O⁶-Methylguanine induces altered proteins at the level of transcription in human cells

O⁶-Methylguanine (O⁶-meG), which is produced in DNA following exposure to methylating agents, instructs human RNA polymerase II to mis-insert bases opposite the lesion during transcription. In this study, we examined the effect of O⁶-meG on transcription in human cells and investigated the subsequent effects on protein function following translation of the resulting mRNA. In HEK293 cells, O⁶-meG induced incorporation of uridine or cytidine in nascent RNA opposite the adduct. In cells containing active O⁶-alkylguanine-DNA alkyltransferase (AGT), which repairs O⁶-meG, 3 misincorporation of uridine was observed opposite the lesion. In cells where AGT function was compromised by addition of the AGT inhibitor O⁶-benzylguanine, ∼58 of the transcripts contained a uridine misincorporation opposite the lesion. Furthermore, the altered mRNA induced changes to protein function as demonstrated through recovery of functional red fluorescent protein (RFP) from DNA coding for a non-fluorescent variant of RFP. These data show that O⁶-meG is highly mutagenic at the level of transcription in human cells, leading to an altered protein load, especially when AGT is inhibited.