Possible cause of G·C→C·G transversion mutation by guanine oxidation product, imidazolone

Abstract

Background: The genome is constantly assaulted by oxidation reactions which are likely to be associated with oxygen metabolism, and oxidative lesions are generated by many types of oxidants. Such genotoxin-induced alterations in the genomic message have been implicated in aging and in several pathophysiological processes, particularly those associated with cancer. The guanine base (G) in genomic DNA is highly susceptible to oxidative stress due to having the lowest oxidation potential. Therefore, G·C→T·A and G·C→C·G transversion mutations frequently occur under oxidative conditions. One typical lesion of G is 8-oxo-7,8-dihydro-guanine (8-oxoG), which can pair with A. This pairing may cause G·C→T·A transversion mutations. Although the number of G·C→C·G transversions is rather high under specific oxidation conditions such as riboflavin photosensitization, the molecular basis of G·C→C·G transversions is not known. Results: To determine which oxidative products are responsible for G·C→C·G transversion mutations, we photooxidized 5′-d(AAAAAAGGAAAAAA)/5′-d(TTTTTTCCTTTTTT) using either riboflavin or anthraquinone (AQ) carboxylate under UV irradiation. Prolonged low-temperature (4°C) enzymatic digestion of photoirradiated sample indicated that under both conditions the amount of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) initially increased with decreasing amounts of 2′-deoxyguanosine (dG), then decreased with the formation of 2-amino-5-[(2-deoxy-β- D- erythro-pentofuranosyl)amino]-4 H-imidazol-4-one (dIz), suggesting that nascent 8-oxoG was further oxidized to 2,5-diamino-4 H-imidazol-4-one (Iz) in duplex DNA. Photoirradiation of an AQ-linked oligomer with a complementary strand containing 8-oxoG indicated that 8-oxoG residues were oxidized to Iz. These results indicate that Iz is formed from 8-oxoG through long-range hole migration. Primer extension experiments using a template containing Iz demonstrated that only dGTP is specifically incorporated opposite Iz suggesting that specific Iz·G base pairs are formed. The ‘reverse’ approach consisting of DNA polymerization using dIzTP showed that dIzTP is incorporated opposite G, further confirming the formation of a Iz·G base pair. Conclusions: HPLC product analysis demonstrated that Iz is a key oxidation product of G through 8-oxoG in DNA photosensitized with riboflavin or anthraquinone. Photoreaction of AQ-linked oligomer confirmed that Iz is formed from 8-oxoG through long-range hole migration. Two sets of primer extension experiments demonstrated that Iz can specifically pair with G in vitro. Specific Iz·G base pair formation can explain the G·C→C·G transversion mutations that appear under oxidative conditions.