The abundant DNA adduct N⁷-methyl deoxyguanosine contributes to miscoding during replication by human DNA polymerase...

Aside from abasic sites and ribonucleotides, the DNA adduct N⁷-methyl deoxyguanosine (N -CH₃ dG) is one of the most abundant lesions in mammalian DNA. Because N -CH₃ 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 -CH₃ 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 -CH₃ 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 N⁷-CH₃ 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 N⁷-CH₃ 2'-F dG and, by inference, N⁷-CH₃ 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.