- Mechanistic studies of the inhibition of MutT dGTPase by the carcinogenic metal Ni(II)
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Promutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-exo-dG) levels are increased in DNA of animals exposed to carcinogenic metals, such as Ni(II). Besides being generated directly in genomic DNA, 8-oxo-dG may be incorporated there from 8-oxo-7, 8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-oxo- dGTP), a product of oxidative damage to the nucleotide pool. The Escherichia coli dGTPase MutT, and analogous dGTPases in rats and humans, have been suggested as a defense against such incorporation because they hydrolyze 8- oxo-dGTP to 8-oxo-7, 8-dihydro-2'-deoxyguanosine 5'-monophosphate (8-oxo- dGMP). MutT and its mammalian counterparts are Mg(II)-dependent enzymes. Ni(II), in turn, is known to interact antagonistically with Mg(II) in biological systems. Thus, we hypothesized that Ni(II) might inhibit the activity of MutT. As an initial examination of this hypothesis, we conducted enzyme kinetic studies of MutT to determine the effect of Ni(II) on MutT activity and the mechanisms involved. As found, Ni(II) inhibited Mutt in a concentration-dependent manner when either dGTP or 8-oxo-dGTP was the nucleotide substrate. Ni(II) was determined to be an uncompetitive inhibitor of MutT with respect to Mg(II) when dGTP was the substrate, with apparent K(i) of 1.2 mM Ni(II), and a noncompetitive inhibitor with respect to Mg(II) when 8-oxo-dGTP was the substrate, with apparent K(i) of 0.9 mM Ni(II). Hence, the two metal cations did not compete with each other for binding at the Mutt active site. This makes it difficult to predict Ni(II) effects on 8- oxo-dGTPases of other species. However, based upon the amino acid sequences of human and rat MutT-like dGTPases, their capacity for Ni(II) binding should be greater than that of MutT. Whether this could lead to stronger inhibition of those enzymes by Ni(II), or not, remains to be investigated.
- Porter, Dale W.,Nelson, Victor C.,Fivash Jr., Matthew J.,Kasprzak, Kazimierz S.
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- Kinetics of Template-Directed Pyrophosphate-Linked Dideoxyguanylate Synthesis as a Function of 2-MeImpdG and Poly(C) Concentration: Insights into the Mechanism
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Aqueous solutions of deoxyguanosine 5′-monophosphate 2-methylimidazolide, 2-MeImpdG, yield primarily deoxyguanosine 5′-monophosphate, 5′dGMP, and pyrophosphate-linked dideoxyguanylate, dG5′ppdG, abbreviated G2p (see Chart 1). The initial rate of G2p formation, d[G2p]/dt in M h-1, determined at 23°C, pH 7.8, 1.0 M NaCl and 0.2 M Mg2+ by timed high-performance liquid chromatography (HPLC) analysis, exhibits a second-order dependence on 2-MeImpdG concentration, [G]0, indicating a bimolecular mechanism of dimerization in the range 0.02 M ≤ [G]0 ≤ 0.09 M. In the presence of polycytidylate, poly(C), G2p synthesis is accelerated and oligodeoxyguanylate products are formed by incorporation of 2-MeImpdG molecules. The kinetics of G2p formation as a function of both monomer and polymer concentration, expressed in C equivalents, were also determined under the above conditions and exhibited a complex behavior. Specifically, at a constant [poly(C)], values of d[G2p]/dt typically increased with [G]0 with a parabolic upward curvature. At a constant [G]0, values of d[G2p]/dt increase with [poly(C)], but level off at the higher poly(C) concentrations. As [G]0 increases this saturation occurs at a higher poly(C) concentration, a result opposite to expectation for a simple complexation of two reacting monomers with the catalyst prior to reaction. Nevertheless, these results are shown to be quantitatively consistent with a template-directed (TD) mechanism of dimerization where poly(C) acts as the template to bind 2-MeImpdG in a cooperative manner and lead, for the first time, to the formulation of principles that govern template-directed chemistry. Analysis of the kinetic data via a proposed TD cooperative model provides association constants for the affinity between polymer and monomer and the intrinsic reactivity of 2-MeImpdG toward pyrophosphate synthesis. To the best of our knowledge, poly(C)/2-MeImpdG is the first system that could serve as a textbook example of a TD reaction under conditions such that the template is fully saturated by monomers and under conditions that it is not.
- Kanavarioti, Anastassia,Gangopadhyay, Sumana
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- Solution structure, mutagenesis, and NH exchange studies of the MutT enzyme-Mg2+-8-oxo-dGMP complex
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The MutT pyrophosphohydrolase from E. coli (129 residues) catalyzes the hydrolysis of nucleoside triphosphates (NTP), including 8-oxo-dGTP, by substitution at Pβ, to yield NMP and pyrophosphate. The product, 8-oxo-dGMP is an unusually tight binding, slowly exchanging inhibitor with a K D=52nM, (ΔG°=-9.8kcal/mol) which is 6.1kcal/mol tighter than the binding of dGMP (ΔG°=-3.7kcal/mol). The higher affinity for 8-oxo-dGMP results from a more favorable ΔHbinding (-32kcal/mol) despite an unfavorable -TΔS°binding (+22kcal/mol). The solution structure of the MutT-Mg2+-8-oxo-dGMP complex shows a narrowed, hydrophobic nucleotide-binding cleft with Asn-119 and Arg-78 among the few polar residues. The N119A, N119D, R78K and R78A single mutations, and the R78K+N119A double mutant all showed largely intact active sites, on the basis of small changes in the kinetic parameters of dGTP hydrolysis and in 1H-15N HSQC spectra. However, the N119A mutation profoundly weakened the active site binding of 8-oxo-dGMP by 4.3kcal/mol (1650-fold). The N119D mutation also weakened 8-oxo-dGMP binding but only by 2.1kcal/mol (37-fold), suggesting that Asn-119 functioned both as a hydrogen bond donor to C8O, and a hydrogen bond acceptor from N7H of 8-oxo-dGMP, while aspartate at position -119 functioned as an acceptor of a single hydrogen bond. Much smaller weakening effects (0.3-0.4kcal/mol) on the binding of dGMP and dAMP were found, indicating specific hydrogen bonding of Asn-119 to 8-oxo-dGMP. While formation of the wild type MutT-Mg2+-8-oxo-dGMP complex slowed the backbone NH exchange rates of 45 residues distributed throughout the protein, the same complex of the N119A mutant slowed the exchange rates of only 11 residues at or near the active site, indicating an increase in conformational flexibility of the N119A mutant. The R78K and R78A mutations weakened the binding of 8-oxo-dGMP by 1.7 and 1.1kcal/mol, respectively, indicating a lesser role of Arg-78 than of Asn-119 in the selective binding of 8-oxo-dGMP, likely donating a single hydrogen bond to its C6O. The R78K+N119A double mutant weakened the binding of 8-oxo-dGMP (KI slope=3.1mM) by 6.5±0.2kcal/mol which overlaps, within error with the sum of the effects of the two single mutants (6.0±0.3kcal/mol). Such additive effects of the two single mutants in the double mutant are most simply explained by the independent functioning of Asn-119 and Arg-78 in the binding of 8-oxo-dGMP. Independent functioning of these two residues in nucleotide binding is consistent with their locations in the MutT-Mg 2+-8-oxo-dGMP complex, on opposite sides of the active site cleft, with a distance of 8.4±0.5A? between their side chain nitrogens.
- Massiah,Saraswat,Azurmendi,Mildvan
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- Strand breakage of a (6-4) photoproduct-containing DNA at neutral pH and its repair by the ERCC1-XPF protein complex
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The (6-4) photoproduct is one of the major UV-induced lesions in DNA. We previously showed that hydrolytic ring opening of the 5′ base and subsequent hydrolysis of the glycosidic bond of the 3′ component occurred when this photoproduct was treated with aqueous NaOH. In this study, we found that another product was obtained when the (6-4) photoproduct was heated at 90 °C for 6 h, in a 0.1 M solution of N,N′-dimethyl-1,2-ethanediamine adjusted to pH 7.4 with acetic acid. An analysis of the chemical structure of this product revealed that the 5′ base was intact, whereas the glycosidic bond at the 3′ component was hydrolyzed in the same manner. The strand break was detected for a 30-mer oligonucleotide containing the (6-4) photoproduct upon treatment with the above solution or other pH 7.4 solutions containing biogenic amines, such as spermidine and spermine. In the case of spermidine, the rate constant was calculated to be 1.4 × 10-8 s-1 at 37 °C. The strand break occurred even when the oligonucleotide was heated at 90 °C in 0.1 M sodium phosphate (pH 7.0), although this treatment produced several types of 5′ fragments. The Dewar valence isomer was inert to this reaction. The product obtained from the (6-4) photoproduct-containing 30-mer was used to investigate the enzymatic processing of the 3′ end bearing the damaged base and a phosphate. The ERCC1-XPF complex removed several nucleotides containing the damaged base, in the presence of replication protein A. The Royal Society of Chemistry.
- Arichi, Norihito,Yamamoto, Junpei,Takahata, Chiaki,Sano, Emi,Masuda, Yuji,Kuraoka, Isao,Iwai, Shigenori
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- Cytotoxicity of guanine-based degradation products contributes to the antiproliferative activity of guanine-rich oligonucleotides
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Guanine-rich oligonucleotides (GROs) have attracted considerable attention as anticancer agents, because they exhibit cancer-selective antiproliferative activity and can form G-quadruplex structures with higher nuclease resistance and cellular uptake. Recently, a GRO, AS1411 has reached phase II clinical trials for acute myeloid leukemia and renal cell carcinoma. The antiproliferative activity of GROs has been associated with various protein targets; however the real mechanisms of action remain unclear. In this study, we showed evidence that antiproliferative activity of GROs (including AS1411) is mainly contributed by the cytotoxicity of their guanine-based degradation products, such as monophosphate deoxyguanosine (dGMP), deoxyguanosine (dG) and guanine. The GROs with lower nuclease resistance exhibited higher antiproliferative activity. Among nucleotides, nucleosides and nucleobases, only guanine-based compounds showed highly concentration-dependent cytotoxicity. Our results suggest that it is necessary to reconsider the cancer-selective antiproliferative activity of GROs. Since guanine-based compounds are endogenous substances in living organisms, systematic studies of the cytotoxicity of these compounds will provide new information for the understanding of certain diseases and offer useful information for drug design.
- Zhang, Nan,Bing, Tao,Liu, Xiangjun,Qi, Cui,Shen, Luyao,Wang, Linlin,Shangguan, Dihua
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p. 3831 - 3838
(2015/06/25)
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- Fully automated continuous meso-flow synthesis of 5′-nucleotides and deoxynucleotides
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The first continuous meso-flow synthesis of natural and non-natural 5′-nucleotides and deoxynucleotides is described, representing a significant advance over the corresponding in-flask method. By means of this meso-flow technique, a synthesis with time consumption and high-energy consumption becomes facile to generate products with great efficiency. An abbreviated duration, satisfactory output, and mild reaction conditions are expected to be realized under the present procedure.
- Zhu, Chenjie,Tang, Chenglun,Cao, Zhi,He, Wei,Chen, Yong,Chen, Xiaochun,Guo, Kai,Ying, Hanjie
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p. 1575 - 1581
(2015/02/19)
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- Immobilized Drosophila melanogaster deoxyribonucleoside kinase (DmdNK) as a high performing biocatalyst for the synthesis of purine arabinonucleotides
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Fruit fly (Drosophila melanogaster) deoxyribonucleoside kinase (DmdNK; EC: 2.7.1.145) was characterized for its substrate specificity towards natural and non-natural nucleosides, confirming its potential in the enzymatic synthesis of modified nucleotides. DmdNK was adsorbed on a solid ion exchange support (bearing primary amino groups) achieving an expressed activity >98%. Upon cross-linking with aldehyde dextran, expressed activity was 30-40%. Both biocatalysts (adsorbed or cross-linked) were stable at pH 10 and room temperature for 24 h (about 70% of retained activity). The cross-linked DmdNK preparation was used for the preparative synthesis of arabinosyladenine monophosphate (araA-MP) and fludarabine monophosphate (FaraAMP). Upon optimization of the reaction conditions (50 mM ammonium acetate, substrate/ATP ratio= 1:1.25, 2 mM MgCl2, 378C, pH 8) immobilized DmdNK afforded the title nucleotides with high conversion (>90%), whereas with the soluble enzyme lower conversions were achieved (78-87%). Arabinosyladenine monophosphate was isolated in 95% yield and high purity (96.5%).
- Serra, Immacolata,Conti, Silvia,Piskur, Jure,Clausen, Anders R.,Munch-Petersen, Birgitte,Terreni, Marco,Ubiali, Daniela
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p. 563 - 570
(2014/05/20)
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- A single nuclease-resistant linkage in DNA as a versatile tool for the characterization of DNA lesions: Application to the guanine oxidative lesion g+34 generated by metalloporphyrin/KHSO5 reagent
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The oxidation of an oligonucleotide containing a single nuclease-resistant phosphodiester link, a stereoisomerically pure methylphosphonate, by manganese (Mn-TMPyP) or iron (Fe-TMPyP) porphyrin associated to KHSO5 allowed the isolation and characterization of a guanine lesion corresponding to an increase of mass of 34 amu as compared to guanine ( G+34 ), namely, 5-carboxamido-5-formamido-2-iminohydantoin. Enzymatic digestion of the damaged oligonucleotide afforded, apart from the undamaged nucleotide monomer pool, a unique dinucleotide doubly modified with a methylphosphonate and an oxidized guanine base that is suitable for NMR analysis. The method can be applied to the study of any DNA lesion. More importantly, the method can be extended to the analysis of DNA damage in a sequence context. Any preselected residue in a DNA sequence may be individually analyzed by the easy introduction of a single nuclease-resistant link at the 3′- or 5′-position.
- Tomaszewska, Agnieszka,Mourgues, Sophie,Guga, Piotr,Nawrot, Barbara,Pratviel, Genevieve
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p. 2505 - 2512
(2013/01/15)
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- Mechanism of the alkali degradation of (6-4) photoproduct-containing DNA
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The (6-4) photoproduct is one of the major damaged bases produced by ultraviolet light in DNA. This lesion is known to be alkali-labile, and strand breaks occur at its sites when UV-irradiated DNA is treated with hot alkali. We have analyzed the product obtained by the alkali treatment of a dinucleoside monophosphate containing the (6-4) photoproduct, by HPLC, NMR spectroscopy, and mass spectrometry. We previously found that the N3-C4 bond of the 5′ component was hydrolyzed by a mild alkali treatment, and the present study revealed that the following reaction was the hydrolysis of the glycosidic bond at the 3′ component. The sugar moiety of this component was lost, even when a 3′-flanking nucleotide was not present. Glycosidic bond hydrolysis was also observed for a dimer and a trimer containing 5-methyl-2-pyrimidinone, which was used as an analog of the 3′ component of the (6-4) photoproduct, and its mechanism was elucidated. Finally, the alkali treatment of a tetramer, d(GT(6-4)TC), yielded 2′-deoxycytidine 5′-monophosphate, while 2′-deoxyguanosine 3′-monophosphate was not detected. This result demonstrated the hydrolysis of the glycosidic bond at the 3′ component of the (6-4) photoproduct and the subsequent strand break by β-elimination. It was also shown that the glycosidic bond at the 3′ component of the Dewar valence isomer was more alkali-labile than that of the (6-4) photoproduct. The Royal Society of Chemistry 2012.
- Arichi, Norihito,Inase, Aki,Eto, Sachise,Mizukoshi, Toshimi,Yamamoto, Junpei,Iwai, Shigenori
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p. 2318 - 2325
(2012/04/10)
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- Two thymidine kinases and one multisubstrate deoxyribonucleoside kinase salvage DNA precursors in Arabidopsis thaliana
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Deoxyribonucleotides are the building blocks of DNA and can be synthesized via de novo and salvage pathways. Deoxyribonucleoside kinases (EC 2.7.1.145) salvage deoxyribonucleosides by transfer of a phosphate group to the 5' of a deoxyribonucleoside. This salvage pathway is well characterized in mammals, but in contrast, little is known about how plants salvage deoxyribonucleosides. We show that during salvage, deoxyribonucleosides can be phosphorylated by extracts of Arabidopsis thaliana into corresponding monophosphate compounds with an unexpected preference for purines over pyrimidines. Deoxyribonucleoside kinase activities were present in all tissues during all growth stages. In the A. thaliana genome, we identified two types of genes that could encode enzymes which are involved in the salvage of deoxyribonucleosides. Thymidine kinase activity was encoded by two thymidine kinase 1 (EC 2.7.1.21)-like genes (AtTK1a and AtTK1b). Deoxyadenosine, deoxyguanosine and deoxycytidine kinase activities were encoded by a single AtdNK gene. T-DNA insertion lines of AtTK1a and AtTK1b mutant genes had normal growth, although AtTK1a AtTK1b double mutants died at an early stage, which indicates that AtTK1a and AtTK1b catalyze redundant reactions. The results obtained in the present study suggest a crucial role for the salvage of thymidine during early plant development. 2012 The Authors Journal compilation
- Clausen, Anders R.,Girandon, Lenart,Ali, Ashfaq,Knecht, Wolfgang,Rozpedowska, Elzbieta,Sandrini, Michael P. B.,Andreasson, Erik,Munch-Petersen, Birgitte,Piskur, Jure
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p. 3889 - 3897
(2013/01/13)
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- An efficient reagent for the phosphorylation of deoxyribonucleosides, DNA oligonucleotides, and their thermolytic analogues
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(Chemical Equation Presented) The phosphoramidite 11 was prepared in three steps from methyl 2-mercaptoacetate and demonstrated efficiency in the synthesis of conventional 5′-/3′-phosphate/thiophosphate monoester derivatives of 2′-deoxyribonucleosides and DNA oligonucleotides. Moreover, the use of 11 has enabled the preparation of the dinucleoside phosphorothioate analogue 26 in high yields (>95%) with minimal cleavage (2%) of the thermolytic thiophosphate protecting group.
- Ausin, Cristina,Grajkowski, Andrzej,Cieslak, Jacek,Beaucage, Serge L.
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p. 4201 - 4204
(2007/10/03)
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- Anabolism of amdoxovir: Phosphorylation of dioxolane guanosine and its 5′-phosphates by mammalian phosphotransferases
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Amdoxovir [(-)-β-D-2,6-diaminopurine dioxolane, DAPD], the prodrug of dioxolane guanosine (DXG), is currently in Phase I/II clinical development for the treatment of HIV-1 infection. In this study, we examined the phosphorylation pathway of DXG using 15 purified enzymes from human (8), animal (6), and yeast (1) sources, including deoxyguanosine kinase (dGK), deoxycytidine kinase (dCK), high Km 5′-nucleotidase (5′-NT), guanylate (GMP) kinase, nucleoside monophosphate (NMP) kinase, adenylate (AMP) kinase, nucleoside diphosphate (NDP) kinase, 3-phosphoglycerate (3-PG) kinase, creatine kinase, and pyruvate kinase. In addition, the metabolism of 14C-labeled DXG was studied in CEM cells. DXG was not phosphorylated by human dCK, and was a poor substrate for human dGK with a high Km (7 mM). Human 5′-NT phosphorylated DXG with relatively high efficiency (4.2% of deoxyguanosine). DXG-MP was a substrate for porcine brain GMP kinase with a substrate specificity that was 1% of dGMP. DXG-DP was phosphorylated by all of the enzymes tested, including NDP kinase, 3-PG kinase, creatine kinase, and pyruvate kinase. The BB-isoform of human creatine kinase showed the highest relative substrate specificity (47% of dGDP) for DXG-DP. In CEM cells incubated with 5 μM DXG for 24 h, 0.015 pmole/106 cells (～7.5 nM) of DXG-TP was detected as the primary metabolite. Our study demonstrated that 5′-nucleotidase, GMP kinase, creatine kinase, and NDP kinase could be responsible for the activation of DXG in vivo.
- Feng, Joy Y.,Parker, William B.,Krajewski, Megan L.,Deville-Bonne, Dominique,Veron, Michel,Krishnan, Preethi,Cheng, Yung-Chi,Borroto-Esoda, Katyna
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p. 1879 - 1888
(2007/10/03)
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- Phosphorylation of isocarbostyril- and difluorophenyl-nucleoside thymidine mimics by the human deoxynucleoside kinases
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The thymidine mimics isocarbostyril nucleosides and difluorophenyl nucleosides were tested as deoxynucleoside kinase substrates using recombinant human cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK), and mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). The isocarbostyril nucleoside compound 1-(2-deoxy-β-D-ribofuranosyl)- isocarbostyril (EN1) was a poor substrate with all the enzymes. The phosphorylation rates of EN1 with TK1 and TK2 were cat/Km) with both TK1 and dGK, but not with TK2. The kcat/Km value for EN2 with TK2 was 12.6% relative to that for Thd. Of the difluorophenyl nucleosides, 5-(1′-(2′-deoxy-β-D-ribofuranosyl))-2,4-difluorotoluene (JW1) and 1-(1′-(2′-deoxy-β-D-ribofuranosyl))-2,4-difluoro-5- iodobenzene (JW2) were substrates for TK1 with phosphorylation efficiencies of about 5% relative to that for Thd. Both analogues were considerably more efficient substrates for TK2, with kcat/Km values of 45% relative to that for Thd. 2,5-Difluoro-4-[1-(2-deoxy-β-L-ribofuranosyl)]- aniline (JW5), a L-nucleoside mimic, was phosphorylated up to 15% as efficiently as deoxycytidine by dCK. These data provide a possible explanation for the previously reported lack of cytotoxicity of the isocarbostyril- and difluorophenyl nucleosides, but potential mitochondrial effects of EN2, JW1 and JW2 should be further investigated.
- Al-Madhoun, Ashraf Said,Eriksson, Staffan,Wang, Zhi-Xian,Naimi, Ebrahim,Knaus, Edward E.,Wiebe, Leonard I.
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p. 1865 - 1874
(2007/10/03)
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- A new method for the phosphorylation of nucleosides
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A new phosphorylating reagent, 2-O-(4,4'-dimethoxytrityl)ethylsulfonylethan-2'-yl-phosphate (1), was developed for the phosphorylation of primary and secondary alcohols of nucleosides. In the many examples studied, yields in the phosphorylation step were excellent (~80 to 95%). There is potential for wide applicability of this procedure, not only in nucleoside and nucleotide chemistry, but also in the phosphorylation of biomolecules such as carbohydrates and amino acids. (C) 2000 Elsevier Science Ltd.
- Taktakishvili,Nair
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p. 7173 - 7176
(2007/10/03)
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- Identification and quantitation of the lesion accompanying base release in bleomycin-mediated DNA degradation
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Interaction of bleomycin, Fe2+, and limiting O2 or bleomycin, Fe3+, and H2O2 with DNA results in the production of free nucleic acid base and an oxidatively damaged sugar lesion that undergoes strand scission subsequent to treatment with alkali. The hexamer d(CGCGCG) has been utilized to develop a protocol to establish the identity of this lesion as a 2'-deoxy-4'-pentulose moiety and to establish its stoichiometric production with respect to free nucleic acid base. The protocol developed has been extended to a variety of heterogeneous DNAs with similar results. The predominant pathway for base release in bleomycin-mediated DNA degradation is the result of chemistry at the C-4' carbon of the sugar moiety.
- Rabow,Stubbe,Kozarich
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p. 3196 - 3203
(2007/10/02)
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- Covalent alkylation of DNA with duocarmycin A. Identification of abasic site structure
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Alkylation of d(CGTATACG) by antitumor antibiotic duocarmycin A was investigated. It was found that N3 of adenine6 (A6) attacks the cyclopropane subunit of duocarmycin A to produce covalently alkylated adduct 5. Upon heating (90 °C, 5 min) the adduct 5 decomposed to modified oligomer 1 with concomitant release of adenine adduct 2.
- Sugiyama,Hosoda,Saito,Asai,Saito
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p. 7197 - 7200
(2007/10/02)
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- Chemical Synthesis of 5'-Phosphorylated DNA Fragments and Their Constituents
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Phosphoryl tris-triazole has been applied to the synthesis of DNA fragments and their constituents bearing 5'-phosphomonoester function and to the preparation of deoxynucleoside 5'- or 3'-diphosphates.
- Jankowska, Jadwiga,Stawinski, Jacek
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