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.
Highlights
Mitochondrial DNA replication [1], and the mitochondrial nucleoside salvage pathway that generates the precursor deoxyribonucleoside triphosphates for mitochondrial DNA replication, have generally been believed to function independently of nuclear DNA replication and cytoplasmic nucleotide metabolism
Older evidence supported the view that mitochondrial nucleotides may be isolated from the corresponding cytoplasmic pools [4], but more recent studies support a metabolic cross-talk between the mitochondria and the cytoplasm and show that nucleotide import from the cytosol very likely contributes to mitochondrial deoxyribonucleoside triphosphates (dNTPs) pools in both cycling and quiescent cells [5,6]
Problems with maintenance of mitochondrial DNA, arising from genetic mutations as well as from antiviral drugs, can lead to debilitating diseases that are often fatal in early life and childhood, or reduced compliance to therapy from patients suffering drug toxicity
Summary
Mitochondrial DNA (mtDNA) replication [1], and the mitochondrial nucleoside salvage pathway that generates the precursor deoxyribonucleoside triphosphates (dNTPs) for mitochondrial DNA replication, have generally been believed to function independently of nuclear DNA (nDNA) replication and cytoplasmic nucleotide metabolism. Older evidence supported the view that mitochondrial nucleotides may be isolated from the corresponding cytoplasmic pools [4], but more recent studies support a metabolic cross-talk between the mitochondria and the cytoplasm and show that nucleotide import from the cytosol very likely contributes to mitochondrial dNTP pools in both cycling and quiescent cells [5,6]. The mechanism of this import is unknown since the discovery that the carrier SLC25A19 (solute carrier family 25, member 19) is a thiamine pyrophosphate transporter and not a deoxyribonucleotide transporter [7]. This is a complex process with some reactions occurring in parallel for the four deoxyribonucleosides, and some reactions using the same enzyme (for example, the first phosphorylation of dT and dC are both catalyzed by TK2) in addition to the presence (not shown in Figure 1A) of feedback mechanisms (for example, dTTP and dCTP inhibition on TK2 [8])
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