Characteristics of a bacteriophage T4-induced complex synthesizing deoxyribonucleotides

A preparation of bacteriophage T4-induced deoxyribonucleotide synthetase complex is described. This very large complex of enzymes can be separated by centrifugation at 100,000 X g, by sucrose step gradient centrifugation, or with molecular exclusion columns. By direct assay and by unidimensional and two-dimensional acrylamide electrophoretic separations the following T4-coded enzymes were shown to be associated with the complex: ribonucleoside diphosphate reductase, dCMP deaminase, dCTP/dUTPase, dCMP hydroxymethylase, dTMP synthetase, and DNA polymerase. Other phage-coded prereplicative proteins related to DNA replication and other phage functions such as the proteins coded by genes 32, 46, rIIA, and rIIB as well as many unidentified proteins were also consistently associated with the isolated fractions. T4 DNA topoisomerase, a membrane-bound enzyme, was found in quantity in all purified fractions of the complex, even in preparations apparently free of membrane and of T4 DNA. The functional integrity of a segment of the complex was followed by measuring the conversion of [5-3H]CDP to the level of 5-hydroxymethyl dCMP. This series of reactions requires the actions of T4-coded ribonucleoside diphosphate reductase and its associated reducing system, dCTP/dUTPase and dCMP hydroxymethylase, 3H being lost to water at the last step. In this reaction sequence an intermediate, [5-3H]dCMP, is maintained at low steady state concentrations, and argument is presented that the synthesis of deoxyribonucleotides is channeled and normally tightly coupled to DNA replication. One of the primary characteristics of this complex is its ready dissociation of dilution into smaller complexes of proteins and to the free forms of the proteins. That the complex is held together by weak electrostatic forces was supported by its sensitivity to dissociation at moderate salt concentrations. Not only the enzymes required in deoxyribonucleotide synthesis but T4 DNA polymerase, T4 DNA topoisomerase, and a number of other proteins dissociate to varying degrees from the larger complexes under these conditions.     

Regulation of ribonucleoside diphosphate reductase synthesis in Escherichia coli: increased enzyme synthesis as a result of inhibition of deoxyribonucleic acid synthesis.

Inhibition of deoxyribonucleic acid (DNA) synthesis in Escherichia coli by chemical inhibitors or by shifting cultures of temperature-sensitive elongation (dnaE and dnaB) or initiation (dnaA) mutants to nonpermissive conditions led to greatly increased synthesis of the enzyme ribonucleoside diphosphate reductase, which catalyzes the first reaction unique to the pathway leading to DNA replication. In contrast to the Gudas and Pardee proposed model for control of the synthesis of DNA repair enzymes, in which both DNA inhibition and DNA degradation are involved, DNA synthesis inhibition in recA, recB, recC, or lex strains results in increased synthesis of ribonucleotide reductase, which suggests that DNA degradation is not required. We propose that inhibition of DNA synthesis causes a cell to accumulate an unknown compound that stimulates the initiation of a new round of DNA replication, and that this same signal is used to induce ribonucleotide reductase synthesis.     

Replicative bacteriophage DNA synthesis in plasmolyzed T4-infected cells: evidence for two independent pathways to DNA.

Bacteriophage T4-infected Escherichia coli rendered permeable to nucleotides by sucrose plasmolysis exhibited two apparently separate pathways or channels to T4 DNA with respect to the utilization of exogenously supplied substrates. By one pathway, individual labeled ribonucleotides, thymidine (tdR), and 5-hydroxymethyl-dCMP could be incorporated into phage DNA. Incorporation of each of these labeled compounds was not dependent upon the addition of the other deoxyribonucleotide precursors, suggesting that a functioning de novo pathway to deoxyribonucleotides was being monitored. The second pathway or reaction required all four deoxyribonucleoside triphosphates or the deoxyribonucleoside monophosphates together with ATP. However, in this reaction, dTTP was not replaced by TdR. The two pathways were also distinguished on the basis of their apparent Mg2+ requirements and responses to N-ethylmaleimide, micrococcal nuclease, and to hydroxyurea, which is a specific inhibitor of ribonucleoside diphosphate reductase. Separate products were synthesized by the two channels, as shown by density-gradient experiments and velocity sedimentation analysis. Each of the pathways required the products of the T4 DNA synthesis genes. Furthermore, DNA synthesis by each pathway appeared to be coupled to the functioning of several of the phage-induced enzymes involved in deoxyribonucleotide biosynthesis. Both systems represent replicative phage DNA synthesis as determined by CsCl density-gradient analysis. Autoradiographic and other studies provided evidence that both pathways occur in the same cell. Further studies were carried out on the direct role of dCMP hydroxymethylase in T4 DNA replication. Temperature-shift experiments in plasmolyzed cells using a temperature-sensitive mutant furnished strong evidence that this gene product is necessary in DNA replication and is not functioning by allowing preinitiation of DNA before plasmolysis.     

Synthesis of 3'-C-methyladenosine and 3'-C-methyluridine diphosphates and their interaction with the ribonucleoside diphosphate reductase from Corynebacterium nephridii.

Two nucleoside diphosphate analogs, 3'-C-methyl-ADP and 3'-C-methyl-UDP, have been tested as substrate and/or allosteric effectors using the adenosylcobalamin-dependent ribonucleoside diphosphate reductase of Corynebacterium nephridii. Neither analog was a substrate for the reductase. However, they did function as allosteric effectors and as inhibitors of the reduction of ADP and UDP, respectively. The nucleotide analogs did not stimulate the hydrogen exchange reaction between [5'-3H2]adenosylcobalamin and the solvent, indicating that the cleavage of the 3'-carbon-hydrogen bond is a prerequisite for the exchange reaction. A reinvestigation of the requirements for the exchange reaction revealed that the deoxyribonucleoside diphosphate products are very effective promoters of this reaction. Indeed, the deoxyribonucleoside diphosphates were found to be more effective in promoting the exchange reaction than the ribonucleoside diphosphate substrates. In contrast, the deoxyribonucleoside triphosphate effectors, dATP, dUTP, and dTTP, were only marginally effective as promoters of this reaction.     

Mosquito has a single multisubstrate deoxyribonucleoside kinase characterized by unique substrate specificity

In mammals four deoxyribonucleoside kinases, with a relatively restricted specificity, catalyze the phosphorylation of the four natural deoxyribonucleosides. When cultured mosquito cells, originating from the malaria vector Anopheles gambiae, were examined for deoxyribonucleoside kinase activities, only a single enzyme was isolated. Subsequently, the corresponding gene was cloned and over-expressed. While the mosquito kinase (Ag-dNK) phosphorylated all four natural deoxyribonucleosides, it displayed an unexpectedly higher relative efficiency for the phosphorylation of purine versus pyrimidine deoxyribonucleosides than the fruit fly multisubstrate deoxyribonucleoside kinase (EC 2.7.1.145). In addition, Ag-dNK could also phosphorylate some medically interesting nucleoside analogs, like stavudine (D4T), 2-chloro-deoxyadenosine (CdA) and 5-bromo-vinyl-deoxyuridine (BVDU). Although the biological significance of multisubstrate deoxyribonucleoside kinases and their diversity among insects remains unclear, the observed variation provides a whole range of applications, as species specific and highly selective targets for insecticides, they have a potential to be used in the enzymatic production of various (di-)(deoxy-)ribonucleoside monophosphates, and as suicide genes in gene therapy.     

Isotope-dilution analysis of the effects of deoxyguanosine and deoxyadenosine on the incorpo?ation of thymidine and deoxycytidine by hydroxyurea-treated thymus cells

It is presumed that the dGTP and dATP needed for replicative DNA synthesis can be formed by way of either `salvage' pathways or biosynthesis de novo. This was examined by adding hydroxyurea to cultures of rat thymus cells to inhibit ribonucleoside diphosphate reductase, a key enzyme of the `de novo' pathway. Most of the inhibition of the incorporation of [Me-³H]thymidine and deoxy[5-³H]cytidine by low concentrations of hydroxyurea (100–500μm) was prevented by substrates of the salvage pathway (400μm-deoxyguanosine and, to a lesser extent, 200μm-deoxyadenosine). However, isotope-dilution studies indicated that the purine deoxyribonucleosides prevented inhibition by decreasing pyrimidine deoxyribonucleotide competitor pools. Evidence was obtained that a hydroxyurea-induced increase in the thymidine-competitor pool (probably dTTP) was prevented to an equal extent by deoxyguanosine and by the inhibitor of thymidylate synthase, deoxy-5-fluorouridine. These compounds had almost identical effects on hydroxyurea dose–response curves and on thymidine isotope-dilution plots. The evidence suggests that exogenous purine deoxyribonucleosides cannot prevent the inhibition by hydroxyurea of thymus-cell DNA synthesis. This could mean that, with respect to the metabolism of purine deoxyribonucleotides, ribonucleoside diphosphate reductase is tightly coupled to DNA polymerase in a multienzyme complex. The complex would not permit entry of exogenous metabolic intermediates into the `de novo' pathway, but would still be subject to the regulatory effects of these intermediates. Thus dGTP and dATP formed from exogenous purine deoxyribonucleosides by salvage pathways might deplete pyrimidine deoxyribonucleotide competitor pools by inhibiting relatively hydroxyurea-insensitive activities of ribonucleoside diphosphate reductase.     

Coupled ribonucleoside diphosphate reduction, channeling, and incorporation into DNA of mammalian cells

There is rapid and specific channeling of ribonucleoside diphosphates into DNA through reactions beginning with ribonucleotide reductase and terminating with DNA polymerase. Lysolecithin-permeabilized Chinese hamster embryo fibroblasts in culture rapidly reduced ribonucleoside diphosphates by ribonucleotide reductase action when dithiothreitol was provided as a reducing agent and incorporated these deoxynucleotides into DNA. The radioactive label provided in ribo-CDP was not diluted by added deoxyribo-CTP during its incorporation into DNA, showing that the ribo-CDP does not pass through a deoxy-CTP pool. Under the conditions that permitted rapid incorporation of ribonucleoside diphosphates, deoxynucleoside triphosphates were very poorly incorporated. Ribonucleotide reductase with the rate-limiting enzyme for the overall process. The Km values for the reductase reaction and the overall process were similar and low enough for saturation by in vivo pools. Natural feedback inhibitors dATP or dTTP inhibited incorporation of labeled ribo-CDP into deoxyribonucleotides and into DNA to the same extent. Ribonucleotide reductase behaved like other enzymes that are associated in a rapidly sedimenting form. It was concentrated in the nucleus during S phase, and most of the enzyme activity in these nuclear extracts was co-sedimented with DNA polymerase on sucrose density gradients. These data support the hypotheses that a physically associated complex of enzymes (replitase) catalyzes the production of deoxynucleotides and their incorporation into DNA in S phase cells.     

In Situ Activity of Enzymes on Polyacrylamide Gels of a Deoxyribonucleic Acid-Membrane Fraction Extracted from Pneumococci

A deoxyribonucleic acid (DNA)-membrane fraction extracted from Diplococcus pneumoniae was subjected to polyacrylamide gel electrophoresis after treatment with 0.16% sodium dodecyl sulfate. At least two DNA polymerase activities were detected by in situ assays with appropriate substrates, templates, and inhibitors, including a co-polymer of deoxyadenylic and thymidylic acid and N-ethylmaleimide. This activity coincided with a fraction in the gel containing 7.5, 9.4, and 24%, respectively of the DNA, phospholipid, and protein present in the DNA-membrane fraction before electrophoresis and sodium dodecyl sulfate treatment. Assays with minced gels showed that several nuclease activities, deoxyribonucleotide kinase activity, and DNA ligase activity also coincided with this fraction. However, ribonucleoside diphosphate reductase activity did not. These results demonstrate that a complex of enzymes involved in DNA replication is firmly bound to the DNA-membrane fraction in pneumococci.     

Protein-DNA interactions in the T4 dNTP synthetase complex dependent on gene 32 single-stranded DNA-binding protein

Our laboratory has reported data suggesting a role for T4 phage gene 32 single-stranded DNA-binding protein in organizing a complex of deoxyribonucleotide-synthesizing enzymes at the replication fork. In this article we examined the effects of gene 32 ablation on the association of these enzymes with DNA-protein complexes. These experiments showed several deoxyribonucleotide-synthesizing enzymes to be present in DNA-protein complexes, with some of these associations being dependent on gene 32 protein. To further understand the role of gp32, we created amber mutations at codons 24 and 204 of gene 32, which encodes a 301-residue protein. We used the newly created mutants along with several experimental approaches--DNA-cellulose chromatography, immunoprecipitation, optical biosensor analysis and glutathione-S-transferase pulldowns--to identify relevant protein-protein and protein-DNA interactions. These experiments identified several proteins whose interactions with DNA depend on the presence of intact gp32, notably thymidylate synthase, dihydrofolate (DHF) reductase, ribonucleotide reductase (RNR) and Escherichia coli nucleoside diphosphate (NDP) kinase, and they also demonstrated direct associations between gp32 and RNR and NDP kinase, but not dCMP hydroxymethylase, deoxyribonucleoside monophosphate kinase, or DHF reductase. Taken together, the results support the hypothesis that the gene 32 protein helps to recruit enzymes of deoxyribonucleoside triphosphates synthesis to DNA replication sites.     

Evidence for the involvement of substrate cycles in the regulation of deoxyribonucleoside triphosphate pools in 3T6 cells

Pool sizes of deoxyribonucleoside triphosphates (dNTPs) in cultured cells are tightly regulated by i.al., the allosteric control of ribonucleotide reductase. We now determine the in situ activity of this enzyme from the turnover of the deoxycytidine triphosphate (dCTP) pool in rapidly growing 3T6 mouse fibroblasts, as well as in cells whose DNA replication was inhibited by aphidicolin or amethopterin, by following under steady state conditions the path of isotope from [5-3H]cytidine into nucleotides, DNA, and deoxynucleosides excreted into the medium. In normal cells as much as 28% of the dCDP synthesized was excreted as deoxynucleoside (mostly deoxyuridine), leading to an accumulation of deoxyuridine in the medium. Inhibition with amethopterin slightly increased ribonucleotide reductase activity, while aphidicolin halved the activity of this enzyme (and thymidylate synthase). In both instances all dCDP synthesized was degraded and excreted as nucleosides. This continued synthesis and turnover in the absence of DNA synthesis is in contrast to the earlier found inhibition of dCTP (and dTTP) turnover when hydroxyurea, an inhibitor of ribonucleotide reductase, was used to block DNA synthesis. To explain our results, we propose that substrate cycles between deoxyribonucleosides and their monophosphates, involving the activities of kinases and phosphatases, participate in the regulation of pool sizes. Within the cycles, a block of the reductase activates net phosphorylation, while inhibition of DNA polymerase stimulates degradation.     

Defect in synthesis of deoxyribonucleotides by a bacteriophage T4 nrdB mutant is suppressed on mutation of T4 DNA topoisomerase gene

Bacteriophage T4 infection is known to induce the formation of a complex of enzymes effecting the de novo synthesis of deoxyribonucleoside triphosphates, which in turn are channeled into T4 DNA replication. The first step in this pathway is catalyzed by a ribonucleoside diphosphate reductase, comprised of subunits coded by T4 genes nrdA and nrdB. Maximum rates of synthesis of the pyrimidine deoxyribonucleotides and of DNA replication in vivo also require a type II DNA topoisomerase encoded by T4 genes 39, 52, and 60. We report the identification of a unique mutant, nrdB93, and the suppression of its defective deoxyribonucleotide synthesis by a gene 39 mutation, 39-01. After infection by 39-01, DNA synthesis and plaque formation were temperature-sensitive, but nearly wild type rates of deoxyribonucleotide synthesis were retained at all temperatures. The nrdB93 mutation had a profound effect on deoxyribonucleotide synthesis at 41 degrees C; even at the permissive temperature of 30 degrees C, synthesis was reduced to 30% of that of wild type or 39-01. However, on infection at 30 degrees C by the double mutant, 39-01 nrdB93, the level of deoxyribonucleotide synthesis again reached that of wild type phage infections; involvement of the comparable host enzyme in the suppression process has been excluded. Suppression of the effect of nrdB93 by 39-01 implicates the gene 39 product in the regulation of nrdB expression. The accompanying paper (Cook, K. S., Wirak, D. O., Seasholtz, A. F., and Greenberg, G. R. (1988) J. Biol. Chem. 263, 6202-6208) examines the nature of the suppression process at the molecular level.     

Ribonucleoside diphosphate reductase is a component of the replication hyperstructure in Escherichia coli

Although the nrdA101 allele codes for a ribonucleoside diphosphate (rNDP) reductase that is essentially destroyed in less than 2 min at 42 degrees C, and chemical inhibition of the enzyme by hydroxyurea stops DNA synthesis at once, we found that incubation at 42 degrees C of an Escherichia coli strain containing this allele allows DNA replication for about 40min. This suggests that mutant rNDP reductase is protected from thermal inactivation by some hyperstructure. If, together with the temperature upshift, RNA or protein synthesis is inhibited, the thermostability time of the mutant rNDP reductase becomes at least as long as the replication time and residual DNA synthesis becomes a run-out replication producing fully replicated chromosomes. This suggests that cessation of replication in the nrdA101 mutant strain is not the result of inactivation of its gene product but of the activity of a protein reflecting the presence of a partially altered enzyme. The absence of Tus protein, which specifically stops the replication complex by inhibiting replicative helicase activity, allows forks to replicate for a longer time at the restrictive temperature in the nrdA101 mutant strain. We therefore propose that rNDP reductase is a component of the replication complex, and that this association with other proteins protects the protein coded by allele nrdA101 from thermal inactivation.     

Functional expression of a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster and its C-terminal deletion mutants

The occurrence of a deoxyribonucleoside kinase in Drosophila melanogaster (Dm-dNK) with remarkably broad substrate specificity has recently been indicated (Munch-Petersen, B., Piskur, J., and S?ndergaard, L. (1998) J. Biol. Chem. 273, 3926-3931). To prove that the capacity to phosphorylate all four deoxyribonucleosides is in fact associated to one polypeptide chain, partially sequenced cDNA clones, originating from the Berkeley Drosophila genome sequencing project, were searched for homology with human deoxyribonucleoside kinases. The total sequence of one cDNA clone and the corresponding genomic DNA was determined and expressed in Escherichia coli as a glutathione S-transferase fusion protein. The purified and thrombin cleaved recombinant protein phosphorylated the four deoxyribonucleosides with high turnover and K(m) values similar to those of the native Dm-dNK, as well as the four ribonucleosides and many therapeutical nucleoside analogs. Dm-dNK has apparently the same origin as the mammalian kinases, thymidine kinase 2, deoxycytidine kinase, deoxyguanosine kinase, and the herpes viral thymidine kinases, but it has a unique C terminus that seems to be important for catalytic activity and specificity. The C-terminal 20 amino acids were dispensable for phosphorylation of deoxyribonucleosides but necessary for full activity with purine ribonucleosides. Removal of the C-terminal 20 amino acids increased the specific activity 2-fold, but 99% of the activity was lost after removal of the C-terminal 30 amino acids.     

Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93)).

To investigate the role of ribonucleoside diphosphate reductase in the deoxyribonucleoside triphosphate synthetase multienzyme complex induced by bacteriophage T4 infection and to study the expression of the T4 nrdA and nrdB genes, we have constructed separate plasmid expression strains overproducing their respective alpha 2 and beta 2 protein products. Because complementation of the two proteins to form an active alpha 2 beta 2 enzyme presented complications, nrdA and nrdB, each with its own tac promoter, were also cloned in tandem into a single expression vector. The resulting plasmid (pnrdAB) overproduces ribonucleoside diphosphate reductase. Phage T4 nrdB93, described by Wirak et al. (D. O. Wirak, K. S. Cook, and G. R. Greenberg, J. Biol. Chem. 263:6193-6201, 1988) contains a lesion in exon II of the gene. The mutation causes not only a temperature-sensitive inactivation of the catalytic structure of the beta 2(93) protein and of its ability to interact with alpha 2 protein to form the alpha 2 beta 2(93) enzyme but also a profound non-temperature-sensitive decrease in the formation of the beta 2(93) protein. An expression vector overproducing active alpha 2 beta 2(93) was constructed by site-directed mutagenesis of the nrdB gene.     

A few amino acid substitutions can convert deoxyribonucleoside kinase specificity from pyrimidines to purines

In mammals, the four native deoxyribonucleosides are phosphorylated to the corresponding monophosphates by four deoxyribonucleoside kinases, which have specialized substrate specificities. These four enzymes are likely to originate from a common progenitor kinase. Insects appear to have only one multisubstrate deoxyribonucleoside kinase (dNK, EC 2.7.1.145), which prefers pyrimidine nucleosides, but can also phosphorylate purine substrates. When the structures of the human deoxyguanosine kinase (dGK, EC 2.7.1.113) and the dNK from Drosophila melanogaster were compared, a limited number of amino acid residues were identified and proposed to be responsible for the substrate specificity. Three of these key residues in Drosophila dNK were then mutagenized and the mutant enzymes were characterized regarding their ability to phosphorylate native deoxyribonucleosides and nucleoside analogs. The mutations converted the dNK substrate specificity from predominantly pyrimidine specific into purine specific. A similar scenario could have been followed during the evolution of kinases. Upon gene duplication of the progenitor kinase, only a limited number of single amino acid changes has taken place in each copy and resulted in substrate-specialized enzymes.     

Effect of 5-fluoro-2′-deoxyuridine and hydroxyurea on the phytohemagglutinin-induced increase of thymidine kinase,replicative DNA polymerase,deoxycytidylate deaminase and CDP reductase activities in human lymphocytes

Summary The inhibitors of DNA synthesis, 5-fluoro-2-deoxyuridine and hydroxyurea, caused an inhibition of thymidine kinase, replicative DNA polymerase and CDP reductase activities in stimulated lymphocytes when they were exposed to the inhibitors during the early transformation period (0–17 hr). However, the enzyme activities were unaffected when the inhibitors were added to cells stimulated for more than 17 hr. As opposed to these enzymes the deoxycytidylate deaminase activity was unaffected by the inhibitors during the entire transformation period (0–28 hr). This indicates a close regulatory mechanism in lymphocytes between DNA synthesis and induction of enzymes involved in DNA replication. The inhibitory mechanism exerted by the inhibitors is for the moment unknown. It might be independent of the well-known inhibition of the target enzymes, thymidylate synthetase and ribonucleoside diphosphate reductase, since there was no immediate apparent correlation in time between depletion of the pool sizes and the inhibition of the enzyme activities.     

Mechanism of inhibition of deoxyribonucleic acid synthesis in Escherichia coli by hydroxyurea

The effects of hydroxyurea on Escherichia coli B/5 physiology (increases in cell mass, number of viable cells, and deoxyribonucleic acid [DNA], RNA, and protein concentrations) were studied in an attempt to find a concentration that completely inhibits DNA synthesis and increase in number of viable cells but has little or no effect on other metabolic processes. These conditions were the most closely approached at an hydroxyurea concentration of 0.026 to 0.033 m. A concentration of 0.026 or 0.033 m was used in subsequent experiments to study the site(s) of inhibition of DNA synthesis in E. coli B/5 by hydroxyurea. Hydroxyurea at a concentration of 10(-2)m was found to inhibit ribonucleoside diphosphate reductase activity completely in crude extracts of E. coli. The synthesis of deoxyribonucleotides was greatly reduced when E. coli cells were grown in the presence of 0.033 m hydroxyurea. Studies on the acid-soluble DNA precursor pools showed that hydroxyurea causes a decrease in the concentration of deoxyribonucleoside diphosphates and deoxyribonucleoside triphosphates and an increase in the total concentration of ribonucleotides. Sucrose density gradient sedimentation of DNA from cells treated with 0.026 m hydroxyurea for 30 min indicated that at this concentration hydroxyurea induces no detectable single- or double-strand breaks. In addition, both replicative and repair syntheses of DNA were found to occur normally in toluene-treated cells in the presence of relatively high concentrations of hydroxyurea. Pulse-chase studies showed that deoxyribonucleotides synthesized prior to the addition of hydroxyurea to cells are utilized normally for DNA synthesis in the presence of hydroxyurea. On the basis of these observations, we have concluded that the primary, if not the only, site of inhibition of DNA synthesis in E. coli B/5 by low concentrations of hydroxyurea is the inhibition of the enzyme ribonucleoside diphosphate reductase.     

Enzyme kinetics of the mitochondrial deoxyribonucleoside salvage pathway are not sufficient to support rapid mtDNA replication

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.