Radiation-induced formation of purine lesions in single and double stranded DNA: revised quantification.
ABSTRACT: The formation of oxidative lesions arising from double stranded DNA damage is of major significance to chemical biology from the perspective of application to human health. The quantification of purine lesions arising from ?-radiation-induced hydroxyl radicals (HO(•)) has been the subject of numerous studies, with discrepancies on the measured 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) lesions reported by different groups. Here we applied an ameliorative protocol for the analysis of DNA damage with quantitative determination of these lesions via isotope dilution liquid chromatography coupled with tandem mass spectrometry. Tandem-type purine lesions were quantified along with 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) and 7,8-dihydro-8-oxo-2'-deoxyadenosine (8-oxo-dA) in single and double stranded DNA, generated during DNA exposure to diffusible HO(•) radicals in the absence or presence of physiological levels of oxygen. The cdA and cdG lesions in absence of oxygen were found ~2 times higher in single than double stranded DNA, with 5'R being ~6.5 and ~1.5 times more predominant than 5'S in cdG and cdA, respectively. Interestingly, in the presence of 5% molecular oxygen the R/S ratios are retained with substantially decreased yields for cdA and cdG, whereas 8-oxo-dA and 8-oxo-dG remain nearly constant. The overall lesion formation follows the order: 8-oxo-dG >> 8-oxo-dA > 5'R-cdG > 5'R-cdA > 5'S-cdA > 5'S-cdG. By this method, there was a conclusive evaluation of radiation-induced DNA purine lesions.
Project description: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.
Project description:Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5',8-cyclopurines (cPu) lesions (i.e., 5'R-cdG, 5'S-cdG, 5'R-cdA and 5'S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82-2.52 lesions/106 nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10-4.11 lesions/106 nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5'R/5'S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O2 indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated.
Project description:Chronic inflammation is estimated to be a causative factor in a variety of diseases. Under inflammatory conditions reactive oxygen species (ROS) and nitrogen species (RNS) are released leading to DNA damage accumulation and genomic instability. Purine 5',8-cyclo-2'-deoxynucleosides (cPu) are oxidative DNA lesions, exclusively derived from the attack of HO• radicals, which are known to have cytotoxic and mutagenic properties. Herein, we have analyzed the presence of cPu in genomic DNA isolated from fresh colon and visceral adipose tissue biopsies collected from inflammatory bowel diseases (IBD)-affected patients and severely obese subjects, respectively, versus what observed in the control specimens. In colon biopsies, characterized by a higher gene expression level of inducible nitric oxide synthase (iNOS), a significant increase of 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) lesions and an accumulation of both diastereomeric cPu have been detected. In contrast, the 8-oxo-dA and 8-oxo-dG levels were extremely lower compared to the colon tissues values and no accumulation of cPu, in the inflamed visceral adipose tissue biopsies isolated from bariatric patients versus the lean counterpart was reported. In addition, in adipose tissue undetectable levels of iNOS have been found. These data suggest a potential involvement of cPu in the colon cancer susceptibility observed in IBD patients.
Project description:5'-R and 5'-S diastereoisomers of 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) containing a base-sugar covalent bond are formed by hydroxyl radicals. R-cdA and S-cdA are repaired by nucleotide excision repair (NER) in mammalian cellular extracts. Here, we have examined seven purified base excision repair enzymes for their ability to repair S-cdG or S-cdA. We could not detect either excision or binding of these enzymes on duplex oligonucleotide substrates containing these lesions. However, both lesions were repaired by HeLa cell extracts. Dual incisions by human NER on a 136-mer duplex generated 24-32 bp fragments. The time course of dual incisions were measured in comparison to cis-anti-B[a]P-N(2)-dG, an excellent substrate for human NER, which showed that cis-anti-B[a]P-N(2)-dG was repaired more efficiently than S-cdG, which, in turn, was repaired more efficiently than S-cdA. When NER efficiency of S-cdG with different complementary bases was investigated, the wobble pair S-cdG·dT was excised more efficiently than the S-cdG·dC pair that maintains nearly normal Watson-Crick base pairing. But S-cdG·dA mispair with no hydrogen bonds was excised less efficiently than the S-cdG·dC pair. Similar pattern was noted for S-cdA. The S-cdA·dC mispair was excised much more efficiently than the S-cdA·dT pair, whereas the S-cdA·dA pair was excised less efficiently. This result adds to complexity of human NER, which discriminates the damaged base pairs on the basis of multiple criteria.
Project description:Age-related macular degeneration (AMD) is the leading cause of blindness among older adults in the developed world. Oxidative damage to mitochondrial DNA (mtDNA) in the retinal pigment epithelium (RPE) may play a key role in AMD. Measurement of oxidative DNA lesions such as 8-oxo-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-2'-deoxyadenosine (8-oxo-dA) in diseased RPE could provide important insights into the mechanism of AMD development. We have developed a liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry method for simultaneous analysis of 8-oxo-dG and 8-oxo-dA in human retinal DNA. The developed method was applied to the analysis of retinal DNA from 5 donors with AMD and 5 control donors without AMD. In mtDNA, the levels of 8-oxo-dG in controls and AMD donors averaged 170 and 188, and 8-oxo-dA averaged 11 and 17 adducts per 10(6) bases, respectively. In nuclear DNA, the levels of 8-oxo-dG in controls and AMD donors averaged 0.54 and 0.96, and 8-oxo-dA averaged 0.04 and 0.05 adducts per 10(6) bases, respectively. This highly sensitive method allows for the measurement of both adducts in very small amounts of DNA and can be used in future studies investigating the pathophysiological role of 8-oxo-dG and 8-oxo-dA in AMD and other oxidative damage-related diseases in humans.
Project description:The hydroxyl radical is a powerful oxidant that generates DNA lesions including the stereoisomeric R and S 5',8-cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) pairs that have been detected in cellular DNA. Unlike some other oxidatively generated DNA lesions, cdG and cdA are repaired by the human nucleotide excision repair (NER) apparatus. The relative NER efficiencies of all four cyclopurines were measured and compared in identical human HeLa cell extracts for the first time under identical conditions, using identical sequence contexts. The cdA and cdG lesions were excised with similar efficiencies, but the efficiencies for both 5'R cyclopurines were greater by a factor of ?2 than for the 5'S lesions. Molecular modeling and dynamics simulations have revealed structural and energetic origins of this difference in NER-incision efficiencies. These lesions cause greater DNA backbone distortions and dynamics relative to unmodified DNA in 5'R than in 5'S stereoisomers, producing greater impairment in van der Waals stacking interaction energies in the 5'R cases. The locally impaired stacking interaction energies correlate with relative NER incision efficiencies, and explain these results on a structural basis in terms of differences in dynamic perturbations of the DNA backbone imposed by the R and S covalent 5',8 bonds.
Project description:Most mammalian cells in nature are quiescent but actively transcribing mRNA for normal physiological processes; thus, it is important to investigate how endogenous and exogenous DNA damage compromises transcription in cells. Here we describe a new competitive transcription and adduct bypass (CTAB) assay to determine the effects of DNA lesions on the fidelity and efficiency of transcription. Using this strategy, we demonstrate that the oxidatively induced lesions 8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine (cdG) and the methylglyoxal-induced lesion N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) strongly inhibited transcription in vitro and in mammalian cells. In addition, cdA and cdG, but not N(2)-CEdG, induced transcriptional mutagenesis in vitro and in vivo. Furthermore, when located on the template DNA strand, all examined lesions were primarily repaired by transcription-coupled nucleotide excision repair in mammalian cells. This newly developed CTAB assay should be generally applicable for quantitatively assessing how other DNA lesions affect DNA transcription in vitro and in cells.
Project description:5',8-Cyclo-2'-deoxyadenosine (cdA) and 5',8-cyclo-2'-deoxyguanosine (cdG) are lesions resulting from hydroxyl radical (HO (·) ) attack on the 5'H of the nucleoside sugar moiety and exist in both 5'R and 5'S diastereomeric forms. Increased levels of cdA and cdG are linked to Nucleotide Excision Repair (NER) mechanism deficiency and mutagenesis. Discrepancies in the damage measurements reported over recent years indicated the weakness of the actual protocols, in particular for ensuring the quantitative release of these lesions from the DNA sample and the appropriate method for their analysis. Herein we report the detailed revision leading to a cost-effective and efficient protocol for the DNA damage measurement, consisting of the nuclease benzonase and nuclease P1 enzymatic combination for DNA digestion followed by liquid chromatography isotope dilution tandem mass spectrometry analysis.
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.
Project description:5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase ? (pol ?) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol ? wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol ? knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol ? plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol ? inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol ?, indicating a mutagenic effect induced by pol ? lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol ? generated an intermediate that failed to be extended by pol ?, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol ? plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol ? bypassing of a cdA lesion.