Cellular exposure to tobacco-specific nitrosamines causes formation of promutagenic O6-[4-oxo-4-(3-pyridyl)but-1-yl]guanine (O6-POB-G) and O6-methylguanine (O6-Me-G) adducts in DNA. These adducts can be directly repaired by O6-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 O6-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 O6-POB-G and O6-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. O6-POB-G at codon 158 decreased the base flipping rate constant by 3.5-fold compared with O6-Me-G at the same position. A similar effect was not observed at other codons.
Bibliographical noteFunding Information:
We thank Dr. Thomas Makris (UMN) for his help with initial kinetics experiments and Dr. Colin Campbell (UMN) for expressing human AGT proteins in . This work was supported by US National Institutes of Health research grants R01 CA095039 (N. T.), R01 GM24689 (J. D. L.), and R35 GM118030 (J. D. L.). E. coli
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- fluorescent probe
- transient kinetics
PubMed: MeSH publication types
- Journal Article