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

Abstract

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.

Scheme 1

(A and B) Postulated hydroxyl radical mediated pathway to Fapy·dG, 8-oxo-dG and Fapy·dA, 8-oxo-dA.

Scheme 2

A general scheme of construction, replication and analysis of the control and modified single-stranded pMS2 vectors.

Figure 1

(A and B) show the constructs with 5′-TGT and 5′-TGA sequence, respectively. Lanes 1 and 2, pMS2 DNA before and after digestion with EcoRV. Lanes 3–5 show pMS2 constructs containing dG, 8-oxo-dG and Fapy·dG, respectively, after enzymatic removal of the scaffold 58mer. Lanes 6 and 7 represent, before and after the removal of the scaffold, respectively, of a ‘mock’ ligation mixture, which did not contain a dodecamer.

Figure 2

anti-8-Oxo-G opposite dCTP (A and C) and syn-8-oxo-G opposite dATP (B and D) in the 5′-TGT and 5′-TGA sequences in the active site of pol β, following MD. Only the base pairs involving 8-oxo-G and the dNTP are shown, along with the base pair on the 3′-side of the 8-oxo-G. The bases are 8-oxo-G (red), A (yellow) and T (green), dCTP (gray) and dATP (purple).

Figure 3

anti-Fapy-G opposite dCTP (A and C) and syn-Fapy-G opposite dATP (B and D) in the 5′-TGT and 5′-TGA sequences in the active site pol β, following MD. Only the base pairs involving Fapy·G and the dNTP are shown, along with the base pair on the 3′-side of the Fapy-G. The bases are Fapy·G (red), A (yellow) and T (green), dCTP (gray) and dATP (purple).

Figure 4

syn-8-Oxo-A (red) opposite dGTP (purple) in the active site pol β. Following MD, a syn-8-oxo-G:dATP structure (5′-TGT sequence) was converted to syn-8-oxo-A:dGTP by simple base replacement. In the base pairing region, overlapping van der Waals contacts are apparent. A wobble is one way to generate a reasonable hydrogen bonded structure (see text). The dGTP (purple) cannot wobble toward the minor groove because of R283 (blue) and Y271 (light blue), so syn-8-oxo-A (red) would have to wobble toward the major groove, in which there is enough room because of the positioning of I33 (green) and H34 (dark green).