Origins of Mitochondrial Thymidine Triphosphate: Dynamic Relations to Cytosolic Pools

Origins of mitochondrial thymidine triphosphate: Dynamic relations to cytosolic pools

Giovanna Pontarin, Lisa Gallinaro, Paola Ferraro, Peter Reichard*, and Vera Bianchi*

Department of Biology, University of Padua, I-35131 Padua, Italy

Contributed by Peter Reichard, August 16, 2003

Nuclear and mitochondrial (mt) DNA replication occur within two physically separated compartments and on different time scales. Both require a balanced supply of dNTPs. During S phase, dNTPs for nuclear DNA are synthesized de novo from ribonucleotides and by salvage of thymidine in the cytosol. Mitochondria contain specific kinases for salvage of deoxyribonucleosides that may provide a compartmentalized synthesis of dNTPs. Here we investigate the source of intra-mt thymidine phosphates and their relationship to cytosolic pools by isotope-flow experiments with [3H]thymidine in cultured human and mouse cells by using a rapid method for the clean separation of mt and cytosolic dNTPs. In the absence of the cytosolic thymidine kinase, the cells (i) phosphorylate labeled thymidine exclusively by the intra-mt kinase, (ii) export thymidine phosphates rapidly to the cytosol, and (iii) use the labeled dTTP for nuclear DNA synthesis. The specific radioactivity of dTTP is highly diluted, suggesting that cytosolic de novo synthesis is the major source of mt dTTP. In the presence of cytosolic thymidine kinase dilution is 100-fold less, and mitochondria contain dTTP with high Fig. 1. Two pathways for mt dTTP: 1, import from cytosol of nucleotides specific radioactivity. The rapid mixing of the cytosolic and mt synthesized in the cytosol; and 2, import of thymidine followed by intra-mt pools was not expected from earlier data. We propose that in phosphorylation. Substrate cycles between thymidine kinases (TKs) and de- proliferating cells dNTPs for mtDNA come largely from import of oxynucleotidases (dNTs) participate in the regulation of both pathways. cytosolic nucleotides, whereas intra-mt salvage of deoxyribo- nDNA, nuclear DNA. nucleosides provides dNTPs in resting cells. Our results are relevant for an understanding of certain genetic mitochondrial diseases. thymidine preferentially into mtDNA, suggesting that the labeled dTMP formed by TK2 was largely sequestered in itochondria contain two separate potential pathways to mitochondria. Mprovide dTTP for mitochondrial (mt) DNA replication (Fig. 1): (i) deoxynucleotide transporters in the membrane Our interest in the regulation of the mt dTTP pool started introduce nucleotides from the cytosol (1, 2), and (ii) thymidine from the discovery of dNT-2 (12), a mt enzyme that specifically kinase 2 (TK2) in the mt matrix phosphorylates thymidine to dephosphorylates dTMP and dUMP. We proposed that this dTMP (3–6), which is further phosphorylated by nucleotide enzyme serves to prevent an accumulation of dTTP inside kinases to dTTP. Preliminary evidence for a third pathway via mitochondria. To distinguish between thymidine metabolism in an intra-mt ribonucleotide reductase (7) has not been followed mitochondria and in the cytosol, we have now developed a up, nor could we confirm it. We do not further consider it here. method for the complete separation of the total mt dTTP pool The first pathway in Fig. 1 relies mainly on de novo synthesis from the cytosolic pool. We used this method to study the of deoxyribonucleoside diphosphates by ribonucleotide reduc- kinetics of [3H]thymidine incorporation into cellular thymidine tase in the cytosol (8) and to a minor extent on the activity of the phosphate (dT-P) pools and into DNA. After separation of mt cytosolic thymidine kinase 1 (TK1) (9). Both enzymes are active and cytosolic dT-P pools, we determined both the total radio- only during the S phase of the cell cycle (10, 11). The first activity in the different compartments and the specific radioac- pathway is therefore absent from terminally differentiated cells. tivity of the two dTTP pools. By comparing the specific radio- The second pathway in Fig. 1 uses thymidine imported from the activity of the administered thymidine and that of the isolated extracellular milieu and is active also outside S phase because dTTP we can estimate the amount of de novo synthesis of TK2 is not cell-cycle regulated. Regulation of this pathway may nonlabeled dTMP. In cells devoid of TK1 the primary phos- Ј Ј occur by an intra-mt substrate cycle involving TK2 and 5 (3 )- phorylation of thymidine occurs inside mitochondria. The ap- deoxyribonucleotidase 2 (dNT-2) (12), similar to the cytosolic pearance of labeled dTTP in the cytosol and of labeled DNA in substrate cycle between TK1 and deoxyribonucleotidase 1 (13). the nucleus then signals transport of dT-P from mitochondria to Several genetic diseases affecting mtDNA replication arise from the cytosol. In cells containing TK1, the phosphorylation of malfunction of enzymes in either mt pathway (14, 15). Also, the thymidine occurs primarily in the cytosol and most of dTTP CELL BIOLOGY genetic loss of the cytosolic thymidine phosphorylase results in recovered in mitochondria originates in the cytosol. From the mutational damage to mtDNA (16). In this case an increased Ϫ ϩ combined results with TK1 and TK1 cells we conclude that in intra-mt dTTP pool is believed to cause the disease (17). Thus, too little and too much dTTP leads to disease. Little is known about the interrelations between the two mt Abbreviations: mt, mitochondrial; TK1, thymidine kinase 1 (cytosolic); TK2, thymidine pathways and dTTP synthesis in the cytosol. Pioneering work by kinase 2 (mt); dNT-2, 5Ј(3Ј)-deoxyribonucleotidase 2; dT-P, thymidine phosphate. Clayton and coworkers (3, 4) initially suggested a rather strict *To whom correspondence may be addressed. E-mail: [email protected] or spatial and metabolic separation. They demonstrated that cul- [email protected]. tured cells lacking TK1, but retaining TK2, incorporated labeled © 2003 by The National Academy of Sciences of the USA

www.pnas.org͞cgi͞doi͞10.1073͞pnas.1635259100 PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12159–12164 Downloaded by guest on September 25, 2021 both human and mouse cultured cells the cytosolic and mt dTTP with 2 vol of 100% methanol and treated the supernatant pools are in rapid exchange. solution after centrifugation as described above for the supernatant from the pellet. Methods Cell Lines and Cell Growth. The established human tumor lines Determination of Specific Radioactivity of dTTP and Total Radioac- OSTTK1Ϫ and HOSTK1ϩ and the mouse fibroblast lines tivity Incorporated into dT-P. We determined the size and specific 3T3TK1Ϫ and 3T3TK1ϩ were available in this laboratory and radioactivity of the dTTP pools by a DNA polymerase assay (13, grown as described earlier (12, 13) in 10-cm Petri dishes to a final 19). Briefly, we used an excess of [␣-32P]dATP and between 0.25 density of Ϸ3 million (3T3) or 4–8 million (OST and HOS) cells. and 12 pmol of [3H]dTTP for a standard curve. In the analyzed At this point, between 30% and 40% of the cells were in S phase. samples, we calculated the amount of dTTP from 32P cpm and We usually used cells from three to five dishes per time point. the specific activity from 3H cpm. For the determination of total Two hours before each labeling experiment we replaced the incorporated radioactivity we did not separate the three thymi- medium with 4 ml of fresh medium containing 20 mM Hepes dine phosphates but determined their total radioactivity after buffer, pH 7.4, and 10% dialyzed FCS. In chase experiments the separation from thymidine by stepwise elution from a 2-ml AG labeled medium was replaced with conditioned medium con- 1-X2 acetate column (mesh 100–200; Bio-Rad). We first eluted taining nonlabeled thymidine (0.3 ␮M for TK1ϩ cells or 1 ␮M thymidine with 34 ml of 50 mM acetic acid, followed by elution for TK1Ϫ cells). We carried out all manipulations of growing of thymidine phosphates with 10 ml of 1.0 M HCl and measured cultures in a thermostated room before rapidly transferring them to the radioactivity of this fraction. Values for the incorporation of a37°C incubator containing 5% CO2 and 95% air. All cultures were isotope are all normalized to one million cells to facilitate periodically checked for mycoplasma contamination. Cell numbers comparison between experiments. To determine the distribution were determined in a Coulter Counter (Beckman Coulter). of radioactivity between the three dT-Ps and the ratio between the three adenosine phosphates, we separated them by HPLC on Isotope Experiments and Separation of mt and Cytosolic dNTPs. We a Nucleosil 100 C18 column (Phenomenex, Belmont, CA) by added [3H]thymidine (20,000 cpm͞pmol; NEN, Milan) to a final isocratic elution (1 ml͞min) with 0.5 M ammonium phosphate, concentration of 1 ␮M (TK1Ϫ cells) or 0.3 ␮M (TK1ϩ cells). pH 3.5. After 25 min, the eluant was replaced by isocratic elution After the indicated times we transferred the dishes on an ice bath with 0.5 M ammonium phosphate͞10% methanol to remove to a cold room, removed the medium, and washed each dish four labeled thymidine from the column. The following retention times with 2 ml of ice-cold PBS with careful draining between times were found: dTTP, 8.3 min; dTDP, 9.5 min; dTMP, 18.1 washes. We removed the cells from the dish by scraping with a min; ATP, 5.3 min; ADP, 5.7 min; AMP, 10.2 min; and thymi- rubber policeman (extraction buffer: 0.21 M mannitol͞0.07 M dine, 37 min. Thymidine phosphates were quantified from their sucrose͞10 mM Tris⅐HCl, pH 7.5͞0.2 mM EGTA͞0.5 mg/ml radioactivity, adenosine phosphates from their peak absorptions. BSA; 1.5 ml per three dishes); transferred them to a round- bottomed, 2-ml Eppendorf tube; and homogenized the suspen- Enzyme Analyses. We measured citrate synthase (20), TK2 (6), 1 sion by aspiration through a 22-gauge 1 ⁄4-inch needle into a and dNT-2 (12) to check for leakage of enzymes from mito- 2.5-ml syringe followed by rapid expulsion. We found micro- chondria. Cytosolic fractions were analyzed directly; whole cells scopically that a single cycle resulted in complete breakage of cell and mt fractions were extracted (12) before analyses. To distin- membranes but incomplete release of mitochondria from nuclei. guish between TK2 and TK1 activity, we used the inhibition of In several experiments we observed that the yield of mitochon- TK2 by deoxycytidine and bromovinyldeoxyuridine (5, 6) and dria improved with increasing (up to 15) cycles, but that mt established in this way that only TK2 activity was present in enzymes (citrate synthase, TK2, and dNT-2) started to leak into TK1Ϫ cells. the cytosol (data not shown). In our standard procedure we therefore settled for one cycle and separated the combined Results mitochondria and nuclei from the cytosol. We centrifuged the Validation of Our Experimental Approach. We used a single cen- homogenate for 20 min at 19,000 ϫ g to remove the cytosol from trifugation of the homogenate at 4°C to separate a mixture of the sedimented nuclei and mitochondria, suspended the pellet in nuclei and mitochondria from the cytosol, followed by one 0.5 ml of extraction buffer, and resedimented it. This washing washing step of the pellet. Because the mt membrane retains step removed any remaining cytosolic dNTPs from the nuclei nucleotides, we assumed that mt dTTP remained in the pellet. and the pellet now contained the mt dNTP pool. To prepare Nuclei are permeable for nucleotides, and any nuclear dTTP is mitochondria in the experiment shown in Fig. 2 A and B, we first in rapid equilibrium with the cytosolic pool. The washing step, sedimented the nuclei from the homogenate by centrifugation at therefore, was expected to remove from the pellet both the 1,400 ϫ g for 5 min and then sedimented mitochondria by included cytosolic dTTP and dTTP present in nuclei. The centrifugation at 19,000 ϫ g for 20 min (18). Each pellet was nucleotides remaining in the second pellet then represent mt washed with 0.5 ml of washing buffer as described above to give nucleotides. nuclear and mt fractions. In all instances it was imperative to Because all of our results depend on this procedure, it was work rapidly and at cold-room temperature to avoid leakage of important to validate its correctness. We did this with three enzymes and dNTPs. different experiments. First, we compared the specific activity of To prepare mt dT-Ps we suspended the pellets in 1 ml of cold mt and cytosolic dTTP prepared by our method with the specific 60% methanol and kept them at Ϫ20°C for at least 1 h. After activity of dTTP in mitochondria and nuclei separated by a centrifugation, we immersed the supernatant solution for 3 min conventional procedure (Fig. 2 A and B). The two methods gave in a boiling water bath to kill any remaining enzyme activity and identical values for mt dTTP, which were different from the then evaporated the methanolic solution by centrifugal vacuum specific activity of cytosolic dTTP. Note that the nuclear and mt evaporation. We dissolved the residue in 0.1 ml of water and used dTTP prepared by the conventional method had identical spe- it for further analyses of nucleotides. The pellet remaining after cific activities, suggesting that the ‘‘nuclear’’ values represent mt methanol extraction was dissolved in 0.5 ml of 0.3 M NaOH and contamination. In a second experiment we found that the used for nuclear DNA analysis. We precipitated portions of the specific activity of dTTP in the wash solution from the first pellet dissolved DNA with 0.3 M HClO4, filtered the precipitate onto was that of cytosolic dTTP and differed from mt dTTP (Fig. 2 glass filters, and determined its radioactivity. To prepare cyto- C and D). A second wash solution contained no dTTP. This solic dT-Ps we precipitated a portion of the cytosolic fraction finding demonstrates that the washing step removed only cyto-

12160 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1635259100 Pontarin et al. Downloaded by guest on September 25, 2021 precursor of dTTP was 20,000 cpm͞pmol in all of our experiments. From Fig. 2 it appears that this value had decreased to between 2,000 and 10,000 in TK1ϩ cells and to between 20 and 100 in TK1Ϫ cells. When we partially inhibited the de novo synthesis of dTMP in TK1Ϫ cells with amethopterin and then labeled the cells with thymidine, the specific activities of both mt and cytosolic dTTP increased 10- to 20-fold. The size of the dTTP pools was much decreased (10-fold in the cytosol and 4-fold in mitochondria), and incorporation of isotope into DNA was inhibited (data not shown), demonstrating the dependence of the cells on de novo synthesis. Our data concerning the mt dTTP pool disagree with an earlier report (21) claiming an increase in the size of this pool during inhibition of dTMP synthesis. Isotope dilution was evidently due to a competition between salvage of labeled thymidine by thymidine kinase and de novo synthesis of dTMP by thymidylate synthase (see Fig. 1). In the absence of TK1, salvage by TK2 was inefficient, resulting in a very low specific activity of dTTP; however, in the presence of TK1, de novo synthesis still accounted for more than half of the newly synthesized dTTP. Fig. 2. Validation of methodology for the rapid separation of cytosolic and ϩ Ϫ mt dTTP. A and C are results from TK1 cells; B and D are results from TK1 cells. In Vivo Turnover of mt and Cytosolic dT-P Pools. We incubated cell In A and B, we divided homogenates from cells incubated for 10 min with 3 labeled thymidine into two parts. We used one part for the preparation of the cultures with [ H]thymidine and used isotope flow experiments combined mt and nuclear fraction (MN) and for a cytosolic fraction (C) by our (13) to determine the time course of isotope incorporation into method, the other part for the separate preparation of mt and nuclear (N) mt dT-P, cytosolic dT-P, and DNA as well as the specific fractions by a conventional method (18). In C and D we also analyzed the radioactivities of the two dTTP pools. In chase experiments we washing (W) from the MN fraction. In all fractions we determined the speciﬁc substituted nonlabeled thymidine for [3H]thymidine after 10 min radioactivity of dTTP. and continued incubation for up to 50 min to follow the disappearance of radioactivity from the dTTP pools. Experi- ments with two different TK1Ϫ lines are shown in Figs. 4 and 5. solic dTTP from the pellet. In a third experiment (Fig. 3) we The cytosol of homogenates from both lines contained Ͻ2% of determined by HPLC the distribution of isotope among the three the thymidine kinase activity of the cells. All phosphorylation of dT-Ps and the ATP to ADP to AMP ratio in mitochondria and [3H]thymidine therefore occurred in mitochondria. Fig. 4 shows in the cytosol. In both cellular compartments the distribution of results of the total isotope incorporation, with 4A giving data for isotope between thymidine phosphates mirrored the molar ratios human OST cells and 4B those for mouse 3T3 fibroblasts. Fig. 5 between adenosine phosphates, with a large preponderance of A and B shows the specific activities of the cytosolic and mt dTTP the triphosphate in the cytosol and a small excess of the pools from the same experiments. diphosphate in mitochondria. Together, the three experiments After a short lag phase, isotope was linearly incorporated into show that our simplified method functions with minimal cross- nuclear DNA (Fig. 4) in both cell lines, indicating a continuous contamination of dT-P between mitochondria and the cytosol. synthesis fed from the cytosolic dTTP pool. This pool reached a plateau after 20 min, demonstrating that between 20 and 60 Competition Between Salvage and de Novo Synthesis of dTTP. The min the loss of dTTP from the pool was replenished by synthesis specific radioactivity of the labeled thymidine provided as a from thymidine. The rapid turnover of the cytosolic dTTP pool is also apparent from the chase experiment in which the pool rapidly lost radioactivity when nonlabeled thymidine was substituted for labeled thymidine in the medium. Phosphorylation of thymidine by TK2 occurred in mitochondria; therefore, the mt dTTP was the precursor of the cytosolic dTTP. The radioactivity in the mt pool reached a steady state after only 10 min (Fig. 4) and afterward was in a dynamic equilibrium with the cytosolic pool. Again, results from the chase substantiate its rapid turnover. Mouse cells (Fig. 4B) incorporated less isotope than human cells (Fig. 4A), but the general behavior of both lines was identical. The specific radioactivities from the same experiment are shown in Fig. 5. The values were now almost the same for human (Fig. 5A) and mouse (Fig. 5B) cells. Both mt and cytosolic dTTP

pools reached their plateaus after 10 min. The radioactivity was CELL BIOLOGY rapidly chased by nonlabeled thymidine. After 2 min the specific radioactivity was 8 times higher in mitochondria than in the cytosol; at later time points the difference decreased to 3-fold, in agreement with a precursor role for mt dTTP. From the Fig. 3. Relative amounts of dTMP͞dTDP͞dTTP and ATP͞ADP͞AMP in cytosol plateau values for cytosolic dTTP and the radioactivity incor- and mitochondria. By using HPLC, we separated the nucleotide fractions from porated into DNA (Fig. 4) we can calculate the rate of DNA mitochondria (A) and cytosol (B) from OSTTK1Ϫ cells incubated for 10 min with synthesis in the two experiments. In human cells the incorpo- labeled thymidine. We quantiﬁed thymidine phosphates from their radioac- ration into DNA between 20 and 60 min was 150 cpm͞min. By tivity and adenosine phosphates from their peak absorbancies. division with 35, which is the average specific activity of cytosolic

Pontarin et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12161 Downloaded by guest on September 25, 2021 Fig. 4. Incorporation of [3H]thymidine into cytosolic dTTP, mt dTTP, and DNA in TK1Ϫ cells: OSTTK1Ϫ (A) and 3T3TK1Ϫ (B). We incubated cells with 1 ␮M [3H]thymidine for the indicated time periods, isolated cytosolic and mt dTTPs and DNA, and determined incorporation of total radioactivity into each fraction. In a chase experiment (broken lines) we removed the medium from cultures, replaced it with conditioned medium containing 1 ␮M nonlabeled thymidine, and continued incubation for the additional indicated times. ‚, Cytosolic dTTP; E, mt dTTP; ᮀ, DNA.

dTTP, we obtain an incorporation of 4.3 pmol of dTMP per min. those with TK1Ϫ cells described in Figs. 4 and 5. In a separate ϩ In a similar calculation the rate of DNA synthesis in mouse 3T3 experiment we found that extracts from TK1 cells contained Ϫ cells came out as 0.8 pmol of dTMP per min. Thus the incor- 100-fold higher thymidine kinase activity than TK1 cells, and ϩ poration of dTTP into DNA was slower in 3T3 cells than in OST TK1 cells also incorporated 100 times more radioactivity (Fig. cells. Nevertheless the interrelation between cytosolic and mt 6). Here, salvage of thymidine competed quite effectively with de dTTP pools remained the same. Moreover, the enormous novo synthesis. In both human and mouse cells the total radio- dilution of isotope indicates that in both types of cells the large activity in the mt and cytosolic dTTP pools reached plateau Ϸ majority of dTTP stemmed from the de novo pathway. values at 20 min, but at very different levels: The plateau values We turn now to TK1ϩ cells. Figs. 6 and 7 show experiments reached up to 30 times higher in the cytosol. Incorporation into with human HOS TK1ϩ and mouse 3T3 TK1ϩ cells patterned on DNA started with a slight lag phase followed by a close to linear phase. The deviation from linearity after 20 min was caused by depletion of the labeled thymidine in the medium, particularly in HOS cells. We had lowered the concentration of thymidine in the medium to 0.3 ␮M to avoid artifactual expansion of the dTTP pool by the effective salvage of the nucleoside. The specific activity of dTTP was much higher in TK1ϩ cells (Fig. 7) than in TK1Ϫ cells, with values of up to 7,000 cpm͞pmol in the cytosol compared with 30 (Fig. 4). Now TK1 in the cytosol was responsible for almost all phosphorylation of thymidine. In consequence the cytosolic dTTP pool had a higher specific activity than the mt pool. This is opposite to TK1Ϫ cells, where phosphorylation occurred in mitochondria and the specific activity always was highest in mt dTTP. Nevertheless the plateau values for the specific activities of mt dTTP were always higher in TK1ϩ cells than in TK1Ϫ cells (40 times in human cells and 10 times in mouse cells). This finding suggests that most of the radioactive mt dTTP was imported from the cytosol. The contribution of TK2 was probably minimal because of the low activity of the enzyme. Discussion Fig. 5. Specific radioactivities of cytosolic and mt dTTP in TK1Ϫ cells after incubation with [3H]thymidine. This is the same experiment as in Fig. 4, but this This work presents the dynamics of a mt dNTP pool and its figure shows the specific radioactivities of the two dTTP pools instead of total connection with the corresponding cytosolic pool. It was made radioactivity. Broken lines indicate chase experiments. ‚, Cytosolic dTTP; E,mt possible by a simple method for the complete separation of mt dTTP. and cytosolic dNTP pools. The validity of this procedure is

12162 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.1635259100 Pontarin et al. Downloaded by guest on September 25, 2021 Fig. 7. Speciﬁc radioactivities of cytosolic and mt dTTP in TK1ϩ cells in the experiment described for Fig. 6. Broken lines indicate chase experiments. ‚, Cytosolic dTTP; E, mt dTTP.

made, because the only phosphorylation of thymidine occurs by TK2, and any radioactivity in the cytosolic pool must originate from the mt pool. In this case it is clear that the mt pool was a precursor of the cytosolic pool. Compared with the specific activity of the precursor thymidine, the specific activities of the cellular pools were very low, indicating that a large majority of dTTP was derived from nonlabeled precursors by de novo synthesis. This conclusion is directly apparent from the large increase in specific activity in amethopterin-treated cells, where de novo synthesis is inhibited. Fig. 6. Incorporation of [3H]thymidine into cytosolic dTTP, mt dTTP, and DNA We could not determine the nature of the nonlabeled material in TK1ϩ cells: HOSTK1ϩ (A) and 3T3TK1ϩ (B). We incubated cells with 0.3 ␮M that contributed to the dilution effect. We found no difference [3H]thymidine for the indicated times and determined incorporation of total between the specific activities of dTMP, dTDP, and dTTP, radioactivity into dTTP pools and DNA. Broken lines indicate chase experi- indicating a rapid equilibration of isotope between these pools. ments conducted with 0.3 ␮M nonlabeled thymidine as described in Fig. 4. ‚, The size of the intra-mt thymidine pool was too small to be Cytosolic dTTP; E, mt dTTP; ᮀ, DNA. determined. We can therefore not decide whether isotope dilution occurred at the level of thymidine or at the level of a specific thymidine phosphate. We can conclude, however, that supported by the experiments in Fig. 2. We obtained similar Ϫ also in the presence of thymidine, cycling TK1 cells synthesize results with both human and mouse cells in culture. In addition ϩ the vast majority of their dTTP de novo and that TK1 cells also to the experiments described here, we performed several exper- ϩ Ϫ strongly depend on de novo synthesis. iments with HeLaTK1 and HeLaTK1 cells with similar results A second point emerging from measurements of the specific (data not shown). We therefore suggest that our results are of radioactivities concerns the turnover of both the cytosolic and mt general validity for cycling cells in culture. pools. We determined earlier (22) a half-life of 4 min during S Our general approach was to determine the flow of tritiated phase for the total cellular dTTP pool of 3T3 cells. The present thymidine through cytosolic and mt dTTP pools into DNA by experiments with nonsynchronized cultures were not designed to measuring both the time-dependent total incorporation of iso- determine half-lives. Nevertheless one can estimate a similar tope into the different compartments and the specific radioac- rapid turnover both from the attainment of isotope equilibrium tivities of mt and cytosolic dTTP. Determinations of specific and from the decay during the chase (Figs. 5 and 7). The radioactivities are often neglected but were of crucial impor- important finding is that the mt and cytosolic pools show the tance for our work. Values are independent of losses during same dynamic behavior, suggesting a rapid exchange between preparation of nucleotides and therefore more reliable than total them.

radioactivity. When the specific radioactivity of dTTP was at a The connection between the two pools appears directly from CELL BIOLOGY plateau after 20 min, the system was in a steady state, and the loss the experiments depicted in Figs. 4–7. In TK1Ϫ cells (Figs. 4 and of radioactivity from the pools through incorporation into DNA 5) thymidine first enters the mt dTTP pool, but radioactivity was balanced by synthesis from thymidine. The attainment of two appears in cytosolic dTTP and is incorporated into DNA after different plateaus for cytosolic and mt dTTP shows that we were only a brief lag period. In TK1ϩ cells (Figs. 6 and 7) thymidine seeing the dynamic behavior of two metabolically distinct pools. is phosphorylated almost exclusively by TK1 in the cytosol but This observation by itself does not distinguish between a mech- then moves rapidly into the mt pool. We did not expect such a anism involving the independent incorporation of radioactivity rapid exchange between the two compartments. Our experi- into two separate pools or the transfer of radioactivity from one ments show that enzymes in the mt membrane catalyze the rapid pool to the other. In TK1Ϫ cells, however, a distinction can be movement of thymidine phosphates (and probably other nucle-

Pontarin et al. PNAS ͉ October 14, 2003 ͉ vol. 100 ͉ no. 21 ͉ 12163 Downloaded by guest on September 25, 2021 otides) in and out of mitochondria and directly demonstrate the symptoms of the genetic absence of the deoxynucleotide trans- function of the first pathway of Fig. 1. In cycling cells, the porter appear during embryogenesis (14), whereas the loss of function of the second pathway, the salvage of thymidine by TK2, TK2 causes disease only after birth (15). was minimal. We suggest that salvage is important only in resting cells in the absence of de novo synthesis in the cytosol without This work was supported by the European Commission (Grant QLRT- sufficient dNTPs for import into mitochondria for mtDNA CT-2000-01004) and the Italian Association for Cancer Research and replication and repair. This difference may explain why the Telethon Italia (Grant GP0140Y01 to V.B.).

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