The abundant DNA adduct

Aside from abasic sites and ribonucleotides, the DNA adduct N(7)-methyl deoxyguanosine (N(7)-CH(3) dG) is one of the most abundant lesions in mammalian DNA. Because N(7)-CH(3) dG is unstable, leading to deglycosylation and ring-opening, its miscoding potential is not well-understood. Here, we employed a 2′-fluoro isostere approach to synthesize an oligonucleotide containing an analog of this lesion (N(7)-CH(3) 2′-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) η, hpol κ, and hpol ι and Dpo4 from the archaeal thermophile Sulfolobus solfataricus. We found that hpol η and Dpo4 can bypass the N(7)-CH(3) 2′-F dG adduct, albeit with some stalling, but hpol κ is strongly blocked at this lesion site, whereas hpol ι showed no distinction with the lesion and the control templates. hpol η yielded the highest level of misincorporation opposite the adduct by inserting dATP or dTTP. Moreover, hpol η did not extend well past an N(7)-CH(3) 2′-F dG:dT mispair. MS-based sequence analysis confirmed that hpol η catalyzes mainly error-free incorporation of dC, with misincorporation of dA and dG in 5–10% of products. We conclude that N(7)-CH(3) 2′-F dG and, by inference, N(7)-CH(3) dG have miscoding and mutagenic potential. The level of misincorporation arising from this abundant adduct can be considered as potentially mutagenic as a highly miscoding but rare lesion.