From the Department of Life Sciences and Chemistry,
Roskilde University, P. O. Box 260, DK 4000 Roskilde, the
¶ Department of Microbiology, Technical University of Denmark, DK
2800 Lyngby, and the
Department of Genetics, Copenhagen
University, DK 1353 Copenhagen, Denmark
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ABSTRACT |
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In mammalian cells, there are three pyrimidine nucleoside salvage enzymes with the capacity to phosphorylate all four deoxynucleosides, the two thymidine kinase isoenzymes, TK1 and TK2, and the deoxycytidine kinase, dCK. TK1 is cell cycle-regulated; TK2 is expressed constitutively and can phosphorylate deoxycytidine to the same extent as thymidine. dCK phosphorylates deoxycytidine, deoxyadenosine, and deoxyguanosine, but not thymidine. In addition, the three kinases can phosphorylate a number of medically important analogs. In cultured Drosophila melanogaster embryonic cells, only one pyrimidine deoxynucleoside kinase was present. This kinase was purified and showed a broad substrate specificity, since it was able to phosphorylate all four deoxynucleosides with high efficiency, as compared with the kinases in mammalian cells. Additionally, a number of nucleoside analogs such as arabinofuranosyl pyrimidines, deoxyuridine, and 5'-fluorodeoxyuridine, were phosphorylated. There was negligible 3'-azidothymidine and no dTMP phosphorylation. The enzyme was active as a monomer of about 30 kDa. We suggest the name D. melanogaster deoxynucleoside kinase for this multifunctional kinase. The substrate specificity, size, and other characteristics show that this enzyme is more related to human TK2 than to the other mammalian deoxyribonucleoside kinases, but is unique with respect to the capacity to phosphorylate all four deoxynucleosides.
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INTRODUCTION |
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The biosynthesis of deoxynucleoside triphosphates in most living organisms is performed by two pathways, the de novo and the salvage pathway. The main enzyme of the de novo pathway is ribonucleotide reductase, which catalyzes the reduction of the 2'-OH group of the nucleoside diphosphates. The enzyme has been extensively studied as to the structure and the complex feedback regulation by the end products, the four deoxynucleoside triphosphates (1, 2).
An alternative pathway for supplying deoxynucleoside triphosphates for
replication and repair of DNA is the salvage pathway. The principal
salvage enzymes are the deoxynucleoside kinases, which phosphorylate
deoxynucleosides to the corresponding deoxynucleoside monophosphates.
In mammalian cells, there are four deoxynucleoside kinases with
different cellular localization and specificities, thymidine kinase 1 (TK1),1 thymidine kinase 2 (TK2), deoxycytidine kinase (dCK), and deoxyguanosine kinase (dGK). TK1
is cytosolic and is expressed only in S-phase cells (3, 4); the
corresponding gene was cloned several years ago (5). TK2, dCK, and dGK
are all constitutively expressed enzymes. TK2 is considered to be
localized in mitochondria (6), but is encoded by a nuclear gene that
was cloned recently (7). dCK is cytosolic, and the corresponding gene
was cloned in 1991 (8). dGK is mitochondrial (9) and has a manyfold
lower phosphorylation capacity compared with the other three pyrimidine
nucleoside kinases. Like TK2, dGK is encoded by a nuclear gene, which
has recently been cloned by two independent groups (10, 11). Homology
studies indicate that mammalian TK1 is closely related to pox-viral TKs and TK from Escherichia coli (12), whereas dCK, dGK, and TK2 share many properties with herpetic TKs (7). The substrate specificities of the mammalian deoxynucleoside kinases toward natural
substrates and a number of therapeutic nucleoside analogs differ
considerably, but with a characteristic pattern useful for distinction
between the enzymes (13). In addition to thymidine (dThd),
2'-deoxyuridine (dUrd), and 5-fluoro-2'-deoxyuridine (FdUrd), TK1 can
phosphorylate 3'-azidothymidine (AZT) and 3'-fluoro-2',3'-dThd (FddThd), and TK2 can phosphorylate deoxycytidine (dCyd) and
1--D-arabinofuranosyl thymine (AraT) (13, 14). dCK can
phosphorylate deoxyadenosine (dAdo), deoxyguanosine (dGuo) and
1-
-D-arabinofuranosyl cytosine (AraC) (15), and dGK can
phosphorylate dAdo (16).
The occurrence of cytosolic and mitochondrial forms of thymidine kinases has also been observed in lower eucaryotes. In the filamentous fungus Achlya ambisexualis, four forms of TK were found, one readily solubilized and three solubilized by detergent. The multiple TK forms were distinctive by their electrophoretic properties, and one of the detergent solubilized forms resembled TK2 in the ability to phosphorylate dCyd (17).
Drosophila melanogaster, the fruit fly, is one of the genetically and developmentally most characterized organisms. However, so far, the metabolism of deoxynucleotides and its regulation and role in the cell proliferation and differentiation during embryogenesis has been only poorly investigated, and to our knowledge there have been no reports about deoxynucleoside kinases in this organism or other insect cells. In the present paper, we describe the purification and characterization of a deoxynucleoside kinase from the fruit fly. The purified kinase is multifunctional and apparently the only deoxynucleoside kinase present in the cultured Drosophila S-2 cells.
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EXPERIMENTAL PROCEDURES |
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Materials
DEAE-Sepharose Fast Flow, Phenyl-Sepharose, the Superose 12 HR
10/30 column, and SDS-polyacrylamide molecular weight standards were
from Pharmacia Biotech Inc. The 3'-dTMP-Sepharose gel-matrix (p-aminophenyl-3'-TMP:CH-Sepharose) was prepared according
to the procedure described by Kowal and Markus (18), using
thymidine-3'-(4-aminophenyl-phosphate) kindly donated by Eger et
al. (19). Fetal calf serum was purchased from Life Technologies,
Inc., and the gel-filtration molecular weight standards, Schneider's
medium, unlabeled nucleotides and nucleosides, ultrapure ammonium
sulfate and CHAPS were purchased from Sigma. 3H-Labeled
thymidine (925 GBq/mmol) and deoxycytidine (740-925 GBq/mmol) were
obtained from Amersham Corp. 3H-Labeled deoxyadenosine
(1106 GBq), deoxyguanosine (226 GBq/mmol), deoxyuridine (629 GBq/mmol),
5-fluoro-deoxyuridine (703 GBq), 2',3'-dideoxy-2',3'-didehydrothymidine
(744 GBq/mmol), 3'-fluoro-2',3'-dideoxythymidine (289 GBq/mmol),
3'-azido-2',3'-dideoxythymidine (740 GBq/mmol), 1--D-arabinofuranosyl thymine (107 GBq/mmol),
1-
-D-arabinofuranosyl cytosine (862 GBq/mmol),
2',3'-dideoxycytidine (185 GBq/mmol), and acyclovir (655 GBq/mmol) were
from Moravek Biochemicals Inc., Brea, CA. When present, ethanol was
evaporated and the analog resuspended in water. All other chemicals
were of the highest purity available. All solutions were made with
ultrafiltered (0.22 µm) and autoclaved water, purified by the
Milli-RO 15 water system from Millipore.
Cells
The D. melanogaster S-2 cell line of embryonic origin
were grown in Schneider's medium supplemented with 10% fetal calf
serum in a Brown Biostat M bioreactor (B. Brown, Melsungen, Germany) as
described previously (20). Cells (8.6 × 1010) were
harvested by centrifugation (2000 × g) and pellets
stored at 80 °C until used for enzyme purification.
Enzyme Assays
The nucleoside kinase activities were determined by initial velocity measurements based on four time samples by the DE-81 filter paper assay using tritium-labeled substrates (14). The standard assay conditions were: 50 mM Tris-HCl, pH 8.0 (22 °C), 2.5 mM MgCl2, 10 mM dithiothreitol, 0.5 mM CHAPS, 3 mg/ml bovine serum albumin, 2.5 mM ATP, and 10 µM radiolabeled substrate, unless otherwise indicated.
The products of the kinase reaction were analyzed by mixing samples of 10 µl from a standard reaction mixture with 20 µl of 5 mM dThd, dTMP, dTDP, and dTTP, and spotting 10 µl of this solution on polyethyleneimine-cellulose plates. After developing the plates ascending in 0.5 M LiCl2, the spots with nucleosides and nucleotides were identified under UV light and cut out. The radioactivity was extracted with 0.2 M KCl, 0.1 M HCl and determined by liquid scintillation.
The variance in duplicate assays was below 5% (CV). The variance in the determination of kinetic constants in two independent experiments was below 20% (CV). One unit of activity is defined as 1 nmol of deoxynucleoside 5'-monophosphate formed/min. Specific activity is expressed as units/mg of protein, where the kinase activity is measured at saturating substrate concentrations.
Enzyme Purification
Buffers-- Buffer A consisted of 20 mM potassium phosphate buffer (pH 7.4), 15% glycerol, 1 mM potassium EDTA, 1 mM dithiothreitol. Buffer B consisted of 20 mM Tris, pH 7.5 (20 °C), 5 mM MgCl2, 10% glycerol, 2 mM dithiothreitol. Buffer C consisted of 10 mM Tris-HCl, pH 8 (20 °C), 5 mM MgCl2, 10% glycerol, 2 mM dithiothreitol, 0.5 mM CHAPS. Buffer D consisted of 50 mM Tris-HCl, pH 7.5 (20 °C), 5 mM MgCl2, 10% glycerol, 2 mM dithiothreitol, 0.5 mM CHAPS. Buffer E consisted of 50 mM potassium phosphate buffer, pH 7.5, 5 mM MgCl2, 10% glycerol, 2 mM dithiothreitol. Buffer F consisted of 50 mM imidazole-HCl, pH 7.5, 5 mM MgCl2, 2 mM dithiothreitol, 0.1 M KCl.
Purification steps I-V were performed at 4 °C according to previously described procedures (14) with minor modifications.Step I: Preparation of Crude Extract-- The pellet containing 8.6 × 1010 Drosophila S-2 cells was suspended in buffer A and homogenized by treatment with a French press. The homogenate was centrifuged 30 min at 12,000 × g (fraction I).
Step II: Streptomycin/Ammonium Sulfate Precipitation and G-25 Chromatography-- The nucleic acids were precipitated with streptomycin sulfate (0.7%), and the resulting supernatant precipitated with ammonium sulfate in two steps (20% and 70%) as described elsewhere (15). All centrifugations were performed at 12,000 × g. The ammonium sulfate pellet was suspended in buffer B, divided in three portions of about 30 ml, and each portion was desalted on a Sephadex G-25 column (bed volume, 50 mm × 200 mm) with buffer B. The peak fractions were collected and pooled (fraction II).
Step III: DEAE Ion Exchange Chromatography-- Fraction II was divided in three portions, and each portion was chromatographed on a DEAE Sepharose Fast Flow column (bed volume, 50 mm × 250 mm), equilibrated with buffer B + 0.5 mM CHAPS. After application, unbound material was washed out with buffer B + 0.5 mM CHAPS (600 ml) and the bound material was eluted with a 0-0.5 M KCl gradient in buffer B (2 × 1.2 liters). The fractions containing TK and dCK activity were pooled (fraction III).
Step IV: 3'-dTMP-Sepharose Chromatography-- Fraction III was divided in three portions, and each portion was applied on a 3'-dTMP-Sepharose column (bed volume, 10 mm × 64 mm) equilibrated with buffer C. The unbound material was washed out with buffer C and then with buffer D, and the bound material was eluted with buffer D containing 2 mM dThd. The fractions containing TK activity were pooled (fraction IV).
Step V: Phenyl-Sepharose Chromatography-- Fraction IV was made 1.5 M with ammonium sulfate and applied on a Phenyl-Sepharose column (10 mm × 12.8 mm) equilibrated with buffer E + 1.5 M ammonium sulfate. The unbound material was washed out with the equilibration buffer. The bound material was eluted in two steps, first with buffer E and thereafter with buffer E + 10 mM CHAPS.
Molecular Weight Determinations
The subunit size of the purified protein was determined by discontinuous SDS-polyacrylamide gel electrophoresis in Tris-HCl (pH 6.8 and 8.8, 22 °C) with a stacking gel of 4.5% and a separating gel of 12%, according to the procedure of Laemmli (21). The protein bands were visualized by silver staining.
The apparent size of the native enzyme was determined by gel-filtration on a prepacked Superose 12 column connected to a Gradifrac system essentially as described (14). The column was equilibrated and the enzyme eluted with buffer F.
Other Methods
The protein concentration was determined according to Bradford (22). Conductivity was measured with the CDM 3 Radiometer conductivity meter using the CDC 314 cell.
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RESULTS |
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Purification of Deoxynucleoside Kinases from Cultured Drosophila Cells-- In the desalted ammonium sulfate fraction (fraction II), all four deoxynucleoside kinase activities were present, although the amounts of dAK and dGK activity were less than 5% of that of TK and dCK, as measured at standard assay conditions. When fraction II was chromatographed on DEAE-Sepharose, only one peak of TK and dCK activity was obtained, the activities coinciding with each other. Furthermore, the dAK and the dGK activities coincided with the TK and dCK activities, and the peak with the four deoxynucleoside kinase activities was eluted at about 80 mM KCl (Fig. 1).
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Molecular Weight Determinations-- The mass of the subunit and the quality of the purification were analyzed by SDS-polyacrylamide gel electrophoresis of the peak fraction from Phenyl-Sepharose chromatography. Only a single band appeared on the gel, and the size was estimated to be about 30 kDa (Fig. 2).
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Kinase Reaction Products-- The products of the kinase reaction were analyzed by polyethyleneimine-cellulose liquid chromatography as described under "Experimental Procedures." No time-dependent increase in the radioactivity in the dTDP and dTTP spots was observed. Hence, it is concluded that Dm-dNK is unable to phosphorylate dTMP.
Temperature Dependence of the Dm-dNK-- When the TK activity of Dm-dNK was examined at various temperatures, a broad maximum was observed ranging from 40 to 60 °C. Therefore, all kinetic investigations were performed at 37 °C to compare the results with those of the mammalian kinases.
Substrate Specificity-- As is shown in Table II, the substrate specificity of Dm-dNK is broad. dUrd and FdUrd were as efficient substrates as dThd and dCyd. Both arabinosyl pyrimidines, AraT and AraC, were also phosphorylated. In addition, the phosphorylation of 2',3'-didehydro-2',3'-dideoxythymidine and FddThd was measurable, although below 1% of the TK activity at the tested substrate concentration. Only the activity with acyclovir was not detected, and progress curves stayed horizontal.
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Substrate Competition-- The ability of the four substrates to compete with each other was examined by determining the inhibition constants shown in Table IV. As it appears, all four substrates interacted with each other. dThd was a strong inhibitor of the dCK, dAK, and dGK kinase activity of Dm-dNK, with Ki values between 1 and 3 µM. dCyd, dAdo, and dGuo were efficient inhibitors of the TK activity as indicated by the Ki values of 1, 20, and 100 µM, respectively. Finally, dAdo and dGuo also appeared to interact, since dAdo inhibited the dGK activity and dGuo the dAK activity with Ki values of 160 and 400 µM, respectively.
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DISCUSSION |
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The present work was initiated with the purpose to investigate the pattern of TK isoenzymes in D. melanogaster, and to compare it with the situation in other organisms. In the initial purification steps, the TK isoenzymes from mammalian cells, TK1 and TK2, were separated from each other and from dCK by DEAE ion exchange chromatography (14). When the desalted ammonium sulfate fraction from cultured Drosophila S-2 cells (fraction II) was chromatographed on DEAE-Sepharose under conditions identical to those applied for the mammalian enzymes, only one peak of TK and dCK activity was obtained, with the activities coinciding (Fig. 1). This indicated the absence of deoxynucleoside kinases equivalent to the mammalian TK1 and dCK.
The single peak obtained from the DEAE chromatography contained all four deoxynucleoside kinase activities, and these activities were co-purified more than 20,000-fold (Table I) to nearly 99% purity, as judged from SDS-gel electrophoresis (Fig. 2). The activities were not separable during the molecular weight determinations. In the SDS gel, there was a single band with a mass of 30 kDa (Fig. 2), and the four kinase activities migrated together on the Superose 12 column with an apparent size of about 33 kDa (Fig. 3). Furthermore, the dAK, dCK, dGK, and TK activities were all bound to the dTMP-Sepharose matrix, were eluted together in the thymidine buffer, and were bound to Phenyl-Sepharose, where the strong binding required more than 5 mM CHAPS to elute the activities. Together, these results indicate that the four deoxynucleoside kinase activities were associated to the same monomeric protein of about 30 kDa. It is proposed to designate this multifunctional kinase as Dm-dNK.
The occurrence of multifunctional deoxynucleoside kinases with two or three of the four deoxynucleoside kinase activities has been observed previously in mammalian cells. TK2 is also a dCyd kinase (14), and dCK can phosphorylate dAdo and dGuo, although with lower efficiency than dCyd (15). However, a kinase with the ability to phosphorylate all four deoxynucleoside kinase has to our knowledge not been found previously. From Lactobacillus acidophilus R-26, two heterodimeric deoxynucleoside kinases, a dGK/dAK and a dCK/dAK have been isolated (23). The two kinases showed different binding affinities, as dCK/dAK bound to dCTP-Sepharose, whereas dGK/dAK bound to dATP-Sepharose. In Drosophila, a similar arrangement of closely related monomeric proteins with the same size, each specific for one or two of the substrates, seems unlikely, since all deoxynucleoside kinase activities of Dm-dNK were bound to dTMP-Sepharose.
The Km values for the four deoxynucleoside substrates (Table III) were compared with those reported for TK1 from human lymphocytes (24), TK2 (14), and dCK (15) from human leukemic spleen, and dGK from human brain (16). With dCyd and dThd, the Dm-dNK Km values were in the low micromolar range around 1 µM, and comparable to Km for dCyd found for human dCK (1 µM) and for dThd found for the high affinity form of human TK1 (0.5 µM). The Dm-dNK Km values for dAdo and dGuo were about 100- and 650-fold higher than those for dCyd and dThd, respectively. However, the Dm-dNK Km value for dAdo was in the same range as that found for the human dCK, and the Dm-dNK Km value for dGuo was about 5.5-fold higher than the one found with the human dCK, and 86-fold higher than the one found with the bovine dGK. The kinetics was clearly classical Michaelis-Menten, in contrast to the kinetics of mammalian TKs and dCK, which exhibited cooperative reaction mechanism with their deoxynucleoside substrates (14, 15, 24)
The specific activities were high for all four Dm-dNK
activities, about 30,000 units/mg for dCyd and dThd, and about 36,000 for dAdo and dGuo, corresponding to kcat values
of 15 s1 for the TK and dCK activities, and 18-19
s
1 for the dAK and dGK activities. When compared with the
kcat values calculated for the mammalian
enzymes, using the subunit masses, those of Dm-dNK were
manyfold higher, about 4-fold higher compared with TK1 (14), 200-, 60-, and 45-fold higher compared with the dCK, dAK, and dGK activities of
human dCK (15), respectively, and 13,000- and 9000-fold higher compared
with the dGK and dAK activities of bovine dGK (16), respectively.
Likewise, the specificity constants
kcat/Km for the
Dm-dNK ranged from 2.6 × 104
s
1 M
1 to 1.6 × 107 s
1 M
1 (Table
III), and were manyfold higher as compared with the corresponding mammalian activities. The lowest specificity constant of
Dm-dNK was that for dGuo, but it was an order of magnitude
higher than that for human dCK-dGuo (2.7 × 103
s
1 M
1) and bovine dGK-dGuo
(2.8 × 103 s
1
M
1). Regarding these relations and the levels
of specific activities, it is likely that the dAK and dGK activities of
the Dm-dNK play a significant role for the supply of purine
deoxynucleotides for replication and repair of DNA in
Drosophila cells.
The substrate inhibition studies indicated that the four substrates competed with each other (Table IV). dThd was a strong inhibitor of the phosphorylation of dCyd, dAdo, and dGuo with Ki values in the range of Km for dThd. dAdo and dGuo were able to compete with each other with Ki values in the range of their Km values. Furthermore, dAdo and dGuo were relatively efficient inhibitors of the phosphorylation of thymidine. Together, these data indicate that the four substrates are phosphorylated at the same site of the enzyme.
The absence of a TK equivalent to the mammalian S-phase specific TK1 was unexpected, as other DNA replication associated enzymes previously have been isolated from Drosophila embryonic cells (25, 26). Additionally, the absence of a dCK equivalent to the mammalian dCK should be noted. On the other hand, as discussed above, the dCK and TK activities of the Dm-dNK are even more efficient in their phosphorylation capacities than the mammalian kinases.
The specificity toward a number of nucleoside substrates showed interesting properties of the Dm-dNK (Table II). Thus, in addition to the four deoxynucleosides, dUrd and FdUrd were phosphorylated to the same degree as dThd and dCyd and, unexpectedly, both arabinosyl furanosyl pyrimidines were phosphorylated. Such a diverse substrate specificity has to our knowledge not been found for other eucaryotic and bacterial deoxynucleoside kinases.
Dm-dNK was eluted from the DEAE matrix at essentially the same KCl concentration as human TK2 (14), but also in other aspects there were similarities. Like TK2 (14), Dm-dNK was a monomer of about 30 kDa with dCK activity, showed negligible capacity to phosphorylate AZT, was able to phosphorylate AraT (Table II), and could use CTP as a phosphate donor. However, mammalian TK2 is unable to phosphorylate dAdo or dGuo.
With respect to the capacity to phosphorylate the other
arabinofuranosyl nucleoside analog, AraC, the Dm-dNK
resembles human dCK that had a kcat value of
7 × 102 s
1 and a specificity constant
of 3.5 × 103 s
1
M
1 (15). The Dm-dNK, however, was
even more efficient with a 390-fold higher kcat
value (27 s
1) and a 280-fold higher specificity constant
(Table III).
From the properties regarding the native and subunit size, substrate specificity, kinetic constants, and substrate interaction, it is rational to propose that the multifunctional efficient deoxynucleoside kinase from Drosophila S-2 cells is an enzyme with unique properties not hitherto found in any organism. Whether the multifunctional Drosophila deoxynucleoside kinase is present in other insects is currently under investigation.
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ACKNOWLEDGEMENT |
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We gratefully acknowledge the skillful and committed technical assistance of Marianne Lauridsen.
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FOOTNOTES |
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* This work was supported by the Danish Research Council, the NOVO Research Foundation, and the Foundation for Protection of Animals.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed. Tel.: 45-46-74-24-18; Fax: 45-46-74-30-11; E-mail: bmp{at}ruc.dk.
1
The abbreviations used are: TK1, cytoplasmic
specific thymidine kinase; TK2, mitochondrial thymidine kinase; AraC,
1--D-arabinofuranosylcytosine; AraT,
1-
-D-arabinofuranosylthymine; AZT,
3'-azido-2',3'-dideoxythymidine; CHAPS,
3-((3-cholamidopropyl)dimethylammonio)-1-propylsulfonic acid;
dAdo, deoxyadenosine; dAK, deoxyadenosine kinase; dCK,
deoxycytidine kinase; dCyd, deoxycytidine; dGK, deoxyguanosine kinase;
dGuo, deoxyguanosine; Dm-dNK, D. melanogaster
deoxynucleoside kinase; dThd, thymidine; dUrd, deoxyuridine; FddThd,
3'-fluoro-2',3'-dideoxythymidine; FdUrd, 5-fluorodeoxyuridine; TK,
thymidine kinase; CV, coefficient of variation.
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REFERENCES |
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