DNA sequence context as a determinant of the quantity and chemistry of guanine oxidation produced by hydroxyl radicals and one-electron oxidants.
ABSTRACT: DNA sequence context has emerged as a critical determinant of the location and quantity of nucleobase damage caused by many oxidizing agents. However, the complexity of nucleobase and 2-deoxyribose damage caused by strong oxidants such as ionizing radiation and the Fenton chemistry of Fe2+-EDTA/H2O2 poses a challenge to defining the location of nucleobase damage and the effects of sequence context on damage chemistry in DNA. To address this problem, we developed a gel-based method that allows quantification of nucleobase damage in oxidized DNA by exploiting Escherichia coli exonuclease III to remove fragments containing direct strand breaks and abasic sites. The rigor of the method was verified in studies of guanine oxidation by photooxidized riboflavin and nitrosoperoxycarbonate, for which different effects of sequence context have been demonstrated by other approaches (Margolin, Y., Cloutier, J. F., Shafirovich, V., Geacintov, N. E., and Dedon, P. C. (2006) Nat. Chem. Biol. 2, 365-366). Using duplex oligodeoxynucleotides containing all possible three-nucleotide sequence contexts for guanine, the method was used to assess the role of DNA sequence context in hydroxyl radical-induced guanine oxidation associated with gamma-radiation and Fe2+-EDTA/H2O2. The results revealed both differences and similarities for G oxidation by hydroxyl radicals and by one-electron oxidation by riboflavin-mediated photooxidation, which is consistent with the predominance of oxidation pathways for hydroxyl radicals other than one-electron oxidation to form guanine radical cations. Although the relative quantities of G oxidation produced by hydroxyl radicals were more weakly correlated with sequence-specific ionization potential than G oxidation produced by riboflavin, damage produced by both hydroxyl radical generators and riboflavin within two- and three-base runs of G showed biases in location that are consistent with a role for electron transfer in defining the location of the damage products. Furthermore, both gamma-radiation and Fe2+-EDTA/H2O2 showed relatively modest effects of sequence context on the proportions of different damage products sensitive to E. coli formamidopyrimidine DNA glycosylase and hot piperidine, although GT-containing sequence contexts displayed subtle biases in damage chemistry (formamidopyrimidine DNA glycosylase/piperidine ratio). Overall, the results are consistent with the known chemistry of guanine oxidation by hydroxyl radical and demonstrate that charge migration plays a relatively minor role in determining the location and chemistry of hydroxyl radical-mediated oxidative damage to guanine in DNA.
Project description:Reactive oxygen and nitrogen species generated during respiration, inflammation, and immune response can damage cellular DNA, contributing to aging, cancer, and neurodegeneration. The ability of oxidized DNA bases to interfere with DNA replication and transcription is strongly influenced by their chemical structures and locations within the genome. In the present work, we examined the influence of local DNA sequence context, DNA secondary structure, and oxidant identity on the efficiency and the chemistry of guanine oxidation in the context of the Kras protooncogene. A novel isotope labeling strategy developed in our laboratory was used to accurately map the formation of 2,2-diamino-4-[(2-deoxy-?-D-erythropentofuranosyl)amino]-?5(2?H)-oxazolone (Z), 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG), and 8-nitroguanine (8-NO2-G) lesions along DNA duplexes following photooxidation in the presence of riboflavin, treatment with nitrosoperoxycarbonate, and oxidation in the presence of hydroxyl radicals. Riboflavin-mediated photooxidation preferentially induced OG lesions at 5' guanines within GG repeats, while treatment with nitrosoperoxycarbonate targeted 3'-guanines within GG and AG dinucleotides. Little sequence selectivity was observed following hydroxyl radical-mediated oxidation. However, Z and 8-NO2-G adducts were overproduced at duplex ends, irrespective of oxidant identity. Overall, our results indicate that the patterns of Z, OG, and 8-NO2-G adduct formation in the genome are distinct and are influenced by oxidant identity and the secondary structure of DNA.
Project description:Guanine is a major target for oxidation in DNA, with 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) as a major product. 8-oxodG is itself significantly more susceptible to oxidation than guanine, with the resulting damage consisting of more than 10 different products. This complexity has hampered efforts to understand the determinants of biologically relevant DNA oxidation chemistry. To address this problem, we have developed a high mass accuracy mass spectrometric method to quantify oxidation products arising site specifically in DNA. We applied this method to quantify the role of sequence context in defining the spectrum of damage products arising from oxidation of 8-oxodG by two oxidants: nitrosoperoxycarbonate (ONOOCO(2)(-)), a macrophage-derived chemical mediator of inflammation, and the classical one-electron oxidant, riboflavin-mediated photooxidation. The results reveal the predominance of dehydroguanidinohydantoin (DGh) in 8-oxodG oxidation by both oxidants. While the relative quantities of 8-oxodG oxidation products arising from ONOOCO(2)(-) did not vary as a function of sequence context, products of riboflavin-mediated photooxidation of 8-oxodG were highly sequence dependent. Several of the 8-oxodG oxidation products underwent hydrolytic conversion to new products with half-lives of 2-7 h. The results have implications for understanding the chemistry of DNA oxidation and the biological response to the damage, with DNA damage recognition and repair systems faced with a complex and dynamic set of damage targets.
Project description:DNA oxidation by reactive oxygen species is nonrandom, potentially leading to accumulation of nucleobase damage and mutations at specific sites within the genome. We now present the first quantitative data for sequence-dependent formation of structurally defined oxidative nucleobase adducts along p53 gene-derived DNA duplexes using a novel isotope labeling-based approach. Our results reveal that local nucleobase sequence context differentially alters the yields of 2,2,4-triamino-2H-oxal-5-one (Z) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (OG) in double stranded DNA. While both lesions are overproduced within endogenously methylated (Me)CG dinucleotides and at 5' Gs in runs of several guanines, the formation of Z (but not OG) is strongly preferred at solvent-exposed guanine nucleobases at duplex ends. Targeted oxidation of (Me)CG sequences may be caused by a lowered ionization potential of guanine bases paired with (Me)C and the preferential intercalation of riboflavin photosensitizer adjacent to (Me)C:G base pairs. Importantly, some of the most frequently oxidized positions coincide with the known p53 lung cancer mutational "hotspots" at codons 245 (GGC), 248 (CGG), and 158 (CGC) respectively, supporting a possible role of oxidative degradation of DNA in the initiation of lung cancer.
Project description:RNA oxidation is important in the etiology of disease and as a tool for studying the structure and folding kinetics of this biopolymer. Nucleobase radicals are the major family of reactive intermediates produced in RNA exposed to diffusible species such as hydroxyl radical. The nucleobase radicals are believed to produce direct strand breaks by abstracting hydrogen atoms from their own and neighboring ribose rings. By independently generating the formal C5 hydrogen atom addition product of uridine in RNA, we provide the first chemical characterization of the pathway for direct strand scission from an RNA nucleobase radical. The process is more efficient under anaerobic conditions. The preference for strand scission in double-stranded RNA over single-stranded RNA suggests that this chemistry may be useful for analyzing the secondary structure of RNA in hydroxyl radical cleavage experiments if they are carried out under anaerobic conditions.
Project description:Our study explores the antioxidant and cytoprotective effects of baicalein and further discusses the possible mechanisms. A methyl thiazolyl tetrazolium (MTT) assay revealed that baicalein could considerably enhance the viability of hydroxyl radical-treated bone marrow-mesenchymal stem cells (bmMSCs) at 37-370 µM. The highest viability rate was 120.4%. In subsequent studies, baicalein was observed to effectively scavenge hydroxyl radical and PTIO• radicals, reducing Fe3+ and Cu2+ ions. In the Fe2+-chelating UV-vis spectra, mixing of baicalein with Fe2+ yielded two evident redshifts (275 ? 279 nm and 324 ? 352 nm) and a broad absorption peak (?max ? 650 nm, ? = 1.6 × 10³ L mol-1·cm-1). Finally, we compared the Fe2+-chelating UV-vis spectra of baicalein and its analogues, including 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, catechol, pyrogallol, and chrysin. This analysis revealed that the 4-keto group of the C-ring played a role. The 5,6,7-trihydroxy-group (pyrogallol group) in the A-ring served as an auxochrome, enhancing the absorbance of the UV-vis spectra and deepening the color of the Fe2+-complex. We concluded that baicalein, as an effective hydroxyl radical-scavenger, can protect bmMSCs from hydroxyl radical-mediated oxidative stress. Its hydroxyl radical-scavenging effects are likely exerted via two pathways: direct scavenging of hydroxyl radicals, possibly through electron transfer, and indirect inhibition of hydroxyl radical generation via Fe2+ chelation through the 4-keto-5,6,7-trihydroxy groups.
Project description:It has been suggested that DNA contains sacrificial nucleobase sequences that protect sensitive regions of the genome from oxidative damage. Oxidation of DNA by loss of an electron generates a radical cation that can migrate long distances by hopping. The radical cation can be trapped irreversibly at certain sites (GG steps) by reaction with H2O or O2 leading to the formation of lesions (oxidative damage). A series of DNA oligomers that contain regularly spaced GG steps and an 8-oxo-7,8-dihydroguanine (8-oxoG), which serves as a proxy for possibly sacrificial protective low oxidation potential sites, was prepared and analyzed. We find that in certain special sequences of DNA nucleobases that 8-oxoG protects remote GG steps from oxidative damage but that this is not a general phenomenon extending to normal mixed sequence DNA. This is a consequence of the change in the relative rate of charge hopping compared with trapping of the radical cation. When hopping is relatively slow, 8-oxoG exerts no protective effect. Thus, it seems unlikely that low oxidation potential sequences play a meaningful part in protecting mixed sequence DNA from damage.
Project description:The chlorine radical is a potent atmospheric oxidant, capable of perturbing tropospheric oxidative cycles normally controlled by the hydroxyl radical. Significantly faster reaction rates allow chlorine radicals to expedite oxidation of hydrocarbons, including methane, and in polluted environments, to enhance ozone production. Here we present evidence, from the CARIBIC airborne dataset, for extensive chlorine radical chemistry associated with Asian pollution outflow, from airborne observations made over the Malaysian Peninsula in winter. This region is known for persistent convection that regularly delivers surface air to higher altitudes and serves as a major transport pathway into the stratosphere. Oxidant ratios inferred from hydrocarbon relationships show that chlorine radicals were regionally more important than hydroxyl radicals for alkane oxidation and were also important for methane and alkene oxidation (>10%). Our observations reveal pollution-related chlorine chemistry that is both widespread and recurrent, and has implications for tropospheric oxidizing capacity, stratospheric composition and ozone chemistry.
Project description:Telomere shortening occurs during oxidative and inflammatory stress with guanine (G) as the major site of damage. In this work, a comprehensive profile of the sites of oxidation and structures of products observed from G-quadruplex and duplex structures of the human telomere sequence was studied in the G-quadruplex folds (hybrid (K(+)), basket (Na(+)), and propeller (K(+) + 50% CH3CN)) resulting from the sequence 5'-(TAGGGT)4T-3' and in an appropriate duplex containing one telomere repeat. Oxidations with four oxidant systems consisting of riboflavin photosensitization, carbonate radical generation, singlet oxygen, and the copper Fenton-like reaction were analyzed under conditions of low product conversion to determine relative reactivity. The one-electron oxidants damaged the 5'-G in G-quadruplexes leading to spiroiminodihydantoin (Sp) and 2,2,4-triamino-2H-oxazol-5-one (Z) as major products as well as 8-oxo-7,8-dihydroguanine (OG) and 5-guanidinohydantoin (Gh) in low relative yields, while oxidation in the duplex context produced damage at the 5'- and middle-Gs of GGG sequences and resulted in Gh being the major product. Addition of the reductant N-acetylcysteine (NAC) to the reaction did not alter the riboflavin-mediated damage sites but decreased Z by 2-fold and increased OG by 5-fold, while not altering the hydantoin ratio. However, NAC completely quenched the CO3(•-) reactions. Singlet oxygen oxidations of the G-quadruplex showed reactivity at all Gs on the exterior faces of G-quartets and furnished the product Sp, while no oxidation was observed in the duplex context under these conditions, and addition of NAC had no effect. Because a long telomere sequence would have higher-order structures of G-quadruplexes, studies were also conducted with 5'-(TAGGGT)8-T-3', and it provided oxidation profiles similar to those of the single G-quadruplex. Lastly, Cu(II)/H2O2-mediated oxidations were found to be indiscriminate in the damage patterns, and 5-carboxamido-5-formamido-2-iminohydantoin (2Ih) was found to be a major duplex product, while nearly equal yields of 2Ih and Sp were observed in G-quadruplex contexts. These findings indicate that the nature of the secondary structure of folded DNA greatly alters both the reactivity of G toward oxidative stress as well as the product outcome and suggest that recognition of damage in telomeric sequences by repair enzymes may be profoundly different from that of B-form duplex DNA.
Project description:Materials that solidify in response to an initiation stimulus are currently utilized in several biomedical and surgical applications; however, their clinical adoption would be more widespread with improved physical properties and biocompatibility. One chemistry that is particularly promising is based on the thiol-ene addition reaction, a radical-mediated step-growth polymerization that is resistant to oxygen inhibition and thus is an excellent candidate for materials that polymerize upon exposure to aerobic conditions. Here, thiol-ene-based hydrogels are polymerized by exposing aqueous solutions of multi-functional thiol and allyl ether PEG monomers, in combination with enzymatic radical initiating systems, to air. An initiating system based on glucose oxidase, glucose, and Fe2+ is initially investigated where, in the presence of glucose, the glucose oxidase reduces oxygen to hydrogen peroxide which is then further reduced by Fe2+ to yield hydroxyl radicals capable of initiating thiol-ene polymerization. While this system is shown to effectively initiate polymerization after exposure to oxygen, the polymerization rate does not monotonically increase with raised Fe2+ concentration owing to inhibitory reactions that retard polymerization at higher Fe2+ concentrations. Conversely, replacing the Fe2+ with horseradish peroxidase affords an initiating system is that is not subject to the iron-mediated inhibitory reactions and enables increased polymerization rates to be attained.
Project description:The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'-oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well-defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, neocarzinostatin and calicheamicin gamma(1)(I), was exploited in control studies, with neocarzinostatin producing 10 2-deoxyribonolactone and 300 nucleoside 5'-aldehyde per 10(6) nt per microM in accord with its established minor 1'- and major 5'-oxidation chemistry. Calicheamicin unexpectedly caused 1'-oxidation at a low level of 10 2-deoxyribonolactone per 10(6) nt per microM in addition to the expected predominance of 5'-oxidation at 560 nucleoside 5'-aldehyde per 10(6) nt per microM. The two hydroxyl radical-mediated DNA oxidants, gamma-radiation and Fe(2+)-EDTA, produced nucleoside 5'-aldehyde at a frequency of 57 per 10(6) nt per Gy (G-value 74 nmol/J) and 3.5 per 10(6) nt per microM, respectively, which amounted to 40% and 35%, respectively, of total 2-deoxyribose oxidation as measured by a plasmid nicking assay. However, gamma-radiation and Fe(2+)-EDTA produced different proportions of 2-deoxyribonolactone at 7% and 24% of total 2-deoxyribose oxidation, respectively, with frequencies of 10 lesions per 10(6) nt per Gy (G-value, 13 nmol/J) and 2.4 lesions per 10(6) nt per microM. Studies in TK6 human lymphoblastoid cells, in which the analytical data were corrected for losses sustained during DNA isolation, revealed background levels of 2-deoxyribonolactone and nucleoside 5'-aldehyde of 9.7 and 73 lesions per 10(6) nt, respectively. Gamma-irradiation of the cells caused increases of 0.045 and 0.22 lesions per 10(6) nt per Gy, respectively, which represents a approximately 250-fold quenching effect of the cellular environment similar to that observed in previous studies. The proportions of the various 2-deoxyribose oxidation products generated by gamma-radiation are similar for purified DNA and cells. These results are consistent with solvent exposure as a major determinant of hydroxyl radical reactivity with 2-deoxyribose in DNA, but the large differences between gamma-radiation and Fe(2+)-EDTA suggest that factors other than hydroxyl radical reactivity govern DNA oxidation chemistry.