Dataset Information

Induction of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in isolated DNA by Fenton-type reagents.

ABSTRACT: Exposure of aqueous solutions of DNA to X- or ?-rays, which induces the hydroxyl radical as one of the major reactive oxygen species (ROS), can result in the generation of a battery of single-nucleobase and bulky DNA lesions. These include the (5'R) and (5'S) diastereomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG), which were also found to be present at appreciable levels in DNA isolated from mammalian cells and tissues. However, it remains unexplored how efficiently the cdA and cdG can be induced by Fenton-type reagents. By employing HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) with the use of the isotope-dilution technique, here we demonstrated that treatment of calf thymus DNA with Cu(II) or Fe(II), together with H2O2 and ascorbate, could lead to dose-responsive formation of both the (5'R) and (5'S) diastereomers of cdA and cdG, though the yields of cdG were 2-4 orders of magnitude lower than that of 8-oxo-7,8-dihydro-2'-deoxyguanosine. This result suggests that the Fenton reaction may constitute an important endogenous source for the formation of the cdA and cdG. Additionally, the (5'R) diastereomers of cdA and cdG were induced at markedly higher levels than the (5'S) counterparts. This latter finding, in conjunction with the previous observations of similar or greater levels of the (5'S) than (5'R) diastereomers of the two lesions in mammalian tissues, furnishes an additional line of evidence to support the more efficient repair of the (5'R) diastereomers of the purine cyclonucleosides in mammalian cells.

SUBMITTER: Guerrero CR

PROVIDER: S-EPMC3822073 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Induction of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in isolated DNA by Fenton-type reagents.

Guerrero Candace R CR Wang Jin J Wang Yinsheng Y

Chemical research in toxicology 20130904 9

Exposure of aqueous solutions of DNA to X- or γ-rays, which induces the hydroxyl radical as one of the major reactive oxygen species (ROS), can result in the generation of a battery of single-nucleobase and bulky DNA lesions. These include the (5'R) and (5'S) diastereomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG), which were also found to be present at appreciable levels in DNA isolated from mammalian cells and tissues. However, it remains unexplored how effic ...[more]

PMID: 23961697

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Project description:8,5'-Cyclopurine deoxynucleosides are unique tandem lesions containing an additional covalent bond between the base and the sugar. These mutagenic and genotoxic lesions are repaired only by nucleotide excision repair. The N-glycosidic (or C1'-N9) bond of 2'-deoxyguanosine (dG) derivatives is usually susceptible to acid hydrolysis, but even after cleavage of this bond of the cyclopurine lesions, the base would remain attached to the sugar. Here, the stability of the N-glycosidic bond and the products formed by formic acid hydrolysis of (5'S)-8,5'-cyclo-2'-deoxyguanosine (S-cdG) were investigated. For comparison, the stability of the N-glycosidic bond of 8,5'-cyclo-2',5'-dideoxyguanosine (ddcdG), 8-methyl-2'-deoxyguanosine (8-Me-dG), 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-Oxo-dG), and dG was also studied. In various acid conditions, S-cdG and ddcdG exhibited similar stability to hydrolysis. Likewise, 8-Me-dG and dG showed comparable stability, but the half-lives of the cyclic dG lesions were at least 5-fold higher than those of dG or 8-Me-dG. NMR studies were carried out to investigate the products formed after the cleavage of the C1'-N9 bond. 2-Deoxyribose generated α and β anomers of deoxyribopyranose and deoxyribopyranose oligomers following acid treatment. S-cdG gave α- and β-deoxyribopyranose linked guanine as the major products, but α and β anomers of deoxyribofuranose linked guanine and other products were also detected. The N-glycosidic bond of 8-Oxo-dG was found exceptionally stable in acid. Computational studies determined that both the protonation of the N7 atom and the rate constant in the bond breaking step control the overall kinetics of hydrolysis, but both varied for the molecules studied indicating a delicate balance between the two steps. Nevertheless, the computational approach successfully predicted the trend observed experimentally. For 8-Oxo-dG, the low pK(a) of O(8) and N3 prevented appreciable protonation, making the free energy for N-glycosidic bond cleavage in the subsequent step very high.

Project description:Reactive oxygen species generate many lesions in DNA, including R and S diastereomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG). Herein, the result of replication of a plasmid containing S-cdA in Escherichia coli is reported. S-cdA was found mutagenic and highly genotoxic. Viability and mutagenicity of the S-cdA construct were dependent on functional pol V, but mutational frequencies (MFs) and types varied in pol II- and pol IV-deficient strains relative to the wild-type strain. Both S-cdA → T and S-cdA → G substitutions occurred in equal frequency in wild-type E. coli, but the frequency of S-cdA → G dropped in pol IV-deficient strain, especially when being SOS induced. This suggests that pol IV plays a role in S-cdA → G mutations. MF increased significantly in pol II-deficient strain, suggesting pol II's likely role in error-free translesion synthesis. Primer extension and steady-state kinetic studies using pol IV, exo-free Klenow fragment (KF (exo(-))), and Dpo4 were performed to further assess the replication efficiency and fidelity of S-cdA and S-cdG. Primer extension by pol IV mostly stopped before the lesion, although a small fraction was extended opposite the lesion. Kinetic studies showed that pol IV incorporated dCMP almost as efficiently as dTMP opposite S-cdA, whereas it incorporated the correct nucleotide dCMP opposite S-cdG 10-fold more efficiently than any other dNMP. Further extension of each lesion containing pair, however, was very inefficient. These results are consistent with the role of pol IV in S-cdA → G mutations in E. coli. KF (exo(-)) was also strongly blocked by both lesions, but it could slowly incorporate the correct nucleotide opposite them. In contrast, Dpo4 could extend a small fraction of the primer to a full-length product on both S-cdG and S-cdA templates. Dpo4 incorporated dTMP preferentially opposite S-cdA over the other dNMPs, but the discrimination was only 2- to 8-fold more proficient. Further extension of the S-cdA:T and S-cdA:C pair was not much different. For S-cdG, conversely, the wrong nucleotide, dTMP, was incorporated more efficiently than dCMP, although one-base extension of the S-cdG:T pair was less efficient than the S-cdG:C pair. S-cdG, therefore, has the propensity to cause G → A transition, as was reported to occur in E. coli. The results of this study are consistent with the strong replication blocking nature of S-cdA and S-cdG, and their ability to initiate error-prone synthesis by Y-family DNA polymerases.

Dataset Information

Induction of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in isolated DNA by Fenton-type reagents.

Publications

Induction of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine in isolated DNA by Fenton-type reagents.

Similar Datasets

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