6-phenylpyrrolocytosine as a fluorescent probe to examine nucleotide flipping catalyzed by a DNA repair protein

Delshanee Kotandeniya, Melanie S. Rogers, Jenna Fernandez, Sreenivas Kanugula, Robert H.E. Hudson, Freddys Rodriguez, John D. Lipscomb, Natalia Tretyakova

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


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.

Original languageEnglish (US)
Article numbere23405
Issue number1
StatePublished - Oct 24 2020

Bibliographical note

Funding 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

Publisher Copyright:
© 2020 Wiley Periodicals LLC.


  • alkylguanine
  • base-flipping
  • dealkylation
  • fluorescent probe
  • transient kinetics


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