Nitrosamine-induced cancer: O4-alkylthymine produces sites of DNA hyperflexibility.

The carcinogenic properties of N-nitroso compounds are associated with their ability to alkylate DNA, in particular to form O6-alkylguanine and O4-alkylthymine. DNA duplexes containing either O6-alkylguanine or O4-alkylthymine were synthesized, and each duplex was ligated to form a set of DNAs of increasing length with the alkylated base out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of the DNA. The DNA contained the sequence 5' CAA 3', which is the 61st codon of the K-ras gene, because this codon is a preferred site of mutation for a number of carcinogens including the methylating carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). O4-Methylthymine or O4-ethylthymine replaced thymine in either of the two A.T base-pairs of this codon (normally CAA), and O6-methylguanine replaced the guanine in the G.C pair. All the sequences containing O4-alkylthymine exhibited anomalous, slow, gel migration and ligated to form circles of unusually small diameter. In general, the effect was seen when the alkylated base-pair was out of phase with the helical repeat as well as when it was in phase, suggesting that the alkylated base-pair confers flexibility which is largely isotropic, i.e., has no preferred direction, rather than anisotropic flexibility or bending. However, at pH 8.3 the 21-base-pair set containing O4-alkylT.A had significantly greater anomalous migration than the 16-base-pair set, suggesting that the flexibility produced by this base-pair has a significant anisotropic component and thus resembles true bending.(ABSTRACT TRUNCATED AT 250 WORDS)     

Preparation of Oligonucleotides Containing Non-natural Base Analogues

Abstract

The preparation of a protected derivative of 2-aza- 2′-deoxyinosine carrying a photolabile protecting group is described. The new derivative is useful to prepare oligonucleotides containing 2-azahypoxanthine. The synthesis of oligonucleotides containing 2-fluorohypoxanthine and O⁴-alkylthymine is also described.     

Active-site alkylation destabilizes human O6-alkylguanine DNA alkyltransferase

O(6)-alkylguanine-DNA alkyltransferase (AGT) repairs pro-mutagenic O(6)-alkylguanine and O(4)-alkylthymine lesions in DNA. The alkylated form of the protein is not reactivated; instead, it is rapidly ubiquitinated and degraded. Here, we show that alkylation destabilizes the native fold of the protein by 0.5-1.2 kcal/mole and the DNA-binding function by 0.8-1.4 kcal/mole. On this basis, we propose that destabilization of the native conformational ensemble acts as a signal for ubiquitination.     

Solid-phase synthesis of oligoribonucleotides using 9-fluorenylmethoxycarbonyl (Fmoc) for 5''-hydroxyl protection.

Efficient solid-phase synthesis of a series of oligoribonucleotides of up to 20 residues is described that utilises the 9-fluorenylmethoxycarbonyl group (Fmoc) for 5'-protection and 4-methoxytetrahydropyran-4-yl (Mthp) for 2'-protection of ribonucleotide monomers and a phosphoramidite coupling procedure. The Fmoc group is removed after each coupling step by treatment with 0.1M DBU in acetonitrile. Oligoribonucleotides are isolated in 2'-protected form in good yield and shown to be readily and efficiently deprotected by mild acidic treatment.     

In vitro miscoding of alkylthymines with DNA and RNA polymerases

A series of poly(dT,RdT) polynucleotides containing O2-, 3- and O4-methyl- or ethyldeoxythymines have been prepared and used as templates for Pol I, Pol alpha and Pol R to investigate the miscoding properties of these modified bases with the different nucleotide polymerising enzymes. Only O4-alkylthymine (O4-RT) leads to a large number of errors (incorporation of dGMP with a mutagenic efficiency approaching unity). O2-RT has a very much lower, but none-the-less significant, mutagenic efficiency and 3-RT does not lead to errors with the DNA polymerases. All alkylthymines lead to significant errors with the RNA polymerase. The results confirm conclusions concerning the promutagenic nature of the alkylthymines and that the reported differences in their promutagenicity with the various polymerases reflect different specificities of the nucleotide polymerising enzymes used.     

Dealkylation rates of O6-alkyldeoxyguanosine, O4-alkylthymidine and related compounds in an alkyl-transfer system

Bacterial O6-alkylguanine-DNA alkyltransferase (AGT) removes alkyl group from O6-alkylguanine and O4-alkylthymine residues in DNA, both of which are considered to be DNA damages most related to the induction of cancer and/or mutation. The repair process involves alkyl-transfer of an O-alkyl group to the active site of the enzyme, where an SH-group of cysteine residue plays the role of alkyl acceptor. In order to elucidate the chemical characteristics of substrates for this enzyme, dealkylation rates of O6-alkyldeoxyguanosine, O4-alkylthymidine and related compounds were measured using an alkyl-transfer system. Thiophenol-triethylamine system was employed as an alkyl acceptor and twenty-one O-alkyl compounds were tested. Dealkylation proceeded with pseudo first order kinetics. The half-life of O6-methyldeoxyguanosine (MedG) was 122 h and no remarkable dependence on N-9 substituents (H, CH3 and deoxyribose) was observed. A compound lacking 2-NH2 group underwent demethylation about three times faster than O6-methylguanines did, while, a compound lacking imidazole moiety underwent demethylation about 2.5 times more slowly. The half-life of O4-methylthymidine (MedT) was 38 h and no remarkable dependence on N-1 (H, CH3 and deoxyribose) and C-5 (H and CH3) substituents was observed. Deethylation proceeded much more slowly than demethylation. Substitution of selenophenol for thiophenol resulted in a 4.5 times faster MedG demethylation rate. Demethylation rates were moderately correlated with values for NMR chemical shift of CH3 group, an indicator of electron density, although the correlation curves of a series of MedG and MedT derivatives were quite different. This result suggests that some different rate-determining factors other than electron density are playing a role. These findings may be of help in resolving the details of the mechanisms of enzymic repair by bacterial and mammalian AGT.     

Solid-phase synthesis and side reactions of oligonucleotides containing O-alkylthymine residues

As part of our studies on the molecular mechanism of mutation [Chambers, R. W. (1982) in Molecular and Cellular Mechanisms of Mutagenesis (Lemontt, J. F., & Generoso, W. M., Eds.) pp 121-145, Plenum, New York and London], we wanted to prepare specific oligonucleotides carrying O2- or O4-alkylthymidine residues. Since O-alkylthymine moieties are known to be alkali labile, side reactions were expected during the deprotection procedures used for synthesis of oligonucleotides on a solid support by the classical phosphoramidite method. We have studied these side reactions in detail. Kinetic data show the deprotection procedures displace most O-alkyl groups at rates that make these procedures inappropriate for synthesis of most oligonucleotides carrying O-alkylthymine moieties. We describe alternative deprotection procedures, using readily accessible reagents, that we have used successfully to synthesize a series of oligonucleotides carrying several different O-alkylthymine moieties. The oligonucleotides synthesized are d(A-A-A-A-G-T-alkT-T-A-A-A-A-C-A-T), where alk = O2-methyl, O2-isopropyl, O4-methyl, O4-isopropyl, and O4-n-butyl. This work extends the previously described procedure for the chemical synthesis of oligonucleotides carrying an O4-methylthymine moiety [Li, B. F., Reese, C. B., & Swann, P. F. (1987) Biochemistry 26, 1086-1093] and reports the first chemical synthesis of an oligonucleotide carrying an O2-alkylthymine. The oligonucleotides synthesized have a sequence corresponding to the minus strand that is complementary to the viral strand at the start of gene G in bacteriophage phi X174 replicative form DNA where the normal third codon has been replaced with the ocher codon, TAA.     

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.     

Increased resistance to the toxic effects of alkylating agents in tobacco expressing the E. coli DNA repair gene ada.

The protein coding region of the E. coli gene ada has been transferred to tobacco plants by a leaf disc transformation procedure involving an Agrobacterium tumefaciens Ti plasmid. Transformed plants were shown to be transgenic for the ada message and had increased levels of O6-alkylguanine DNA alkyltransferase activity. The N-methyl-N-nitrosourea- or taurinechlorethylnitrosourea-induced inhibition of growth of calluses or of cells in suspension was considerably lower in ada-transformed than in non-transformed plants. This indicates that O6-alkylguanine, O4-alkylthymine or phosphotriesters are growth-inhibitory lesions in tobacco.     

Theoretical discussion on the mechanism of binding CO2 by DBU and alcohol

Quantum chemical studies on the reaction of binding CO₂ by amidine base diazabicyclo [5.4.0]-undec-7-ene (DBU) and alcohol were carried out at the B3LYP/6-31g(d) level in order to find the reaction mechanism. The structures of reactants and product were optimised, and thermodynamic analyses were also carried out using the single point energy calculation and frequency analyses. It is noted that the reaction of binding CO₂ by DBU and propanol is thermodynamically feasible and qualitatively in accordance with the experimental observations. The results of thermodynamic and kinetic analyses demonstrate that the possible reaction mechanisms can be a two-step bimolecular reaction and a one-step trimolecular reaction. In the two-step bimolecular mechanism, the first step is the formation of intermediate by DBU and CO₂, and the second step is the nucleophilic attack of propanol on the intermediate. In the one-step trimolecular mechanism, O and H atoms of hydroxyl in propanol form an O–C bond with CO₂ and an H–N bond with DBU, respectively. The one-step trimolecular reaction seems a more reasonable mechanism because of the consideration of kinetic parameters.     

7-deaza-2'-deoxyxanthosine: nucleobase protection and base pairing of oligonucleotides

Oligonucleotides containing 7-deaza-2'-deoxyxanthosine (1) and 2'-deoxyxanthosine (2) were prepared. The 2-(4-nitrophenyl)ethyl group is applicable for 7-deazaxanthine protection that is removed with DBU by beta-elimination, while the deprotection of the allyl residue with Pd (0) catalyst failed. Contrarily, the allyl group was found to be an excellent protecting group for 2'-deoxyxanthosine (2). The base pairing of nucleosides 1 and 2 with the four canonical DNA constituents as well as with 3 within the 12-mer duplexes is studied.     

Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA

The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.     

Comparison of procedures for directly obtaining protected peptide acids from peptide-resins.

The preparation of small-sized protected peptide acids related to cholecystokinin and gomesin was attempted using peptide-Kaiser oxime resins (KOR) as starting materials. For comparison, peptide-2-Cl-trityl resin (CLTR) was also employed. While peptide detachment from KOR was achieved through the oxime ester bond hydrolysis mediated by DBU, hydroxide ion or Ca(+2) ion, peptide release from CLTR was accomplished through acid-catalysed hydrolysis of the peptide-resin ester linkage. Amino acid analysis of the peptide-resins before and after peptide release allowed the calculation of the reaction yields. RP-HPLC and LC/ESI-MS of the resulting crude peptides allowed estimation of their quality. The data collected indicated that: (i) among the procedures used for peptide displacement from KOR, the one employing DBU was the most efficient since it furnished all model protected peptide acids with the highest quality in a very short time; (ii) although slow, Ca(+2)-assisted peptide detachment from KOR was selective and was suitable for generating high-quality protected peptide acids containing up to five residues; (iii) even though the protocols employed for peptide release from CLTR have shown to be appropriate, the one employing 1% TFA/DCM was the most productive; (iv) in terms of product quality, DBU-catalysed peptide detachment from KOR was similar to TFA-catalysed peptide release from CLTR although the latter was more productive. These findings are relevant to peptide chemists in general, but especially to those interested in preparing acyl donors for convergent peptide syntheses by the t-Boc chemistry.     

Synthesis and characterization of oligodeoxynucleotides containing 4-O-methylthymine

The carcinogenicity of N-nitroso compounds is believed to result from the alkylation of DNA, particularly on O-6 of the guanine and O-4 of the thymine residues. In order to study the base-pairing properties of 4-O-methylthymidine (T*) residues and the structural changes produced in DNA by the presence of this alkylated nucleoside, the oligodeoxyribonucleotides T*GCG, CGCAAGCTT*GCG, CGCGAGCTT*GCG, and CGCAAGCTTGCG were synthesized by the phosphotriester approach in solution. The 4-O-methylthymidine required for oligonucleotide synthesis was prepared by treating the 4-(3-nitro-1,2,4-triazolo) derivative of 3',5'-bis-O-(methoxyacetyl)thymidine with 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU) in methanol solution. The susceptibility of the 4-O-methyl group of T* toward nucleophiles enables this group of 4-O-methylthymidine-containing oligomers to be labeled by a direct exchange reaction with [13C]- or [14C]methanol in the presence of DBU. Although it has been previously suggested that 4-O-methylthymine forms stable base pairs with guanine, the thermal melting profiles of the double helices formed by these dodecamers suggest that the presence of 4-O-methylthymine paired to either adenine or guanine destablizes the helix. The melting curve of the sequence containing a 4-O-methylthymine residue base paired to guanine was biphasic and similar to that of an analogous sequence containing 6-O-methylguanine paired to thymine.     

Solid-phase synthesis of backbone-modified DNA analogs by the boranophosphotriester method using new protecting groups for nucleobases

Backbone-modified DNA analogs were synthesized in good yields by the boranophosphotriester method on a solid support. The oligodeoxyribonucleoside boranophosphates, protected with 2-(azidomethyl)benzoyl groups for nucleobases, were converted into DNA and its backbone-modified analogs via the corresponding H-phosphonate intermediates. A new protecting group for the O6 position of 2'-deoxyguanosine, 4-azidobenzyl (ABn) group, was also developed. The ABn group can be quickly removed by treatment with MePPh2 and H2O in the presence of 2-mercaptoethanol.     

Interactions of human O6-alkylguanine-DNA alkyltransferase (AGT) with short single-stranded DNAs

The O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. Here we characterize the binding of AGT to single-stranded DNAs ranging in length from 5 to 78 nucleotides (nt). Binding is moderately cooperative (37.9 +/- 3.0 相似文献   

Synthesis of 2′-Deoxynucleoside 5′-Methylenebis-(phosphonate)s Using 2-(4-Nitrophenyl)ethyl Methylenebis(phosphonate) as the Phosphonylating Agent.

Abstract

2-(4-Nitrophenylethyl) methylenebis(phosphonate) (1) has been prepared by reaction of 2-(4-nitrophenyl)ethyl alcohol with methylenebis(phosphonyl) tetrachloride. Compound 1 was treated with diisopropylcarbodiimide (DIC) to give bicyclic intermediate 2, which in reaction with suitably protected 2′-deoxynucleosides 3 gave P¹,P²-disubstituted methylenebis(phosphonate)s 4. Removal of the nitrophenylethyl group by β-elimination with DBU afforded the corresponding 2′-deoxynucleoside 5′-methylenebis(phosphonate) analogues 5.

     

Protected 1-3 segment of the peptaibol antibiotics alamethicin and hypelcin. Solid-state and solution study of a stereochemically constrained linear peptide

A study of the modes of folding and self-association of Z-Aib-L-Pro-Aib-OMe (the protected 1-3 segment of the peptaibol antibiotics alamethicin and hypelcin) in the solid state was performed using i.r. absorption and X-ray diffraction. The stereochemically constrained tripeptide molecules adopt a 4 leads to 1 intramolecularly H-bonded form (beta-turn), where the single intramolecular H-bond is found between the peptide N-H group of the Aib3 residue and the urethane C = O group of the N-blocking benzyloxycarbonyl moiety. This folded structure is stabilized by an intermolecular H-bond between the urethane N-H group of the Aib1 residue and the peptide C = O group of the Pro2 residue of a symmetry related molecule. According to the i.r. absorption data, in CH2Cl2 and TMP solutions the same intramolecularly H-bonded form occurs as that found in solid state. Compared to the situation in the solid state, in CH2Cl2 and TMP solvation of the urethane N-H group replaces self-association (through the same N-H group). The results are also discussed in relation to those obtained for other protected -Aib-X-Aib- (X = Aib, L-Ala, L-Val) tripeptide segments of peptaibol antibiotics.     

Mutations that probe the cooperative assembly of O⁶-alkylguanine-DNA alkyltransferase complexes

O(6)-Alkylguanine-DNA alkyltransferase (AGT) repairs mutagenic O(6)-alkylguanine and O(4)-alkylthymine adducts present in DNA that has been exposed to alkylating agents. AGT binds DNA cooperatively, and models of cooperative complexes predict that residues 1-7 of one protein molecule and residues 163-169 of a neighboring protein are closely juxtaposed. To test these models, we used directed mutagenesis to substitute triplets of alanine for triplets of native residues across these two sequences. Six of eight designed mutants expressed AGT at detectable levels. All mutant AGTs that were expressed were folded compactly, bound DNA with stoichiometries equivalent to that of the wild-type protein, and were able to protect Escherichia coli to varying degrees from the potent alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All mutations attenuated DNA binding cooperativity, but unexpectedly, they also reduced the affinity of AGT for DNA. This suggests that the protein-protein and protein-DNA interactions of AGT are strongly coupled. When normalized for differences in AGT expression, cells expressing mutants KDC(3-5)-AAA, DCE(4-6)-AAA, and KEW(165-167)-AAA were significantly more susceptible to MNNG than wild-type cells. This is the first evidence, to the best of our knowledge, of a role for residues at the protein-protein interface and, by implication, cooperative protein-protein interactions in the cell-protective mechanisms of AGT.     

Methyl transferases induced during chemical adaptation of M. luteus.

Three peaks of methyltransferase activity specific for MNNG alkylated DNA have been identified from extracts of chemically adapted M. luteus. They are designated as TI to TIII in order to their elution from a Sephadex G-75 column. The first one of these peaks has been purified to homogeneity. TI, is an inducible, unusually salt resistant, heat labile protein which corrects O6-methylguanine in alkylated DNA by the transfer of the O6-alkyl group to a cysteine amino acid in the TI protein. There is a stoichiometric relationship between the loss of O6-methylguanine from the DNA and the production of S-methylcysteine. Partially purified TII & TIII proteins show specificity for O4-alkylthymine and methyl phosphotriesters respectively. The mode of repair by the isolated methyltransferases is similar yet there is no competition for substrate specificity. The apparent molecular weights of TI, TII & TIII proteins are 31Kd, 22Kd, and 13Kd respectively.