Mapping three guanine oxidation products along DNA following exposure to three types of reactive oxygen species.

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-ß-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.

Influence of C-5 substituted cytosine and related nucleoside analogs on the formation of benzo[a]pyrene diol epoxide-dG adducts at CG base pairs of DNA.

Endogenous 5-methylcytosine ((Me)C) residues are found at all CG dinucleotides of the p53 tumor suppressor gene, including the mutational 'hotspots' for smoking induced lung cancer. (Me)C enhances the reactivity of its base paired guanine towards carcinogenic diolepoxide metabolites of polycyclic aromatic hydrocarbons (PAH) present in cigarette smoke. In the present study, the structural basis for these effects was investigated using a series of unnatural nucleoside analogs and a representative PAH diolepoxide, benzo[a]pyrene diolepoxide (BPDE). Synthetic DNA duplexes derived from a frequently mutated region of the p53 gene (5'-CCCGGCACCC GC[(15)N(3),(13)C(1)-G]TCCGCG-3', + strand) were prepared containing [(15)N(3), (13)C(1)]-guanine opposite unsubstituted cytosine, (Me)C, abasic site, or unnatural nucleobase analogs. Following BPDE treatment and hydrolysis of the modified DNA to 2'-deoxynucleosides, N(2)-BPDE-dG adducts formed at the [(15)N(3), (13)C(1)]-labeled guanine and elsewhere in the sequence were quantified by mass spectrometry. We found that C-5 alkylcytosines and related structural analogs specifically enhance the reactivity of the base paired guanine towards BPDE and modify the diastereomeric composition of N(2)-BPDE-dG adducts. Fluorescence and molecular docking studies revealed that 5-alkylcytosines and unnatural nucleobase analogs with extended aromatic systems facilitate the formation of intercalative BPDE-DNA complexes, placing BPDE in a favorable orientation for nucleophilic attack by the N(2) position of guanine.

Synthesis of DNA oligodeoxynucleotides containing structurally defined N6-(2-hydroxy-3-buten-1-yl)-adenine adducts of 3,4-epoxy-1-butene.

3,4-Epoxy-1-butene (EB) is generated by cytochrome P450-mediated epoxidation of 1,3-butadiene (BD), an important environmental and industrial chemical classified as a probable human carcinogen. The ability of EB to induce point mutations at GC and AT base pairs has been attributed to its reactions with DNA to form covalent nucleobase adducts. Guanine alkylation is preferred at the endocyclic N7 nitrogen, while adenine can be modified at the N1-, N3-, N7-, and the N6 positions. For each of these sites, a pair of regioisomeric 2-hydroxy-3-buten-1-yl and 1-hydroxy-3-buten-2-yl adducts is produced as a result of epoxide ring opening at the terminal C-4 or the internal C-3 carbon position of EB, respectively. The N6-EB-adenine adducts are of particular interest because of their stability in DNA, potentially leading to their accumulation in vivo. In the present work, synthetic DNA oligomers containing structurally defined N6-(2-hydroxy-3-buten-1-yl)-dA (N6-HB-dA) adducts were prepared for the first time by a postoligomerization approach that involved coupling 6-chloropurine-containing DNA with synthetic 1-amino-3-buten-2-ol. N6-HB-dA-containing DNA oligomers were isolated by reversed phase HPLC, and the presence of N6-HB-dA in their structure was confirmed by molecular weight determination from HPLC-ESI- -MS of the intact strands and by HPLC-ESI+-MS/MS and MS/MS/MS analyses of the enzymatic digests using synthetic N6-HB-dA as an authentic standard. N6-HB-dA-containing oligomers generated in this study will be used for structural and biological studies.