Oxidation of Guanine by Carbonate Radicals Derived from Photolysis of Carbonatotetramminecobalt(III) Complexes and the pH Dependence of Intrastrand DNA Cross‐Links Mediated by Guanine Radical Reactions

Abstract

The carbonate radical anion CO3•− is a decomposition product of nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, an important biological byproduct of the inflammatory response. The selective oxidation of guanine in DNA by CO3•− radicals is known to yield spiroiminodihydantoin (Sp), guanidinohydantoin (Gh), and a novel intrastrand cross-linked product, 5’-d(CCATCG*CT*ACC) between guanine C8 (G*) and thymine N3 (T*) atoms in the oligonucleotide (Crean et al., Nucleic Acids Res., 2008, 36, 742–755). Involvement of the T-N3 (pKa of N3-H is 9.67) suggests that the formation of 5’-d(CCATCG*CT*ACC) might be pH – dependent. This hypothesis was tested generating CO3•− radicals by the photodissociation of carbonatotetramminecobalt(III) complexes by steady-state UV irradiation that allowed for studies of product yields in the pH 5.0 – 10.0 range. The yields of 5’-d(CCATCG*CT*ACC) is ~ 45 times greater at pH 10.0 than at pH 5.0, which is consistent with the proposed mechanism that requires N3(H) thymine proton dissociation followed by nucleophilic addition to the C8 guanine radical.

DNA Sequence Context as a Determinant of the Quantity and Chemistry of Guanine Oxidation Produced by Hydroxyl Radicals and One-electron Oxidants

“…5) are consistent with the higher proportion of Fpg-sensitive lesions associated with riboflavin compared with ␥-radiation (68). The major lesions observed to date with riboflavin consist of Fpg-sensitive 8-oxo-G and piperidine-sensitive Ox (39, 40, 68 -70), along with smaller amounts of spiroiminodihydantoin, which is sensitive to Fpg (46,71) and partially labile in piperidine (47). The major stable G oxidation products in hydroxyl radical-induced DNA damage (7,72,73) include 8-oxo-G and Ox, along with Fapy-G, which is sensitive to both Fpg and piperidine (40).…”

Oxidatively Generated Guanine(C8)-Thymine(N3) Intrastrand Cross-links in Double-stranded DNA Are Repaired by Base Excision Repair Pathways

“…4). In contrast, denaturing gel electrophoresis showed that the standard hot piperidine treatment cleaves 5Ј-CCA-TCG*CT*ACC at G* and T* sites with the release of 5Ј-CCATCp and 5Ј-pACC fragments according to MALDI-TOF/MS analysis (27). This is clear evidence that hot alkali is unable to hydrolyze the G*[C8-N3]T* bond to form the intact T that is generated by the bifunctional DNA glycosylases and AP endonucleases (Figs.…”

“…3 and 4). The G*[C8-N3]T* bond is also resistant to nuclease P1, which generates cross-linked dinucleotide d(G*-T*) and d(G*pT*) fragments after complete hydrolysis of the single-stranded DNA containing G*CT* and G*T* IntraCL, respectively (10,27). The phosphodiesterases 1 and 2 do not cleave the phosphodiester bonds between the crosslinked nucleotides (Fig.…”

“…The phosphodiesterases 1 and 2 do not cleave the phosphodiester bonds between the crosslinked nucleotides (Fig. 2), and the combined action of these enzymes generates only d(G*pCpT*) and d(G*pT*) fragments (10,27).…”

“…Since the absorption spectra of these cross-linked and normal duplexes are identical, it is not straightforward to verify the presence of these lesions (10). While mass spectrometry combined with two-dimensional 13 C NMR yields definitive characterization (10), a more simple approach is to monitor the stepwise degradation of the single-stranded oligonucleotides using exonucleases that stall at the sites of the cross-linked nucleotides (27). The MALDI-TOF/MS analysis of the digestion products of G*CT* and G*T* duplexes generated by snake venom phosphodiesterase 1 (digestion from the 3Ј-end) and calf spleen phosphodiesterase 2 (digestion from the 5Ј-end) clearly demonstrated the presence and integrity of the intrastrand crosslinks in these duplexes (Fig.…”