Insertion of dGMP and dAMP during in vitro DNA synthesis opposite an oxidized form of 7,8-dihydro-8-oxoguanine

Oxidative damage to DNA bases commonly results in the formation of oxidized purines, particularly 7,8-dihydro-8-oxoguanine (8-oxoG) and 7,8-dihydro-8- oxoadenine (8-oxoA), the former being a well-known mutagenic lesion. Since 8-oxoG is readily subject to further oxidation compared with normal bases, the insertion of a base during DNA synthesis opposite an oxidized form of 8 -oxoG was investigated in vitro, A synthetic template containing a single 8 -oxoG lesion was first treated with different one-electron oxidants or unde r singlet oxygen conditions and then subjected to primer extension catalyze d by Klenow fragment exo- (Kf exo-), calf thymus DNA polymerase a (pol alph a) or human DNA polymerase beta (pol beta), Consistent with previous report s, dAMP and dCMP are inserted selectively opposite 8-oxoG with all three DN A polymerases, Interestingly, oxidation of 8-oxoG was found to induce dAMP and dGMP insertion opposite the lesion by Kf exo- with transient inhibition of primer extension occurring at the site of the modified base, Furthermor e, the lesion constitutes a block during DNA synthesis by pol alpha and pol beta, Experiments with an 8-oxoA-modified template oligonucleotide show th at both 8-oxoA and an oxidized form of 8-oxoA direct insertion of dTMP by K f exo-, Mass spectrometric analysis of 8-oxoG-containing oligonucleotides b efore and after oxidation with IrCl62- are consistent with oxidation of pri marily the 8-oxoG site, resulting in formation of a guanidinohydantoin moie ty as the major product, No evidence for formation of abasic sites was obta ined. These results demonstrate that an oxidized form of 8-oxoG, possibly g uanidinohydantoin, may direct misreading and misinsertion of dNTPs during D NA synthesis. If such a process occurred in vivo, it would represent a poin t mutagenic lesion leading to G-->T and G-->C transversions, However, the c orresponding oxidized form of 8-oxoA primarily shows correct insertion of T during DNA synthesis with Kf exo-.