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

Aside from abasic sites and ribonucleotides, the DNA adduct N-7-methyl deoxyguanosine (N-7-CH3 dG) is one of the most abundant lesions in mammalian DNA. Because N-7-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-7-CH3 2 '-F dG) and examined its miscoding potential with four Y-family translesion synthesis DNA polymerases (pols): human pol (hpol) eta, hpol kappa, and hpol iota and Dpo4 from the archaeal thermophile Sulfolobus solfataricus. We found that hpol eta and Dpo4 can bypass the N-7-CH3 2 '-F dG adduct, albeit with some stalling, but hpol kappa is strongly blocked at this lesion site, whereas hpol iota showed no distinction with the lesion and the control templates. hpol eta yielded the highest level of misincorporation opposite the adduct by inserting dATP or dTTP. Moreover, hpol eta did not extend well past an N-7-CH3 2 '-F dG:dT mispair. MS-based sequence analysis confirmed that hpol eta catalyzes mainly error-free incorporation of dC, with misincorporation of dA and dG in 5-10% of products. We conclude that N-7-CH3 2 '-F dG and, by inference, N-7-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.