8-hydroxyguanine and oxaluric-acid

The DNA oxidation product 7,8-dihydro-8-oxoguanine (8-oxoG) forms several mutagenic oxidation products, including a metastable oxaluric acid (Oa) derivative. We report here that a synthetic oligonucleotide containing Oa hydrolyzes under simulated "in vivo" conditions to form a mutagenic urea (Ua) lesion. Using the Oa 2'-deoxyribonucleoside as a model, the hydrolysis rate depended strongly upon the concentrations of bicarbonate and divalent magnesium. In buffered solutions containing physiologically relevant levels of these species, the half-life of Oa nucleoside was approximately 40 h at 37 degrees C. The mutagenic properties of Ua in DNA were investigated using a M13mp7L2 bacteriophage genome containing Ua at a specific site. Transfection of the lesion-containing genome into wild-type AB1157 Escherichia coli allowed determination of the mutation frequency and DNA polymerase bypass efficiency from the resulting progeny phage. Ua was bypassed with an efficiency of 11% as compared to a guanine control and caused a 99% G-->T mutation frequency, assuming the lesion originated from G, which is at least an order of magnitude higher than the mutation frequency of 8-oxoG under the same conditions. SOS induction of bypass DNA polymerase(s) in the bacteria prior to transfection caused the mutation frequency and type to shift to 43% G-->T, 46% G-->C, and 10% G-->A mutations. We suggest that Ua is instructional, meaning that the shape of the lesion and its interactions with DNA polymerases influence which nucleotide is inserted opposite the lesion during replication and that the instructional nature of the lesion is modulated by the size of the binding pocket of the DNA polymerase. Replication past Ua, when formed by hydrolysis of the 8-oxoG oxidation product Oa, denotes a pathway that nearly quantitatively generates point mutations in vivo.

Topics: DNA; DNA Damage; Escherichia coli; Guanine; Hydrolysis; Mutagenesis; Mutagens; Oxamic Acid; Oxidation-Reduction; Point Mutation; SOS Response, Genetics; Transfection; Urea

Oxidative reactions within DNA commonly result in base modifications. Among the four DNA bases, guanine is the most susceptible to various oxidants, and its related oxidized form, 8-oxo-7,8-dihydroguanine, has been extensively studied in terms of repair and mutagenicity. However, 8-oxo-7,8-dihydroguanine is readily subjected to further oxidation, and this has become a point of interest. We recently found that singlet oxygen oxidation of 8-oxo-7,8-dihydroguanine led to the predominant formation of oxaluric acid as the final product. We report herein on the biological features of oxaluric acid dealing with in vitro DNA synthesis and its removal from DNA by repair enzymes. Nucleotide insertion opposite oxaluric acid, catalyzed by Kf exo(-) and Taq indicates, that oxaluric acid induces G to T and G to C transversions. On the other hand, oxaluric acid represents a block when synthesis is performed with pol beta. Interestingly, DNA repair experiments carried out with formamidopyrimidine DNA N-glycosylase (Fpg) and endonuclease III (endo III) show that oxaluric acid is a substrate for both enzymes. Values of k(cat)/K(m) for the Fpg-mediated removal of oxidative guanine lesions revealed that 8-oxoGua is only a slightly better substrate than oxaluric acid. Interestingly, the results obtained with endo III suggest that oxaluric acid is a much better substrate than is 5-hydroxycytosine (5-OHC), an oxidized pyrimidine base.

Topics: Animals; Cattle; Deoxyribonuclease (Pyrimidine Dimer); DNA; DNA Damage; DNA Polymerase beta; DNA Polymerase I; DNA Repair; Endodeoxyribonucleases; Escherichia coli Proteins; Free Radicals; Guanine; Kinetics; Mutagenesis; Oligonucleotides; Oxamic Acid; Oxidation-Reduction; Reactive Oxygen Species; Substrate Specificity; Taq Polymerase

8-Oxoguanine (8-oxo-G) is one of the most common DNA lesions present in normal tissues due to exposure to reactive oxygen species. Studies at this and other laboratories suggest that 8-oxo-G is highly susceptible to secondary oxidation, making it a likely target for endogenous oxidizing agents, such as peroxynitrite (ONOO-). Synthetic oligonucleotides containing 8-oxoguanine were treated with ONOO-, and the reaction products were analyzed by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI--MS). CCACAACXCAAA, CCAAAGGXAGCAG, CCAAAXGGAGCAG, and TCCCGAGCGGCCAAAGGXAGCAG (X is 8-oxo-G) were found to readily react with peroxynitrite via the same transformations as those observed for free 8-oxo-2'-deoxyguanosine. The composition of the reaction mixtures was a function of ONOO- concentration and of the storage time after exposure. The oligonucleotide products isolated at low [ONOO-]/[DNA] ratios (<5) were tentatively assigned as containing 3a-hydroxy-5-imino-3,3a,4,5-tetrahydro-1H-imidazo[4, 5d]imidazol-2-one, 5-iminoimidazolidine-2,4-dione, and its hydrolytic product, oxaluric acid. At a [ONOO-]/[DNA] ratio of >10, 2,4,6-trioxo[1,3,5]triazinane-1-carboxamidine- and cyanuric acid-containing oligomers were the major products. The exact location of a modified base within a DNA sequence was determined using exonuclease digestion of oligonucleotide products followed by LC/ESI--MS analysis of the fragments. For all 8-oxo-G-containing oligomers, independent of the sequence, the reactions with ONOO- took place at the 8-oxo-G residues. These results suggest that 8-oxo-G, if present in DNA, is rapidly oxidized by peroxynitrite and that oxaluric acid is a likely secondary oxidation product of 8-oxo-G under physiological conditions.

Topics: Chromatography, Liquid; DNA; Guanine; Indicators and Reagents; Mass Spectrometry; Nitrates; Oligonucleotides; Oxamic Acid; Oxidants