Structure and in vitro replication of DNA templates containing 7,8-dihydro-8-oxoadenine.

Adenine residues in DNA are oxidized under the action of ionizing radiation at the C-8 position to give 7,8-dihydro-8-oxoadenine. The formation of this lesion can be considered a cause of mutations and carcinogenesis. Oligodeoxyribonucleotides 39 and 47 bases long containing a single 7,8-dihydro-8-oxoadenine (8-hydroxyadenine) residue were synthesized by using nucleoside phosphoramidites. They were used as templates to study the copies obtained in vitro by the Klenow fragment and the thermostable Taq DNA polymerase. 7,8-Dihydro-8-oxoadenine does not block the replication and thymine is incorporated opposite the damage. The modifications of the DNA duplex conformation provoked by 7,8-dihydro-8-oxoadenine are minor. 1H-NMR spectroscopy shows that the duplex is in a B form, the sugar in a normal position in the helix and the modified base in the anti position. NMR confirms that 7,8-dihydro-8-oxoadenine exists predominantly in the keto form.     

Structure-activity relationship of phosmidosine: importance of the 7,8-dihydro-8-oxoadenosine residue for antitumor activity

To study the structure-activity relationship of phosmidosine, a variety of phosmidosine derivatives 9a-g were synthesized by condensation of N-diisopropyl N'-(N-tritylprolyl)phosphorodiamidite 6 with appropriately protected nucleoside derivatives 7a-g. As the result, replacement of the 7,8-dihydro-8-oxoadenine base by adenine and 6-N-acetyladenine did not affect the antitumor activity. However, phosmidosine derivatives containing uracil, cytosine, and guanine in place of the 7,8-dihydro-8-oxoadenine base did not show significant activity. A plausible explanation for the selective expression of phosmidosine compared with that of phosmidosine analogs having other amino acids in place of proline is also discussed. These results suggest that phosmidosine serves as an inhibitor of prolyl adenosine 5'-phosphate (prolyl-AMP) to inhibit the peptide synthesis in cancer-related cells.     

Further studies of KMnO4 oxidation of synthetic DNAs containing oxidatively damaged bases.

Recently we found that KMnO4 oxidation of DNA oligomers containing a 7,8-dihydro-8-oxoguanine (8-oxo-G) residue induces damage to the neighboring base residues; other modified bases, 7,8-dihydro-8-oxoadenine (8-oxo-A) and 5-hydroxyuracil (5-oh-U), show similar behavior in DNA. The present study indicated that the ability to induce damage, which could also occur by the oxidation of a 5-oh-C residue, was low as in the case of 5-oh-U. On the other hand, in order to examine the pathways and the intermediates for the oxidative degradation of 8-oxo-A, we have carried out the KMnO4 oxidation using an 8-oxo-2'-deoxyadenosine derivative as a model and have determined the structures of the three major products.     

Gel electrophoretic detection of 7,8-dihydro-8-oxoguanine and 7, 8-dihydro-8-oxoadenine via oxidation by Ir (IV).

Two gel electrophoretic methods are described for detection of 7, 8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine based on their further oxidation with one-electron oxidants including IrCl62-and IrBr62-. The products of nucleobase oxidation lead to enhanced piperidine-sensitive cleavage and to highly visible stop points in a primer extension assay. 8-oxoG and 8-oxoA lesions may be distinguished by the latter's inability to be oxidized by IrBr62-compared to IrCl62-Comparison is also made to oxidation by MnO4-.     

Oxidative DNA base modifications and polycyclic aromatic hydrocarbon DNA adducts in squamous cell carcinoma of larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.     

Peroxynitrite Mediated Oxidation of Purine Bases of Nucleosides and Isolated DNA

Reaction of nitric oxide with superoxide anion produces the highly reactive species peroxynitrite (ONOO^?). This compound has been shown to be a strong oxidant of lipids and proteins. However, no data are available on its effect on DNA, with the exception of the induction of strand breaks. We report the result of studies on the reactions of peroxynitrite with the adenine and guanine moieties of nucleosides and isolated DNA. The samples were analyzed for 8-oxo-7,8-dihydro-2′-deoxyguano-sine (8-oxo-dGuo), 2,2-diamino-4–[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5–(2H)-oxazolone (oxazolone) and 8-oxo-7,8-dihydro-2′-deoxyadenosine (8-oxo-dAdo). The effects of peroxynitrite treatment were compared with those of ionizing radiation in aerated aqueous solution, chosen as a source of hydroxyl radicals. At the nucleoside level, both oxidizing conditions led to the formation of oxazolone and 8-oxo-dAdo. In addition, evidence was provided for the formation of the 4R* and 4S* diastereoisomers of 4-hydroxy-8-oxo-4,8-dihydro-2′-deoxyguanosine. The latter dGuo oxidation products were chosen as markers of the release of singlet oxygen (¹O₂) upon reaction of peroxynitrous acid with hydrogen peroxide. Oxidation of purine bases was then studied within isolated DNA. A significant increase in the level of 8-oxp-dGuo, oxazolone and 8-oxo-dAdo was observed within double stranded DNA upon exposure to γ-radiation. Oxazolone and 8-oxo-dAdo were formed upon peroxynitrite treatment but no significant increase in the amount of 8-oxo-dGuo was detected. These results showed that peroxynitrite exhibits oxidizing properties toward purine moieties both in nucleosides and isolated DNA. However, the significant differences in the oxidative damage distribution within DNA observed after exposure to γ radiation by comparison with peroxynitrite treatment questions the involvement of hydroxyl radicals as the main oxidizing species released by decomposition of peroxynitrous acid.     

Method for the synthesis of uric acid derivatives

A general procedure to obtain tetra-substituted uric acid by stepwise N-alkylation is described. 2,6-Dichloropurine (1) was condensed with 1-propanol by Mitsunobu reaction to give 9-propyl congener (2). Treatment of 2 with ammonia gave adenine derivative (4a), which was converted to the 8-oxoadenine (5b) in 3 steps. Methylation of 5b proceeded site-specifically to give 6-amino-2-chloro-7,8-dihydro-7-methyl-9-propylpurin-8-one (6) as a sole product. Compound 6 was successively treated with NaNO2 and iodomethane to give 2-chloro-1,6,7,8-tetrahydro-1,7-dimethyl-9-propylpurin-6,8-dione (9) accompanied by the O6-methyl product (8) in 75% and 6.9%, respectively. After nucleophilic substitution of 9 with NaOAc, the product (11) was reacted with iodomethane to give the uric acid (12) and the 2-methoxy product (13) in 46% and 15.5%, respectively. However, the reaction of 11 with the benzylating agents gave only O-benzyl products (14a,b).     

Oxidative DNA Base Modifications and Polycyclic Aromatic Hydrocarbon DNA Adducts in Squamous Cell Carcinoma of Larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.     

Measurement of 8-hydroxy-2′-deoxyguanosine in DNA by high-performance liquid chromatography-mass spectrometry: comparison with measurement by gas chromatography-mass spectrometry

Measurement of 8-hydroxy-2′-deoxyguanosine (8-OH-dGuo) in DNA by high-performance liquid chromatography/mass spectrometry (LC/MS) was studied. A methodology was developed for separation by LC of 8-OH-dGuo from intact and modified nucleosides in DNA hydrolyzed by a combination of four enzymes: DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionization-electrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10⁶ DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10⁶ DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/isotope-dilution MS with selected-ion monitoring (GC/IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8-dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/IDMS-SIM and GC/IDMS-SIM can provide similar results.     

Mutagenic target for hydroxyl radicals generated in Escherichia coli mutant deficient in Mn- and Fe- superoxide dismutases and Fur,a repressor for iron-uptake systems

We previously reported that mutations in Mn- and Fe-superoxide dismutases and Fur, a repressor for iron uptake systems, simulated generation of hydroxyl radicals, and caused hypermutability in Escherichia coli. The predominant type of spontaneous mutation was GC --> TA, followed by AT --> CG, suggesting the involvement of 7,8-dihydro-8-oxoguanine (8-oxoG) and 1,2-dihydro-2-oxoadenine (2-oxoA) in DNA as well as 7,8-dihydro-8-oxodeoxyguanosine triphosphate (8-oxodGTP) and 1,2-dihydro-2-oxodeoxyadenosine triphosphate (2-oxodATP) in the nucleotide pool. To determine the targets contributing to oxidative mutagenesis, DNA or nucleotides, we characterized spontaneous mutations and compared the distribution to those in mutMY and mutT strains, in which GC --> TA and AT --> CG were predominantly induced, respectively. The hotspots and sequence contexts where AT --> CG occurred frequently in sodAB fur strain were almost identical to those in mutT strain,whereas, those where GC --> TA occurred frequently in sodAB fur strain were quite different from those in mutMY strain. These observations suggested that AT --> CG is due to 8-oxodGTP, while GC --> TA is produced by some other lesion(s). The 2-oxodATP is also a major oxidative lesion in nucleotides, and strongly induces GC --> TA. The expression of cDNA for MTH1, which can hydrolyze 2-oxodATP as well as 8-oxodGTP, partially but significantly, suppressed the GC --> TA mutator phenotype of the sodAB fur strain, whereas, it did not for the mutMY strain. Additionally, the sequence contextby 2-oxodATP in E. coli was similar to that in sodAB fur strain. These results suggested that the targets contributing to oxidative mutagenesis in sodAB fur strain are nucleotides such as dGTP and dATP, rather than DNA.     

Mammalian 8-oxoguanine DNA glycosylase 1 incises 8-oxoadenine opposite cytosine in nuclei and mitochondria,while a different glycosylase incises 8-oxoadenine opposite guanine in nuclei

Cells are continuously exposed to oxidative species, which cause several types of oxidative DNA lesions. Repair of some of these lesions has been well characterized but little is known about the repair of many DNA lesions. The oxidized adenine base, 7,8-dihydro-8-oxoadenine (8-oxoA), is a relatively common DNA lesion, which is believed to be mutagenic in mammalian cells. This study investigates repair of 8-oxoA in nuclear and mitochondrial mammalian extracts. In nuclei, 8-oxoA:C and 8-oxoA:G base pairs are recognized and cleaved; in contrast, only 8-oxoA:C base pairs are cleaved in mitochondria. High stability of the DNA helix increased the efficiency of incision of 8-oxoA, and the efficiency decreased at DNA bends and condensed regions of the helix. Using liver extracts from mice knocked out for 8-oxoguanine DNA glycosylase 1 (OGG1), we demonstrated that OGG1 is the only glycosylase that incises 8-oxoA, when base-paired with cytosine in mitochondria and nuclei, but a different enzyme incises 8-oxoA when base-paired with guanine in the nucleus. Consistent with this result, a covalent DNA-protein complex was trapped using purified human OGG1 or human nuclear or mitochondrial extracts with a DNA substrate containing an 8-oxoA:C base pair.     

Genetic effects of oxidative DNA damage: comparative mutagenesis of 7,8-dihydro-8-oxoguanine and 7,8-dihydro-8-oxoadenine in Escherichia coli.

A single 7,8-dihydro-8-oxoguanine (G8-OXO; 8-hydroxyguanine) adduct in the lacZ alpha gene of bacteriophage M13 DNA induces a targeted G-->T transversion after replication in Escherichia coli (Biochemistry, 29, 7024-7031 (1990)). This mutation is thought to be due to the facile formation during DNA synthesis of a G8-OXO.base pair, where G8-OXO is in the syn conformation about the deoxyglycosyl bond. A related modified purine, 7,8-dihydro-8-oxoadenine (A8-OXO; 8-hydroxyadenine), is an abundant product found in irradiated and oxidized DNAs. Similar to G8-OXO, as a mononucleoside A8-OXO assumes the syn conformation. This work has assessed the relative mutagenicities of A8-OXO and G8-OXO in the same experimental system. A deoxypentanucleotide containing A8-OXO [d(GCT-A8-OXOG)] was synthesized. After 5'-phosphorylation with [gamma-32P] ATP, the oligomer was ligated into a duplex M13mp19-derived genome at a unique NheI restriction site. Genomes containing either A8-OXO (at position 6275, [+] strand) or G8-OXO (position 6276) were denatured with heat and introduced into E.coli DL7 cells. Analysis of phage DNA from mutant plaques obtained by plating immediately after transformation (infective centers assay) revealed that G8-OXO induced G-->T transversions at an apparent frequency of approximately 0.3%. The frequency and spectrum of mutations observed in DNA sequences derived from 172 mutant plaques arising from the A8-OXO-modified DNA were almost indistiguishable from those generated from transfection of an adenine-containing control genome. We conclude that A8-OXO is at least an order of magnitude less mutagenic than G8-OXO in E.coli cells with normal DNA repair capabilities.     

Recognition and kinetics for excision of a base lesion within clustered DNA damage by the Escherichia coli proteins Fpg and Nth

Ionizing radiation and radiomimetic anticancer agents induce clustered DNA damages that are thought to lead to deleterious biological consequences, due to the challenge that clustered damage may present to the repair machinery of the cell. Specific oligonucleotides, containing either dihydrothymine (DHT) or 7,8-dihydro-8-oxoguanine (8-oxoG) opposite to specific lesions at defined positions on the complementary strand, have been used to determine the kinetic constants, K(M), k(cat), and specificity constants, for excision of DHT and 8-oxoG by the Escherichia coli base excision repair proteins, endonuclease III (Nth) and formamidopyrimidine glycosylase (Fpg), respectively. For excision of DHT opposite to 8-oxoadenine by Nth or Fpg proteins, or 8-oxoG opposite to 8-oxoG by Fpg, the major change in the specificity constant occurs when the second lesion on the complementary strand is one base to the site opposite to DHT or 8-oxoG. The specificity constants for excision of DHT or 8-oxoG by both proteins are reduced by up to 2 orders of magnitude when an abasic site or a strand break is opposite on the complementary strand. Whereas the values of K(M) are only slightly affected by the presence of a second lesion, the major change is seen as a reduction in the values of k(cat). The binding of Fpg protein to oligonucleotides containing 8-oxoG is inhibited, particularly when a single strand break is near to 8-oxoG on the complementary strand. It is inferred that not only the binding affinity of Fpg protein to the base lesion but also the rate of excision of the damaged base is reduced by the presence of another lesion, particularly a single strand break or an AP site on the complementary strand.     

Role of iron and superoxide for generation of hydroxyl radical, oxidative DNA lesions, and mutagenesis in Escherichia coli

We measured the generation of hydroxyl radical (OH(.)) and oxidative DNA lesions in aerobically grown Escherichia coli cells lacking in both superoxide dismutases (SodA SodB) and repressor of iron uptake (Fur) using electroparamagnetic resonance and gas chromatography-mass spectrometry with a selected-ion monitoring method. A specific signal corresponding to OH(.) generation and an increase in oxidative DNA lesions such as 7,8-dihydro-8-oxoguanine and 1,2-dihydro-2-oxoadenine were detected in the strain deficient in sodA sodB fur. We showed that iron metabolism deregulation in fur mutant produced a 2.5-fold iron overload. The sodA sodB fur strain was about 100-fold higher mutability than the wild-type strain. The mutation spectrum in the strain was found to induce GC --> TA and AT --> CG transversions predominantly. The hypermutability of the strain was suppressed by the tonB mutation which reduces iron transport. Thus, excess iron and excess superoxide were responsible for OH(.) generation, oxidative DNA lesion formation, and hypermutability in E. coli.     

Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine.

Oxidative damage to DNA bases commonly resultsin the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8-oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8-oxoG was investigated in vitro. A synthetic template containing a single 8-oxoG lesion was first treated with different one-electron oxidants or under singlet oxygen conditions and then subjected to primer extension catalyzed by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase alpha (pol alpha) or human DNA polymerase beta (pol beta). Consistent with previous reports, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DNA polymerases. Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base. Furthermore, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta. Experiments with an 8-oxoA-modified template oligonucleotide show that both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by Kf exo-. Mass spectrometric analysis of 8-oxoG-containing oligonucleotides before and after oxidation with IrCl62-are consistent with oxidation of primarily the 8-oxoG site, resulting in formation of a guanidinohydantoin moiety as the major product. No evidence for formation of abasic sites was obtained. These results demonstrate that an oxidized form of 8-oxoG, possibly guanidinohydantoin, may direct misreading and misinsertion of dNTPs during DNA synthesis. If such a process occurred in vivo, it would represent a point mutagenic lesion leading to G-->T and G-->C transversions. However, the corresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.     

Novel DNA Damage Mediated by Oxidation of Various Modified Nucleotide Residues

Abstract

Permanganate reaction of DNA oligomers containing an 8-oxoadenine or 5hydroxyuracil residue was studied, and the results were compared with those for an 8-oxoguanine-containing oligomers. We obtained similar results and found that the nucleotide residues neighboring the modified base were damaged and that the novel damage was induced by the oxidation of the modified base.     

Mutagenic potentials of damaged nucleic acids produced by reactive oxygen/nitrogen species: approaches using synthetic oligonucleotides and nucleotides: survey and summary

DNA and DNA precursors (deoxyribonucleotides) suffer damage by reactive oxygen/nitrogen species. They are important mutagens for organisms, due to their endogenous formation. Damaged DNA and nucleotides cause alterations of the genetic information by the mispairing properties of the damaged bases, such as 8-hydroxyguanine (7,8-dihydro- 8-oxoguanine) and 2-hydroxyadenine. Here, the author reviews the mutagenic potentials of damaged bases in DNA and of damaged DNA precursors formed by reactive oxygen/nitrogen species, focusing on the results obtained with synthetic oligonucleotides and 2′-deoxyribonucleoside 5′-triphosphates.     

Base excision and DNA binding activities of human alkyladenine DNA glycosylase are sensitive to the base paired with a lesion

The human alkyladenine DNA glycosylase has a broad substrate specificity, excising a structurally diverse group of damaged purines from DNA. To more clearly define the structural and mechanistic bases for substrate specificity of human alkyladenine DNA glycosylase, kinetics of excision and DNA binding activities were measured for several different damaged and undamaged purines within identical DNA sequence contexts. We found that 1,N(6)-ethenoadenine (epsilonA) and hypoxanthine (Hx) were excised relatively efficiently, whereas 7,8-dihydro-8-oxoguanine, O(6)-methylguanine, adenine, and guanine were not. Single-turnover kinetics of excision of Hx and epsilonA paired with T showed that excision of Hx was about four times faster than epsilonA, whereas binding assays showed that the binding affinity was about five times greater for epsilonA than for Hx. The opposing pyrimidine base had a significant effect on the kinetics of excision and DNA binding affinity of Hx but a small effect on those for epsilonA. Surprisingly, replacing a T with a U opposite Hx dramatically reduced the excision rate by a factor of 15 and increased the affinity by a factor of 7-8. The binding affinity of human alkyladenine DNA glycosylase to a DNA product containing an abasic site was similar to that for an Hx lesion.     

7,8-Dihydro-8-oxo-1,N6-ethenoadenine: an exclusively Hoogsteen-paired thymine mimic in DNA that induces A→T transversions in Escherichia coli

This work investigated the structural and biological properties of DNA containing 7,8-dihydro-8-oxo-1,N⁶-ethenoadenine (oxo-ϵA), a non-natural synthetic base that combines structural features of two naturally occurring DNA lesions (7,8-dihydro-8-oxoadenine and 1,N⁶-ethenoadenine). UV-, CD-, NMR spectroscopies and molecular modeling of DNA duplexes revealed that oxo-ϵA adopts the non-canonical syn conformation (χ = 65º) and fits very well among surrounding residues without inducing major distortions in local helical architecture. The adduct remarkably mimics the natural base thymine. When considered as an adenine-derived DNA lesion, oxo-ϵA was >99% mutagenic in living cells, causing predominantly A→T transversion mutations in Escherichia coli. The adduct in a single-stranded vector was not repaired by base excision repair enzymes (MutM and MutY glycosylases) or the AlkB dioxygenase and did not detectably affect the efficacy of DNA replication in vivo. When the biological and structural data are viewed together, it is likely that the nearly exclusive syn conformation and thymine mimicry of oxo-ϵA defines the selectivity of base pairing in vitro and in vivo, resulting in lesion pairing with A during replication. The base pairing properties of oxo-ϵA, its strong fluorescence and its invisibility to enzymatic repair systems in vivo are features that are sought in novel DNA-based probes and modulators of gene expression.     

Method for the Synthesis of Uric Acid Derivatives

Abstract

A general procedure to obtain tetra-substituted uric acid by stepwise N-alkylation is described. 2,6-Dichloropurine (1) was condensed with 1-propanol by Mitsunobu reaction to give 9-propyl congener (2). Treatment of 2 with ammonia gave adenine derivative (4a), which was converted to the 8-oxoadenine (5b) in 3 steps. Methylation of 5b proceeded site-specifically to give 6-amino-2-chloro-7,8-dihydro-7-methyl-9-propylpurin-8-one (6) as a sole product. Compound 6 was successively treated with NaNO₂ and iodomethane to give 2-chloro-1,6,7,8-tetrahydro-1,7-dimethyl-9-propylpurin-6,8-dione (9) accompanied by the O ⁶-methyl product (8) in 75% and 6.9%, respectively. After nucleophilic substitution of 9 with NaOAc, the product (11) was reacted with iodomethane to give the uric acid (12) and the 2-methoxy product (13) in 46% and 15.5%, respectively. However, the reaction of 11 with the benzylating agents gave only O-benzyl products (14a,b).