Regulation and Functional Contribution of Thymidine Kinase 1 in Repair of DNA Damage

Cellular supply of dNTPs is essential in the DNA replication and repair processes. Here we investigated the regulation of thymidine kinase 1 (TK1) in response to DNA damage and found that genotoxic insults in tumor cells cause up-regulation and nuclear localization of TK1. During recovery from DNA damage, TK1 accumulates in p53-null cells due to a lack of mitotic proteolysis as these cells are arrested in the G₂ phase by checkpoint activation. We show that in p53-proficient cells, p21 expression in response to DNA damage prohibits G₁/S progression, resulting in a smaller G₂ fraction and less TK1 accumulation. Thus, the p53 status of tumor cells affects the level of TK1 after DNA damage through differential cell cycle control. Furthermore, it was shown that in HCT-116 p53^−/− cells, TK1 is dispensable for cell proliferation but crucial for dTTP supply during recovery from DNA damage, leading to better survival. Depletion of TK1 decreases the efficiency of DNA repair during recovery from DNA damage and generates more cell death. Altogether, our data suggest that more dTTP synthesis via TK1 take place after genotoxic insults in tumor cells, improving DNA repair during G₂ arrest.     

Cell cycle regulation of thymidine kinase: residues near the carboxyl terminus are essential for the specific degradation of the enzyme at mitosis.

The level of human thymidine kinase (TK) polypeptide is subject to cell cycle regulation. The enzyme is barely detectable in G1 phase but increases 10- to 20-fold by M phase. The low level of human TK in G1 phase is due primarily to the specific degradation of the protein during cell division. Substitution of heterologous promoters, removal of the introns, and deletion of all of the 3' untranslated region from the human TK gene do not affect cell cycle regulation of the enzyme. However, deletion of the carboxyl-terminal 40 amino acids or fusion of beta-galactosidase to the carboxyl terminus of human TK completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle. These alterations do not significantly alter the specific enzymatic activity of TK. Changing the carboxyl terminus or deletion of the last 10 amino acids does not alter cell cycle regulation. These data demonstrate that residues near the carboxyl terminus of TK are essential for the cell cycle phase-specific degradation of the enzyme.     

Mitochondrial thymidine kinase and the enzymatic network regulating thymidine triphosphate pools in cultured human cells

In non-proliferating cells mitochondrial (mt) thymidine kinase (TK2) salvages thymidine derived from the extracellular milieu for the synthesis of mt dTTP. TK2 is a synthetic enzyme in a network of cytosolic and mt proteins with either synthetic or catabolic functions regulating the dTTP pool. In proliferating cultured cells the canonical cytosolic ribonucleotide reductase (R1-R2) is the prominent synthetic enzyme that by de novo synthesis provides most of dTTP for mt DNA replication. In non-proliferating cells p53R2 substitutes for R2. Catabolic enzymes safeguard the size of the dTTP pool: thymidine phosphorylase by degradation of thymidine and deoxyribonucleotidases by degradation of dTMP. Genetic deficiencies in three of the participants in the network, TK2, p53R2, or thymidine phosphorylase, result in severe mt DNA pathologies. Here we demonstrate the interdependence of the different enzymes of the network. We quantify changes in the size and turnover of the dTTP pool after inhibition of TK2 by RNA interference, of p53R2 with hydroxyurea, and of thymidine phosphorylase with 5-bromouracil. In proliferating cells the de novo pathway dominates, supporting large cytosolic and mt dTTP pools, whereas TK2 is dispensable, even in cells lacking the cytosolic thymidine kinase. In non-proliferating cells the small dTTP pools depend on the activities of both R1-p53R2 and TK2. The activity of TK2 is curbed by thymidine phosphorylase, which degrades thymidine in the cytoplasm, thus limiting the availability of thymidine for phosphorylation by TK2 in mitochondria. The dTTP pool shows an exquisite sensitivity to variations of thymidine concentrations at the nanomolar level.     

Mitotic control of dTTP pool: a necessity or coincidence?

The fidelity of DNA replication in eukaryotic cells requires a balanced dNTP supply in the S phase. During the cell cycle progression, the production of dTTP is highly regulated to coordinate with DNA replication. Intracellular thymidine is salvaged to dTTP by cytosolic thymidine kinase (TK1) and thymidylate kinase (TMPK), both of which expression increase in the G1/S transition and diminish in the mitotic phase via proteolytic destruction. Anaphase promoting complex/cyclosome (APC/C)-mediated ubiquitination targets TK1 and TMPK to undergo proteasomal degradation in mitosis, by which dTTP pool is minimized in the early G1 phase of the next cell cycle. In this review, we will focus on regulation of TK1 in the post-S phase and the importance of mitotic proteolysis in controlling dNTP balance, replication stress and genomic stability. Finally, we discuss how thymidine pool and oligomeric forms of TK1 can affect mitotic control of dTTP. This article is for the special issue of IMB 20^th anniversary.     

Cell cycle regulation and novel structural features of thymidine kinase,an essential enzyme in Trypanosoma brucei

Thymidine kinase (TK) is a key enzyme in the pyrimidine salvage pathway which catalyzes the transfer of the γ‐phosphate of ATP to 2′‐deoxythymidine (dThd) forming thymidine monophosphate (dTMP). Unlike other type II TKs, the Trypanosoma brucei enzyme (TbTK) is a tandem protein with two TK homolog domains of which only the C‐terminal one is active. In this study, we establish that TbTK is essential for parasite viability and cell cycle progression, independently of extracellular pyrimidine concentrations. We show that expression of TbTK is cell cycle regulated and that depletion of TbTK leads to strongly diminished dTTP pools and DNA damage indicating intracellular dThd to be an essential intermediate metabolite for the synthesis of thymine‐derived nucleotides. In addition, we report the X‐ray structure of the catalytically active domain of TbTK in complex with dThd and dTMP at resolutions up to 2.2 Å. In spite of the high conservation of the active site residues, the structures reveal a widened active site cavity near the nucleobase moiety compared to the human enzyme. Our findings strongly support TbTK as a crucial enzyme in dTTP homeostasis and identify structural differences within the active site that could be exploited in the process of rational drug design.     

The contribution of mitochondrial thymidylate synthesis in preventing the nuclear genome stress

In quiescent fibroblasts, the expression levels of cytosolic enzymes for thymidine triphosphate (dTTP) synthesis are down-regulated, causing a marked reduction in the dTTP pool. In this study, we provide evidence that mitochondrial thymidylate synthesis via thymidine kinase 2 (TK2) is a limiting factor for the repair of ultraviolet (UV) damage in the nuclear compartment in quiescent fibroblasts. We found that TK2 deficiency causes secondary DNA double-strand breaks formation in the nuclear genome of quiescent cells at the late stage of recovery from UV damage. Despite slower repair of quiescent fibroblast deficient in TK2, DNA damage signals eventually disappeared, and these cells were capable of re-entering the S phase after serum stimulation. However, these cells displayed severe genome stress as revealed by the dramatic increase in 53BP1 nuclear body in the G1 phase of the successive cell cycle. Here, we conclude that mitochondrial thymidylate synthesis via TK2 plays a role in facilitating the quality repair of UV damage for the maintenance of genome integrity in the cells that are temporarily arrested in the quiescent state.     

Thymidine Kinase 1 Deficient Cells Show Increased Survival Rate After UV-Induced DNA Damage

Balanced deoxynucleotide pools are known to be important for correct DNA repair, and deficiency for some of the central enzymes in deoxynucleotide metabolism can cause imbalanced pools, which in turn can lead to mutagenesis and cell death. Here we show that cells deficient for the thymidine salvage enzyme thymidine kinase 1 (TK1) are more resistant to UV-induced DNA damage than TK1 positive cells although they have thymidine triphosphate (dTTP) levels of only half the size of control cells. Our results suggest that higher thymidine levels in the TK- cells caused by defect thymidine salvage to dTTP protects against UV irradiation.     

Transfer of a mutant viral thymidine kinase gene results in temperature-sensitive mouse cells.

Temperature-sensitive cell lines were obtained by DNA-mediated transfer of the thymidine kinase (TK) gene from a mutant, ts1117, of herpes simplex virus type 1. The cells died at 39 degrees C in selective medium which contained low levels (1 microgram/ml) of thymidine. In this lethal condition, no revertants were detected among 10(8) cells. It was shown by in vitro analysis of the TK activity that the temperature-sensitive cell line contains an enzyme whose activity is temperature sensitive and relatively unaffected by dTTP. The viral enzyme has these properties. The effect of the lethal growth conditions in the cell line was characterized by cell cycle analysis and rescue experiments which involved a shift to the permissive conditions. The successful transfer of the mutant viral TK activity to cells provides an additional selective marker for gene transfer.     

Purification and Characterization of Wild-Type and Mutant Tk1 Type Kinases From Caenorhabditis Elegans

Caenorhabditis elegans has a single deoxynucleoside kinase-like gene. The sequence is similar to that of human TK1, but besides accepting thymidine as a substrate, the C. elegans TK1 (CeTK1) also phosphorylates deoxyguanosine. In contrast to human TK1, the CeTK1 exclusively exists as a dimer with a molecular mass of ～60 kDa, even if incubated with ATP. Incubation with ATP induces a transition into a more active enzyme with a higher k_cat but unchanged K_m. This activation only occurs at an enzyme concentration in the incubation buffer of 0.5 μg/ml (8.42 nM) or higher. C-terminal deletion of the enzyme results in lower catalytic efficiency and stability.     

X-irradiation effects on thymidine kinase (TK): II. The significance of deoxythymidine triphosphate for inhibition of TK1 activity

Abstract. The purpose of this study was to investigate the mechanism behind the high sensitivity of thymidine kinase 1 (TK1) to X-irradiation. The deoxythymidine triphosphate (dTTP) pool was studied in mouse ascites tumour cells 1–24 h after X-irradiation with 5 Gy. Irradiation changed the Michaelis-Menten kinetics of TK1 from linear to biphasic, showing a negative co-operativity. These changes were closely related to changes in the dTTP pool. Addition of dTTP to the cell extract of non-irradiated cells, or thymidine (dTdR) to the culture medium, resulted in changes very similar to the kinetics found in the irradiated cells. Addition of 5¢-amino-5¢-deoxythymidine (5¢-AdTdR), a thymidine analogue that eliminated the inhibitory effect of dTTP on TK1 activity, completely abolished the irradiation-induced inhibition of TK1 activity. We suggest that the reduced TK1 activity is mainly due to an elevated intracellular concentration of dTTP.     

Studies of intracellular thymidine nucleotides. Relationship between the synthesis of deoxyribonucleic acid and the thymidine triphosphate pool in Escherichia coli K12.

The two types of mutant strains which show resistance to T-even phage infection have been isolated and been shown to have either a higher or lower ratio of dTDP-sugar to dTTP than that of the parent strains. The one with a higher ratio of dTDP-sugar to dTTP than the parents has a large dTDP-sugar pool and small dTTP pool, and a high level of dTDPG pyrophosphorylase activity. The other one, with a lower ratio of dTDP-sugar to dTTP than the parents, has a small dTDP-sugar pool and large dTTP pool, and a low or deficient level of this enzyme activity. They form an entirely mucoid colony in the synthetic agar plate. Mutant cells (Ter-6 and Ter-21) which have deficient dTDPG pyrophosphorylase activity show 2 -- 3 times higher activity of UDPG pyrophosphoyrlase than that of parent cells. The dTDPG pyrophosphorylase-deficient mutants (Ter-15 and Ter-21) have a 3 -- 4 times higher concentration of dTTP and a faster rate of DNA synthesis and cell division than those of parent strains in growth with external thymine. The dTDPG pyrophosphorylase constitutive mutant (Ter-4) has a 0.5 -- 0.33 smaller dTTP pool and a slower rate of DNA synthesis and cell division than those of parent cells grown in the same medium. In the Ter-15 and Ter-21 mutants, the intracellular dTTP-dependent DNA synthesis rapidly disappeared in thymine suboptimal concentration, but the Ter-4 mutant maintained its dTTP-dependent DNA synthesis over a 20 muM concentration of external thymine. In high concentration (100 muM) of external thymidine, the thymidine effects on the intracellular dTTP concentration do not significantly appear in these enzyme-deficient mutants (Ter-15 and Ter-21). Also, the concentration of intracellular dTTP in the cell growth with external thymidine is 2.5 times greater than that with external thymine in these enzyme-deficient mutants (Ter-15 and Ter-21).     

DNA polymerase activity in phytohemagglutinin-stimulated and non-stimulated human lymphocytes

Requirements and optimal conditions have been studied for the activity of DNA polymerase from phytohemagglutinin-stimulated and non-stimulated human lymphocytes. Differences were found in thermal stability and inhibitory effect of KC1 and p-chloromercuribenzoate. The relationship was determined between DNA polymerase activity, cellular pools of dATP, dTTP and incorporation of deoxythymidine into DNA during transformation. The increase in polymerase activity was paralleled by a similar increase in the pools of dATP and dTTP. The enzyme activity and the pool sizes of both nucleotides reached a maximum simultaneously with the peak of deoxythymidine incorporation into DNA. Studies in which protein synthesis was limited by cycloheximide showed that both the DNA polymerase activity and the rise in the pool sizes of both nucleotides were abolished. This implies that the de novo synthesis is required for the enzymes involved.     

Cell cycle related studies on thymidine kinase and its isoenzymes in Ehrlich ascites tumours

Thymidine kinase (TK) activity was measured in relation to the cell cycle of in vivo growing ascites tumour cells. The cells were synchronized by means of centrifugal elutriation and the cell cycle composition of the cell fractions was determined by flow cytometry. TK activity was low in G1, increased during S phase and declined in G2. A half-life of TK activity of about 45 min was found throughout the cell cycle. Four isoenzymes at pI values of 4.1, 5.3, 6.9 and 8.3, denoted as isoenzymes 1-4, were identified using isoelectric focusing. Isoenzymes 3 and 4 were responsible for the profound cell cycle related changes in the TK activity. Corresponding isoenzymes were also found in the fetal mouse liver. In the adult mouse liver isoenzyme 2 was the dominating isoenzyme. The half-life of the isoenzymes was in the same range as for the total TK activity. We conclude that the low TK activity in G1 is due to degradation of the enzyme in G2 at a normal rate combined with an arrest in the synthesis of TK. We also conclude that isoenzyme 4 and the intermediate isoenzyme 3, which had earlier been suggested to be a mitochondrial form of TK, in fact represent cytoplasmatic forms of TK. According to cell cycle and pI studies, isoenzyme 2 belongs to the mitochondrial form. Studies with various phosphor donors and specific substrates, however, indicate that it also contains a cytoplasmic component.     

Transgene Expression of Drosophila melanogaster Nucleoside Kinase Reverses Mitochondrial Thymidine Kinase 2 Deficiency

A strategy to reverse the symptoms of thymidine kinase 2 (TK2) deficiency in a mouse model was investigated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in TK2-deficient mice that have been shown to present with a severe phenotype caused by mitochondrial DNA depletion. The Dm-dNK^+/− transgenic mice were shown to be able to rescue the TK2-deficient mice. The Dm-dNK^+/−TK2^−/− mice were normal as judged by growth and behavior during the observation time of 6 months. The Dm-dNK-expressing mice showed a substantial increase in thymidine-phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause specific mitochondrial DNA mutations or deletions when 6-month-old mice were analyzed. The mitochondrial DNA was also detected at normal levels. In conclusion, the Dm-dNK^+/−TK2^−/− mouse model illustrates how dTMP synthesized in the cell nucleus can compensate for loss of intramitochondrial dTMP synthesis in differentiated tissue. The data presented open new possibilities to treat the severe symptoms of TK2 deficiency.     

Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour

Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [3H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined. Four isoenzymes at pI 4.1, 5.3, 6.9 and 8.3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pI 8.3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other. All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.     

SEMIQUANTITATIVE CELL CYCLE MODEL FOR SLOW GROWING ESCHERICHIA COLI

A cell cycle model for Escherichia coli is discussed. It is assumed that the DNA synthesis starts when a threshold dTTP concentration is reached, that the dTTP synthesis is subject to product inhibition and that the duration of DNA replication is given by the time necessary to synthesize the required quantity of dTTP. The model is compared with data for slow growth rates. The constancy of cell volume and of the quotient of presynthetic period per generation cycle is reproduced by the model. The calculated upper limit for the cell volume and lower limit for the quotient are about ten times higher and lower respectively than the real values.     

Lack of correlation between thymidine kinase activity and changes of DNA synthesis with tumour age: an in vivo study in Ehrlich ascites tumour

Abstract. Thymidine kinase (TK) and its isoenzymes were studied in relation to age of Ehrlich ascites tumour cells growing in vivo. Various steps of the pathway of thymidine through deoxynucleotide metabolism were studied: [³H]-thymidine cellular uptake and incorporation into DNA; the cellular nucleotide pools; and the concentration of thymidine in ascites. In addition, the proportion of cells in the various parts of the cell cycle and the bromodeoxyuridine labelling index were determined.
Four isoenzymes at pi 41, 5-3, 6–9 and 8-3 were identified using isoelectric focusing. The TK activity declined with age of the tumour by about 90%, mostly due to a decrease of the isoenzyme at pi 8-3. However, this decline was neither related to the changes in DNA synthesis rate of the cells with tumour age, nor to the proportion of cells in S-phase or the bromodeoxyuridine (BrdU) labelling index. In contrast, the contribution of DNA synthesis via the thymidine salvage pathway relative to the total DNA synthesis increased from less than 1% at exponential growth to about 15% at plateau phase of growth. Blocking of DNA synthesis by aphidicolin did not change the TK activity. We therefore conclude that changes in TK activity and changes in cell growth are epiphenomena rather than causally related to each other.
All nucleotide pools decreased with tumour age. The inhibition of TK by an increase in the deoxythymidine triphosphate pool could therefore be excluded. With a decrease of the TK activity during tumour growth, increasing amounts of TdR were excreted by the cells and accumulated in the ascites fluid. To explain our results on TK activity we propose a substrate cycle in which thymidine monophosphate supplied by de novo synthesis is dephosphorylated and is then either phosphorylated by TK to thymidine monophosphate or excreted by the cell.     

Cell cycle related [3H]thymidine uptake and its significance for the incorporation into DNA

Cellular uptake of [3H]thymidine [( 3H]TdR) and incorporation into DNA of Ehrlich ascites tumour cells were studied in relation to the cell cycle by measuring the activity in the acid-soluble and insoluble parts of the cell material. Cells were synchronized at various stages of the cell cycle using centrifugal elutriation. The degree of synchrony of the various cell fractions was measured by flow-cytofluorometric DNA analysis. From the cellular uptake, the TdR triphosphate (dTTP) concentration of a mean cell in an unseparated cell population was calculated to be 20 X 10(-18) mol/cell. The pool activity of G1 cells was unmeasurable but rose to maximum values at the border of the G1-S phase. It decreased again during G2. The [3H]TdR incorporation into DNA was low during early S phase, reached a maximum value at two-thirds of the S phase and decreased again during late S phase. These changes in DNA synthesis were not due to changes in the dTTP pool being a limiting factor. During maximum DNA synthesis, 10% X min-1 of the dTTP pool was utilized, at which time the pool size also decreased by about 30%. Changes in pool size during the cell cycle have to be taken into account when the results of incorporation of radioactive TdR into DNA are discussed.     

Characterization of an Epstein-Barr virus-induced thymidine kinase.

Previous work from our laboratory suggested that the selective inhibition of Epstein-Barr virus (EBV) replication by 1-beta-D-arabinofuranosylthymine in human lymphoid cell lines involved the induction of a new thymidine kinase (TK) able to phosphorylate the thymidine analog. We further characterized this enzyme induced in various EBV-positive cell lines after viral genome activation with a combination of sodium butyrate and 12-O-tetradecanoylphorbol-13-acetate. The following results confirmed the existence of an EBV-specific deoxypyrimidine kinase: induction of EBV-related TK was connected with the appearance of viral early antigens in EBV-carrying cells; unexpected behaviors of the enzyme activity upon different fractionating treatments led to the conclusion that EBV-induced TK was extracted as a complex molecular form, larger than other known cellular or viral isozymes; enzymatic properties distinguished EBV-induced TK from host lymphoid cell isozymes but made it resemble other herpesvirus-specific deoxypyrimidine kinases, i.e., by partial inhibition by dTTP or ammonium sulfate, insensitiveness to dCTP, and nonstringent specificity for normal TK substrates. Genetic evidence is required to definitively ensure that EBV-specific TK actually is virus coded in EBV-transformed human lymphoid cells.