Cellular exposure to tobacco-specific nitrosamines causes formation of promutagenic O -[4-oxo-4-(3-pyridyl)but-1-yl]guanine (O -POB-G) and O -methylguanine (O -Me-G) adducts in DNA. These adducts can be directly repaired by O -alkylguanine-DNA alkyltransferase (AGT). Repair begins by flipping the damaged base out of the DNA helix. AGT binding and base-flipping have been previously studied using pyrrolocytosine as a fluorescent probe paired to the O -alkylguanine lesion, but low fluorescence yield limited the resolution of steps in the repair process. Here, we utilize the highly fluorescent 6-phenylpyrrolo-2′-deoxycytidine (6-phenylpyrrolo-C) to investigate AGT-DNA interactions. Synthetic oligodeoxynucleotide duplexes containing O -POB-G and O -Me-G adducts were placed within the CpG sites of codons 158, 245, and 248 of the p53 tumor suppressor gene and base-paired to 6-phenylpyrrolo-C in the opposite strand. Neighboring cytosine was either unmethylated or methylated. Stopped-flow fluorescence measurements were performed by mixing the DNA duplexes with C145A or R128G AGT variants. We observe a rapid, two-step, nearly irreversible binding of AGT to DNA followed by two slower steps, one of which is base-flipping. Placing 5-methylcytosine immediately 5′ to the alkylated guanosine causes a reduction in rate constant of nucleotide flipping. O -POB-G at codon 158 decreased the base flipping rate constant by 3.5-fold compared with O -Me-G at the same position. A similar effect was not observed at other codons.© 2020 Wiley Periodicals LLC.