6‐phenylpyrrolocytosine as a fluorescent probe to examine nucleotide flipping catalyzed by a DNA repair protein
| Autor: | Sreenivas Kanugula, Melanie S. Rogers, Delshanee Kotandeniya, Natalia Y. Tretyakova, Jenna Fernandez, Robert H. E. Hudson, Freddys Rodriguez, John D. Lipscomb |
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| Rok vydání: | 2020 |
| Předmět: |
Alkylation
DNA Repair Guanine Biophysics Guanosine Cytidine 010402 general chemistry 01 natural sciences Biochemistry Article Biomaterials Cytosine DNA Adducts O(6)-Methylguanine-DNA Methyltransferase chemistry.chemical_compound Pyrroles A-DNA Nucleotide Base Pairing Fluorescent Dyes chemistry.chemical_classification 010405 organic chemistry Organic Chemistry General Medicine Molecular biology 0104 chemical sciences Kinetics chemistry CpG site Biocatalysis Mutagenesis Site-Directed CpG Islands Tumor Suppressor Protein p53 DNA Alkyltransferase |
| Zdroj: | Biopolymers |
| ISSN: | 1097-0282 0006-3525 |
| Popis: | Cellular exposure to tobacco-specific nitrosamines causes formation of promutagenic O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]guanine (O(6)-POB-G) and O(6)-methylguanine (O(6)-Me-G) adducts in DNA. These adducts can be directly repaired by O(6)-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(6)-alkyl-guanine 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(6)-POB-G and O(6)-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(6)-POB-G at codon 158 decreased the base flipping rate constant by 3.5-fold compared with O(6)-Me-G at the same position. A similar effect was not observed at other codons. |
| Databáze: | OpenAIRE |
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