Structure-function analysis of a bacterial deoxyadenosine kinase reveals the basis for substrate specificity

Deoxyribonucleoside kinases (dNKs) catalyze the transfer of a phosphoryl group from ATP to a deoxyribonucleoside (dN), a key step in DNA precursor synthesis. Recently structural information concerning dNKs has been obtained, but no structure of a bacterial dCK/dGK enzyme is known. Here we report the structure of such an enzyme, represented by deoxyadenosine kinase from Mycoplasma mycoides subsp. mycoides small colony type (Mm-dAK). Superposition of Mm-dAK with its human counterpart's deoxyguanosine kinase (dGK) and deoxycytidine kinase (dCK) reveals that the overall structures are very similar with a few amino acid alterations in the proximity of the active site. To investigate the substrate specificity, Mm-dAK has been crystallized in complex with dATP and dCTP, as well as the products dCMP and dCDP. Both dATP and dCTP bind to the enzyme in a feedback-inhibitory manner with the dN part in the deoxyribonucleoside binding site and the triphosphates in the P-loop. Substrate specificity studies with clinically important nucleoside analogs as well as several phosphate donors were performed. Thus, in this study we combine structural and kinetic data to gain a better understanding of the substrate specificity of the dCK/dGK family of enzymes. The structure of Mm-dAK provides a starting point for making new anti bacterial agents against pathogenic bacteria.     

Non-homologous recombination of deoxyribonucleoside kinases from human and Drosophila melanogaster yields human-like enzymes with novel activities

In antiviral and cancer therapy, deoxyribonucleoside kinases (dNKs) are often the rate-limiting step in activating nucleoside analog (NA) prodrugs into their cytotoxic, phosphorylated forms. We have constructed libraries of hybrid enzymes by non-homologous recombination of the pyrimidine-specific human thymidine kinase 2 and the broad-specificity dNK from Drosophila melanogaster; their low sequence identity has precluded engineering by conventional, homology-dependent shuffling techniques. From these libraries, we identified chimeras that phosphorylate nucleoside analogs with higher activity than either parental enzyme, and that possess new activity towards the anti-HIV prodrug 2',3'-didehydro-3'-deoxythymidine (d4T). These results demonstrate the potential of non-homologous recombination within the dNK family for creating enzymes with new and improved activities towards nucleoside analogs. In addition, our results exposed a previously unknown role for the C-terminal regions of these dNKs in determining substrate selectivity.     

Deoxyribonucleoside kinases belonging to the thymidine kinase 2 (TK2)-like group vary significantly in substrate specificity, kinetics and feed-back regulation.

In eukaryotic cells deoxyribonucleoside kinases belonging to three phylogenetic sub-families have been found: (i) thymidine kinase 1 (TK1)-like enzymes, which are strictly pyrimidine deoxyribonucleoside-specific kinases; (ii) TK2-like enzymes, which include pyrimidine deoxyribonucleoside kinases and a single multisubstrate kinase from Drosophila melanogaster (Dm-dNK); and (iii) deoxycytidine/deoxyguanosine kinase (dCK/dGK)-like enzymes, which are deoxycytidine and/or purine deoxyribonucleoside-specific kinases. We cloned and characterized two new deoxyribonucleoside kinases belonging to the TK2-like group from the insect Bombyx mori and the amphibian Xenopus laevis. The deoxyribonucleoside kinase from B. mori (Bm-dNK) turned out to be a multisubstrate kinase like Dm-dNK. But uniquely for a deoxyribonucleoside kinase, Bm-dNK displayed positive cooperativity with all four natural deoxyribonucleoside substrates. The deoxyribonucleoside kinase from X. laevis (Xen-PyK) resembled closely the human and mouse TK2 enzymes displaying their characteristic Michaelis-Menten kinetic with deoxycytidine and negative cooperativity with its second natural substrate thymidine. Bm-dNK, Dm-dNK and Xen-PyK were shown to be homodimers. Significant differences in the feedback inhibition by deoxyribonucleoside triphosphates between these three enzymes were found. The insect multisubstrate deoxyribonucleoside kinases Bm-dNK and Dm-dNK were only inhibited by thymidine triphosphate, while Xen-PyK was inhibited by thymidine and deoxycytidine triphosphate in a complex pattern depending on the deoxyribonucleoside substrate. The broad substrate specificity and different feedback regulation of the multisubstrate insect deoxyribonucleoside kinases may indicate that these enzymes have a different functional role than the other members of the TK2-like group.     

Depletion of mitochondrial DNA by down-regulation of deoxyguanosine kinase expression in non-proliferating HeLa cells

Purine deoxyribonucleotides required for mitochondrial DNA replication are either imported from the cytosol or derived from phosphorylation of deoxyadenosine or deoxyguanosine catalyzed by mitochondrial deoxyguanosine kinase (DGUOK). DGUOK deficiency has been linked to mitochondrial DNA depletion syndromes suggesting an important role for this enzyme in dNTP supply. We have generated HeLa cell lines with 20-30% decreased levels of DGUOK mRNA by the expression of small interfering RNAs directed towards the DGUOK mRNA. The cells with decreased expression of the enzyme showed similar levels of mtDNA as control cells when grown exponentially in culture. However, mtDNA levels rapidly decreased in the cells when cell cycle arrest was induced by serum starvation. DNA incorporation of 9-beta-d-arabino-furanosylguanine (araG) was lower in the cells with decreased deoxyguanosine kinase expression, but the total rate of araG phosphorylation was increased in the cells. The increase in araG phosphorylation was shown to be due to increased expression of deoxycytidine kinase. In summary, our findings show that DGUOK is required for mitochondrial DNA replication in resting cells and that small changes in expression of this enzyme may cause mitochondrial DNA depletion. Our data also suggest that alterations in the expression level of DGUOK may induce compensatory changes in the expression of other nucleoside kinases.     

The global distribution and evolution of deoxyribonucleoside kinases in bacteria

Deoxyribonucleoside kinases (dNKs) are important to DNA metabolism, especially in environments where nucleosides are freely available to be absorbed and used for the salvage pathway. Little has previously been known about the complement of dNKs in different bacterial genomes. However, it was believed that Gram-negative bacteria had a single dNK, while Gram-positive bacteria possessed several. An analysis of 992 fully sequenced bacterial genomes, including both Gram-positive and Gram-negative organisms, was conducted to investigate the phylogenetic relationship of all TK1-like and non-TK1-like dNKs. It was illustrated that both gene families evolved through a number of duplications and horizontal gene transfers, leading to the presence of multiple dNKs in different types of bacteria. The findings of this study provide a backbone for further studies into the evolution of the interplay between the de novo and salvage pathways in DNA synthesis with respect to environmental availability of deoxyribonucleosides and metabolic processes generating the provisions of different dNTPs.     

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.     

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.     

Identification of purine deoxyribonucleoside kinases from human leukemia cells: substrate activation by purine and pyrimidine deoxyribonucleosides

Cell extracts from human leukemic T lymphoblasts and myeloblasts were chromatographed on DEAE-cellulose columns to separate purine deoxyribonucleoside, deoxyadenosine (dAdo) and deoxyguanosine (dGuo), phosphorylating activities. Three distinct purine deoxyribonucleoside kinases, a deoxycytidine (dCyd) kinase, an adenosine (Ado) kinase, and a deoxyguanosine (dGuo) kinase (the latter appears to be localized in mitochondria), were resolved. dCyd kinase contained the major phosphorylating activity for dAdo, dGuo, and 9-beta-D-arabinofuranosyladenine (ara-A). Ado kinase represented a second kinase for dAdo and ara-A while a third kinase for dAdo was found in mitochondria. dCyd kinase was purified about 2000-fold with ion-exchange, affinity, and hydrophobic chromatographies. On gel electrophoresis, both dCyd and dAdo phosphorylating activities comigrated, indicating that the activities are associated with the same protein. The enzyme showed a broad pH optimum ranging from pH 6.5 to pH 9.5. Divalent cations Mg2+, Mn2+, and Ca2+ stimulated dCyd kinase activity; Mg2+ produced the maximal activity. dCyd kinase from either lymphoid or myeloid cells showed broad substrate specificity. The enzyme used several nucleoside triphosphates, but ATP, GTP, and dTTP were the best phosphate donors. dCyd was the best nucleoside substrate, since dCyd kinase had an apparent Km of 0.3, 85, 90, and 1400 microM for dCyd, dAdo, dGuo, and ara-A, respectively. The enzyme exhibited substrate activation with both pyrimidine and purine deoxyribonucleosides, suggesting that there is more than one substrate binding site on the kinase. These studies show that, in lymphoblasts and myeloblasts, purine deoxyribonucleosides and their analogues are phosphorylated by dCyd kinase, Ado kinase, and dGuo kinase.     

Acute cytotoxicity of arabinofuranosyl nucleoside analogs is not dependent on mitochondrial DNA

The nucleoside analogs 9-β-D-arabinofuranosylguanine (araG) and 1-β-d-arabinofuranosylthymine (araT) are substrates of mitochondrial nucleoside kinases and have previously been shown to be predominantly incorporated into mtDNA of cells, but the pharmacological importance of their accumulation in mtDNA is not known. Here, we examined the role of mtDNA in the response to araG, araT and other anti-cancer and anti-viral agents in a MOLT-4 wild-type (wt) T-lymphoblastoid cell line and its petite mutant MOLT-4 ρ⁰ cells (lacking mtDNA). The mRNA levels and activities of deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), thymidine kinase 1 (TK1) and thymidine kinase 2 (TK2) were determined in the two cell lines. Compared to that in the MOLT-4 wt cells the mRNA level of the constitutively expressed TK2 was higher (p < 0.01) in the ρ⁰ cells, whereas the TK1 mRNA level was lower (p < 0.05). The enzyme activity of the S-phase restricted TK1 was also lower (p < 0.05) in the MOLT-4 ρ⁰ cells, whereas the activities of dGK, dCK and TK2 were similar in MOLT-4 wt and ρ⁰ cell lines. The sensitivities to different cytotoxic nucleoside analogs were determined and compared between the two cell lines. Interestingly, we found that the acute cytotoxicity of araG, araT and other anti-viral and anti-cancer agents is independent of the presence of mtDNA in MOLT-4 T-lymphoblastoid cells.     

Mitochondrial Thymidine Kinase 2 but Not Deoxyguanosine Kinase Is Up-Regulated During the Stationary Growth Phase of Cultured Cells

Mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) catalyze the initial phosphorylation of pyrimidine and purine deoxyribonucleosides, and are essential for maintaining mitochondrial dNTP pools for mitochondrial DNA replication. Here the expression of mitochondrial TK2 and dGK in relation to cell growth phases in cultured cells was investigated. TK2 and dGK protein levels in isolated mitochondria and TK2 activity in total cell extracts from U2OS and TK1 deficient L929 cells were determined. We found that TK2 levels were negatively correlated with cell growth rates and there was an exponential increase in TK2 levels in cells entering stationary phase. The expression of dGK did not change and appeared to be constitutive.     

Biosynthesis reaction mechanism and kinetics of deoxynucleoside triphosphates, dATP and dGTP

The enzyme reaction mechanism and kinetics for biosyntheses of deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) from the corresponding deoxyadenosine diphosphate (dADP) and deoxyguanosine diphosphate (dGDP) catalyzed by pyruvate kinase were studied. A kinetic model for this synthetic reaction was developed based on a Bi-Bi random rapid equilibrium mechanism. Kinetic constants involved in this pyruvate kinase catalyzed phosphorylation reactions of deoxynucleoside diphosphates including the maximum reaction velocity, Michaelis-Menten constants, and inhibition constants for dATP and dGTP biosyntheses were experimentally determined. These kinetic constants for dATP and dGTP biosyntheses are of the same order of magnitude but significantly different between the two reactions. Kinetic constants involved in ATP and GTP biosyntheses as reported in literature are about one order of magnitude different from those involved in dATP and dGTP biosyntheses. This enzyme reaction requires Mg2+ ion and the optimal Mg2+ concentration was also determined. The experimental results showed a very good agreement with the simulation results obtained from the kinetic model developed. This kinetic model can be applied to the practical application of a pyruvate kinase reaction system for production of dATP and dGTP. There is a significant advantage of using enzymatic biosyntheses of dATP and dGTP as compared to the chemical method that has been in commercial use.     

Giardia intestinalis thymidine kinase is a high-affinity enzyme crucial for DNA synthesis and an exploitable target for drug discovery

Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogs. Here, we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes deoxy-UMP a dead-end product that is potentially harmful if converted to deoxy-UTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against WT as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis–infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.     

Potato tuber isoapyrases: substrate specificity, affinity labeling, and proteolytic susceptibility

Apyrase/ATP-diphosphohydrolase hydrolyzes di- and triphosphorylated nucleosides in the presence of a bivalent ion with sequential release of orthophosphate. We performed studies of substrate specificity on homogeneous isoapyrases from two potato tuber clonal varieties: Desiree (low ATPase/ADPase ratio) and Pimpernel (high ATPase/ADPase ratio) by measuring the kinetic parameters K(m) and k(cat) on deoxyribonucleotides and fluorescent analogues of ATP and ADP. Both isoapyrases showed a broad specificity towards dATP, dGTP, dTTP, dCTP, thio-dATP, fluorescent nucleotides (MANT-; TNP-; ethene-derivatives of ATP and ADP). The hydrolytic activity on the triphosphorylated compounds was always higher for the Pimpernel apyrase. Modifications either on the base or the ribose moieties did not increase K(m) values, suggesting that the introduction of large groups (MANT- and TNP-) in the ribose does not produce steric hindrance on substrate binding. However, the presence of these bulky groups caused, in general, a reduction in k(cat), indicating an important effect on the catalytic step. Substantial differences were observed between potato apyrases and enzymes from various animal tissues, concerning affinity labeling with azido-nucleotides and FSBA (5'-p-fluorosulfonylbenzoyl adenosine). PLP-nucleotide derivatives were unable to produce inactivation of potato apyrase. The lack of sensitivity of both potato enzymes towards these nucleotide analogues rules out the proximity or adequate orientation of sulfhydryl, hydroxyl or amino-groups to the modifying groups. Both apyrases were different in the proteolytic susceptibility towards trypsin, chymotrypsin and Glu-C.     

High-level expression and purification of human thymidine kinase 1: quaternary structure, stability, and kinetics

Human cytosolic thymidine kinase (hTK1) is the key enzyme of the pyrimidine salvage pathway and phosphorylates thymidine to thymidine monophosphate, a precursor building block of the DNA. Wild-type hTK1 (hTK1W) as well as a truncated form of the enzyme (hTK1M) carrying deletions at the N- and C-terminal regions were cloned as His(6)-tagged fusion proteins. Expression, isolation, and purification protocols have been established, leading to high yields of soluble and active wild type (approximately 35 mg) and truncated hTK1 (approximately 23 mg) per liter of culture. The protein was purified to near homogeneity. The chaperone DnaK was identified to be the major contaminant that could be removed by applying an additional ATP-MgCl(2) incubation and washing step. hTK1W was a permanent tetramer in solution, whereas the truncated construct hTK1M appears to be a dimer in absence and presence of substrates. Both hTK1W and hTK1M exhibit pronounced thermal stability with transition temperatures (T(m)) of 71.7 and 73.4 degrees C, respectively, when measured without adding substrates. The presence of substrates stabilized both hTK1W (DeltaT(m) ranging from 5.6 to 12.5 degrees C) and hTK1M (DeltaT(m) ranging from 0.8 to 5.3 degrees C). Both enzymes show high activity over a broad range of pH, temperature, and ionic strength. Kinetic studies determined a K(M) of 0.51 microM and a k(cat) of 0.28 s(-1) for wild-type hTK1. The truncated hTK1M has a K(M) of 0.87 microM and k(cat) of 1.65 s(-1), thus exhibiting increased catalytic efficiency. The availability of recombinant human TK1 will facilitate further biochemical and crystallographic studies.     

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.     

Structure of a type II thymidine kinase with bound dTTP

The structure of human cytosolic thymidine kinase in complex with its feedback inhibitor 2'-deoxythymidine-5'-triphosphate was determined. This structure is the first representative of the type II thymidine kinases found in several pathogens. The structure deviates strongly from the known structures of type I thymidine kinases such as the Herpes simplex enzyme. It contains a zinc-binding domain with four cysteines complexing a structural zinc ion. Interestingly, the backbone atoms of the type II enzyme bind thymine via hydrogen-bonds, in contrast to type I, where side chains are involved. This results in a specificity difference exploited for antiviral therapy. The presented structure will foster the development of new drugs and prodrugs for numerous therapeutic applications.     

Molecular Mechanisms of Mitochondrial DNA Depletion Diseases Caused by Deficiencies in Enzymes in Purine and Pyrimidine Metabolism

Mitochondrial DNA depletion syndrome (MDS), a reduction of mitochondrial DNA copy number, often affects muscle or liver. Mutations in enzymes of deoxyribonucleotide metabolism give MDS, for example, the mitochondrial thymidine kinase 2 (TK2) and deoxyguanosine kinase (dGK) genes. Sixteen TK2 and 22 dGK alterations are known. Their characteristics and symptoms are described. Levels of five key deoxynucleotide metabolizing enzymes in mouse tissues were measured. TK2 and dGK levels in muscles were 5- to 10-fold lower than other nonproliferating tissues and 100-fold lower compared to spleen. Each type of tissue apparently relies on de novo and salvage synthesis of DNA precursors to varying degrees.     

Binding of ATP to TK1-like enzymes is associated with a conformational change in the quaternary structure

Human thymidine kinase 1 (hTK1) and structurally related TKs from other organisms catalyze the initial phosphorylation step in the thymidine salvage pathway. Though ATP is known to be the preferred phosphoryl donor for TK1-like enzymes, its exact binding mode and effect on the oligomeric state has not been analyzed. Here we report the structures of hTK1 and of the Thermotoga maritima thymidine kinase (TmTK) in complex with the bisubstrate inhibitor TP4A. The TmTK-TP4A structure reveals that the adenosine moiety of ATP binds at the subunit interface of the homotetrameric enzyme and that the majority of the ATP-enzyme interactions occur between the phosphate groups and the P-loop. In the hTK1 structure the adenosine group of TP4A exhibited no electron density. This difference between hTK1 and TmTK is rationalized by a difference in the conformation of their quaternary structure. A more open conformation, as seen in the TmTK-TP4A complex structure, is required to provide space for the adenosine moiety. Our analysis supports the formation of an analogous open conformation in hTK1 upon ATP binding.     

Active site mutants of Drosophila melanogaster multisubstrate deoxyribonucleoside kinase.

The multisubstrate deoxyribonucleoside kinase of Drosophila melanogaster (Dm-dNK) is sequence-related to three human deoxyribonucleoside kinases and to herpes simplex virus type-1 thymidine kinase. Dm-dNK phosphorylates both purine and pyrimidine deoxyribonucleosides and nucleoside analogues although it has a preference for pyrimidine nucleosides. We performed site-directed mutagenesis on residues that, based on structural data, are involved in substrate recognition. The aim was to increase the phosphorylation efficiency of purine nucleoside substrates to create an improved enzyme to be used in suicide gene therapy. A Q81N mutation showed a relative increase in deoxyguanosine phosphorylation compared with the wild-type enzyme although the efficiency of deoxythymidine phosphorylation was 10-fold lower for the mutant. In addition to residue Q81 the function of amino acids N28, I29 and F114 was investigated by different substitutions. All of the mutated enzymes showed decreased efficiency of thymidine phosphorylation in comparison with the wild-type enzyme supporting their importance for substrate binding and/or catalysis as proposed by the recently solved structure of Dm-dNK.     

Modification of radiation-induced genetic damage in Drosophila melanogaster male germ cells with nucleic acid precursors II. Effects on post-meiotic cells

Following the observation that the nucleoside pre-treatment reduced the radiation-induced dominant lethality in the post-meiotic germ cells, similar experiments were conducted using the same treatment conditions to study the influence of the nucleoside(s) pre-treatment on the radiation-induced (1.2 kR) incidence of sex-linked recessive lethals and translocation events in the post-meiotic male germ cells of 1-day-old D. melanogaster. The nucleoside pre-treatment reduced the translocation frequency (not statistically significant) and the lethal mutation frequency (statistically significant) in the post-meiotic cells (pre-injection DNA synthesis cells) especially in the mature sperms sampled in brood a (br a). The radio-protective effect of the nucleosides on the mature sperms was confirmed using 7-day-old virgin males and different radiation doses (2.4 kR and 3.6 kR).The frequency of lethal mutation was lowest when irradiation was preceded by the injection of an equimolar solution of thymidine (TdR), deoxyadenosine (AdR), deoxycytidine (CdR) and deoxyguanosine (GdR). However, when the nucleosides were injected after irradiation (within 10–30 min) there was no change in the yield of radiation-induced lethals.The possible mechanisms for the radioprotective action of the nucleosides in the post-meiotic germ cells such as (a) “protection” by a radiochemical action of nucleosides competing for short-lived radicals that might otherwise cause damage to DNA and (b) biochemical-physiological mechanisms such as metabolic events increasing the radioresistance of the cells, providing excess energy for repair or favoring and partaking in the DNA repair synthesis were discussed. Further studies were felt necessary to elucidate this phenomenon.