Dynamics of Pyrimidine Deoxynucleoside Triphosphate
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Proc. Nati Acad. Sci. USA Vol. 80, pp. 1347-1351, March 1983 Cell Biology Dynamics of pyrimidine deoxynucleoside triphosphate pools in relationship to DNA synthesis in 3T6 mouse fibroblasts (nucleoside incorporation/compartmentation/amethopterin block) BjORN NICANDER AND PETER REICHARD Medical Nobel Institute, Department of Biochemistry I, Karolinska Institutet, Box 60400, S-104 01 Stockholm, Sweden Contributed by Peter A. Reichard, November 18, 1982 ABSTRACT The 3H-labeled nucleosides cytidine, deoxycyti- tivity into DNA to this value (1, 2). Such a procedure becomes dine, and thymidine are rapidly incorporated into DNA via dCTP imperative when one wishes to distinguish between effects of or dTTP pools. Between 30 and 60 minafter addition of tracer different manipulations-e. g., pharmacological interference- amounts ofa labeled nucleoside to the medium ofrapidly growing on precursor synthesis and DNA replication. Knowing the spe- 3T6 cells, dNTPpools attained a constant specific activity resulting cific activity of a dNTP under steady-state conditions may in from a steady-state equilibrium between incorporation of nucleo- addition permit determination of absolute rather than relative side, de novo synthesis, and linear incorporation of isotope into rates of DNA synthesis. DNA. Removal oflabeled deoxycytidine or thymidine depleted the The of dNTP pools ofisotope within a few minutes and incorporation into possibility compartmentation of dNTP pools poses DNA stopped. When de novo synthesis ofdTTP was blocked with additionalicomplications (3). Fractionation ofcells in nonaque- amethopterin, the intracellular dTTP pool rapidly reached the ous media led to -the suggestion of separate cytoplasmic and. specific activity of thymidine of' the medium and isotope-incor- nuclear dNTP pools in Chinese hamster ovary cells (4). Kinetic poration into DNA then measured absolute rates of DNA synthe- experiment with HeLa cells were interpreted to indicate pref- sis. In experiments with and without. amethopterin, we found no erential incorporation of labeled thymidine into DNA via a kinetic evidence for the existence ofmore than one dTTP pool and dTTP pool that was not in equilibrium with dTTP formed de the decay ofthe pool suggested that all dTTP served as precursor novo (5). The suggested channeling of DNA precursors via a ofDNA. In contrast, experiments with deoxycytidine and cytidine postulated multienzyme "replitase" also leads to compartmen- suggested the presence of separate dCTP'pools with preferential tation of dNTPs (6). DNA synthesis from the pool labeled from cytidine. The aim ofthe present work was to relate the synthesis and breakdown ofpyrimidine dNTP pools to DNA synthesis in 3T6 Incorporation ofradioactive thymidine or, less frequently, other cells. When dTTP was labeled by using thymidine and de novo labeled nucleosides into DNA is often used to measure the rate synthesis was blocked with amethopterin, the specific activity of DNA synthesis. With thymidine and other deoxynucleo- ofthe pool rapidly approached that ofthe radioactive thymidine' sides, incorporation involves a series of phosphorylation steps in the medium, permitting determination of absolute rates of leading directly to dNTPs; with ribonucleosides, reduction of DNA synthesis. In parallel experiments, we measured incor- the ribose moiety is required in addition. dNTPs are also formed poration of labeled thymidine, deoxycytidine, or cytidine into de novo from small molecules. These interrelationships for the DNA and dNTP pools in the absence of amethopterin. From nucleosides thymidine, deoxycytidine, and cytidine are out- dataobtained under steady-state conditionswith thymidine and lined below. cytidine, we could calculate rates of DNA synthesis very close to those obtained with thymidine in the presence ofamethop- Thymidine Deoxycytidine Cytidine terin, while the results with labeled deoxycytidine gave much lower values. These experiments suggest that, under steady- dTMP +- dUMP +- dCMP CMP state conditions, DNA in 3T6 cells is synthesized from dTTP I 1 I I and dCTP pools in rapid equilibrium with labeled thymidine dTDP de novo dCDP - CDP or cytidine. On the other hand, isotope from deoxycytidine ap- I synthesis I T pears to be preferentially incorporated into a dCTP pool that, dTTP dCTP de novo is not used directly for DNA synthesis. We also found that the synthesis decay ofthe labeled dTTPpoolclosely matched the rate ofDNA \DNA / synthesis; Clearly, incorporation of a labeled nucleoside into DNA de- MATERIALS AND METHODS pends on other factors in addition to the rate of the final poly- Materials. [5-3H]Cytidine, [5-3H]deoxycytidine, and [meth- merization of dNTPs. yl-3H]thymidine with specific activities of 20-30 Ci/mmol (1 In spite of this, incorporation of thymidine has been used Ci = 3.7 GBq) and 3H- and a-32P-labeled dNTPs were obtained widely, and often successfully, to monitor relative rates ofDNA from Amersham. Care was taken to use the radioactive com- synthesis, neglecting possible errors introduced by changes in pounds shortly after delivery or to redetermine their specific specific radioactivities ofdTTP. A safer, but more cumbersome, activity by HPLC. approach would be to measure the specific activity of the im- Incorporation of 3H-Labeled Nucleosides into Cells. 3T6 mediate precursor dNTP and relate incorporation of radioac- mouse fibroblasts (105 cells) were explanted onto 5-cm dishes and grown for 44-48 hr at 37°C in 5 ml of Dulbecco's modified The publication costs ofthis article were defrayed in part by page charge Eagle's medium/10% inactivated horse serum to a density of payment. This article must therefore be hereby marked "advtertise- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation: HAT, hypoxanthine/amethopterin/thymidine. 1347 Downloaded by guest on September 29, 2021 1348 Cell Biology: Nicander and Reichard Proc. Natl. Acad..Sci. USA 80 (1983) 0.6-1.0 X 106 cells. Three hours before addition.ofisotope, the rate. The observed specific activity ofagiven dNTP is the result medium was reduced to 2.5 ml and buffered with 1 M Hepes ofa balance between phosphorylation ofthe labeled nucleoside (pH 7.4) (final concentration, 25 ,uM). In experiments measur- and de novo synthesis. The rate of DNA synthesis can be cal- ing the decay of dNTPs, the cells' were transferred in a warm culated from -the incorporation at 60-120 min. After that time (370C) room from the incubator to tightly sealed boxes.contain- (arrow in Fig. 1), radioactive medium was replaced with pre- ing 5% C02/95% air atmosphere and all further manipulations warmed conditioned medium containing 0.3 A&M unlabeled in the warm room outside the boxes were as brief as possible. nucleoside. With [3H]thymidine and [3H]deoxycytidine, this A 3H1-labeled nucleoside without dilution was added to the resulted in rapid depletion ofthe intracellular acid-soluble ra- medium to afinal concentration ofabout 0.33uM and incubation dioactivity, indicating rapid -turnover of the dNTP pools, with was continued for the indicated times. To measure the decay half-lives of-about 5 min. With [3H]cytidine, about 90% ofthe of label, medium was removed by aspiration and immediately acid-soluble radioactivity waspresentasCTPand, aftermedium replaced with 2.5 ml ofconditioned medium (from parallel cul- change, isotope continued to flow from this pool into the much tures without isotope) containing 0.3 ,uM unlabeled nucleoside. smaller dCTP pool. In this case, we could therefore not deter- Incubations were terminated and isotope incorporation into mine the turnover of the dCTP pool. DNA was determined as described (7) except that 2 ml of cold Decay ofdNTP Pools. A more careful analysis ofthe events 60% methanol (8) was used in place ofHCI04. The mixture was during the first 10 amin of the decay of dTTP and dCTP pools centrifuged at 22,000 x g for 45 min, the supernatant was evap- labeled by using thymidine or deoxycytidine, respectively, was orated to dryness at reduced pressure, and the residue so ob- made in a second experiment. In Fig. 2 the logarithms of the tained was dissolved in 0.2 ml of 10 mM Tris buffer (pH 7.0) specific activities ofthe two dNTPpools are plotted as afunction and used for the determination of dNTPs (8, 9) and their specif- oftime. The linearity ofthe firstpart ofthe curves suggests that, ic activity (refs. 1, 2; unpublished data). during this part of the experiment, the reaction followed first- All values in the figures and tables are normalized to 106 cells. order kinetics with half-lives of5.5 (dTTP) and 6'(dCTP) min. From these values and the pool sizes for dTTP (80 pmol) and dCTP (115 pmol), one can calculate that the initial decay of ra- RESULTS dioactivity corresponded to turnover rates of 10 and 13 pmol/ Attainment of Steady-State Conditions. Tracer amounts of min per 106 cells, respectively. highly 3H-labeled thymidine, deoxycytidine, and cytidine were Thymidine Incorporation Measures the True Rate ofDNA added to separate sets ofcultures ofrapidly growing 3T6 cells. Synthesis. Rates ofDNAsynthesis duringbriefexperiments can After 60 min at 370C, equilibrium was attained in each case (Fig. be computed from the incorporation of isotope into DNA and 1). The total acid-soluble radioactivity, as well as the specific the specific activity ofa dNTP pool labeled under steady-state activity ofthe labeled dNTP pools, had reached a plateau value conditions as described in Fig. 1. This approach assumes that and isotope incorporation into DNA was occurring at a linear the measured value for the dNTP pool represents the specific activity of the immediate precursor for DNA synthesis. How- ever, a given dNTP may be present in more than one pool and have a different specific activity in each. If then DNA is syn- + 16 thesizedpreferentially from only one such pool-derived, e.g., by de novo synthesis via reduction ofribonucleotides-the spe- -14 cific activity ofa given dNTP pool labeled by using thymidine or deoxycytidine via the salvage pathway may not represent the 12 relevant value for measurement of the rate of DNA synthesis.