide pools, where the salvage pathway serves as a backup (46). Thus, the up-regulation of nucleotide pools via the p53 induction after UV damage may allow more bypass of thymine dimers.…”