Genetic effects of oxidative DNA damages: comparative mutagenesis of the imidazole ring-opened formamidopyrimidines (Fapy lesions) and 8-oxo-purines in simian kidney cells.

Fapy.dG and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) are formed in DNA by hydroxyl radical damage. In order to study replication past these lesions in cells, we constructed a single-stranded shuttle vector containing the lesion in 5'-TGT and 5'-TGA sequence contexts. Replication of the modified vector in simian kidney (COS-7) cells showed that Fapy.dG is mutagenic inducing primarily targeted Fapy.G-->T transversions. In the 5'-TGT sequence mutational frequency of Fapy.dG was approximately 30%, whereas in the 5'-TGA sequence it was approximately 8%. In parallel studies 8-oxo-dG was found to be slightly less mutagenic than Fapy.dG, though it also exhibited a similar context effect: 4-fold G-->T transversions (24% versus 6%) occurred in the 5'-TGT sequence relative to 5'-TGA. To investigate a possible structural basis for the higher G-->T mutations induced by both lesions when their 3' neighbor was T, we carried out a molecular modeling investigation in the active site of DNA polymerase beta, which is known to incorporate both dCTP (no mutation) and dATP (G-->T substitution) opposite 8-oxo-G. In pol beta, the syn-8-oxo-G:dATP pair showed greater stacking with the 3'-T:A base pair in the 5'-TGT sequence compared with the 3'-A:T in the 5'-TGA sequence, whereas stacking for the anti-8-oxo-G:dCTP pair was similar in both 5'-TGT and 5'-TGA sequences. Similarly, syn-Fapy.G:dATP pairing showed greater stacking in the 5'-TGT sequence compared with the 5'-TGA sequence, while stacking for anti-Fapy.G:dCTP pairs was similar in the two sequences. Thus, for both lesions less efficient base stacking between the lesion:dATP pair and the 3'-A:T base pair in the 5'-TGA sequence might cause lower G-->T mutational frequencies in the 5'-TGA sequence compared to 5'-TGT. The corresponding lesions derived from 2'-deoxyadenosine, Fapy.dA and 8-oxo-dA, were not detectably mutagenic in the 5'-TAT sequence, and were only weakly mutagenic (<1%) in the 5'-TAA sequence context, where both lesions induced targeted A-->C transversions. To our knowledge this is the first investigation using extrachromosomal probes containing a Fapy.dG or Fapy.dA site-specifically incorporated, which showed unequivocally that in simian kidney cells Fapy.G-->T substitutions occur at a higher frequency than 8-oxo-G-->T and that Fapy.dA is very weakly mutagenic, as is 8-oxo-dA.