Oxidative Generation of Guanine Radicals by Carbonate Radicals and Their Reactions with Nitrogen Dioxide to Form Site Specific 5-Guanidino-4-nitroimidazole Lesions in Oligodeoxynucleotides

Abstract

A simple photochemical approach is described for synthesizing site specific, stable 5-guanidino-4-nitroimidazole (NIm) adducts in single- and double-stranded oligodeoxynucleotides containing single and multiple guanine residues. The DNA sequences employed, 5'-d(ACC CG(1)C G(2)TC CG(3)C G(4)CC) and 5'-d(ACC CG(1)C G(2)TC C), were a portion of exon 5 of the p53 tumor suppressor gene, including the codons 157 (G(2)) and 158 (G(3)) mutation hot spots in the former sequence with four Gs and the codon 157 (G(2)) mutation hot spot in the latter sequence with two Gs. The nitration of oligodeoxynucleotides was initiated by the selective photodissociation of persulfate anions to sulfate radicals induced by UV laser pulses (308 nm). In aqueous solutions, of bicarbonate and nitrite anions, the sulfate radicals generate carbonate anion radicals and nitrogen dioxide radicals by one electron oxidation of the respective anions. The guanine residue in the oligodeoxynucleotide is oxidized by the carbonate anion radical to form the neutral guanine radical. While the nitrogen dioxide radicals do not react with any of the intact DNA bases, they readily combine with the guanine radicals at either the C8 or the C5 positions. The C8 addition generates the well-known 8-nitroguanine (8-nitro-G) lesions, whereas the C5 attack produces unstable adducts, which rapidly decompose to NIm lesions. The maximum yields of the nitro products (NIm + 8-nitro-G) were typically in the range of 20-40%, depending on the number of guanine residues in the sequence. The ratio of the NIm to 8-nitro-G lesions gradually decreases from 3.4 in the model compound, 2',3',5'-tri-O-acetylguanosine, to 2.1-2.6 in the single-stranded oligodeoxynucleotides and to 0.8-1.1 in the duplexes. The adduct of the 5'-d(ACC CG(1)C G(2)TC C) oligodeoxynucleotide containing the NIm lesion in codon 157 (G(2)) was isolated in HPLC-pure form. The integrity of this adduct was established by a detailed analysis of exonuclease digestion ladders by matrix-assisted laser desorption ionization with time-of-flight detection MS techniques.

Oxidative DNA Damage Associated with Combination of Guanine and Superoxide Radicals and Repair Mechanisms via Radical Trapping

“…The duplex solutions were irradiated for a longer time (40 s) because the two-photon ionization of 2AP in the duplex is less efficient than in the single-stranded oligonucleotide. The reversed-phase HPLC separation of the end products proved difficult because of the formation of secondary structures (42). Nevertheless, we were able to isolate Formation of the Iz lesions was confirmed by the complete enzymatic digestion of the irradiated samples followed by HPLC-MS/MS analysis.…”

DNA Polymerase V Allows Bypass of Toxic Guanine Oxidation Products in Vivo

“…Additionally, 8-nitroguanine forms directly from G and is used as a biomarker of inflammation (52). Given that 8-nitroguanine and NI form in double-stranded DNA in approximately equal proportions in vitro and appear to arise mechanistically from the same neutral G radical precursor (53), NI may form in vivo under the conditions that also generate 8-nitroguanine.…”

In Vivo Bypass Efficiencies and Mutational Signatures of the Guanine Oxidation Products 2-Aminoimidazolone and 5-Guanidino-4-nitroimidazole

“…Oxazolone is bypassed efficiently and exhibits a mutation frequency similar to that of Iz in vivo (86%) but preferentially generates G3 T transversion mutations (31). In distinct contrast, NI has been observed only as a result of ONOO Ϫ -related chemistry (28,48,49). NI is a chemically stable lesion (when heated to 90°C for 6 h at pH 7, ϳ10 -15% decomposition of an ODN containing NI occurs) (49) and is refractory to repair by Fpg glycosylase (MutM) and endonuclease III in vitro (48).…”