Specific Incorporation of an Artificial Nucleotide Opposite a Mutagenic DNA Adduct by a DNA Polymerase
| Autor: | Ursina Suter, Arman Nilforoushan, Fritz Eichenseher, Shana J. Sturla, Laura A. Wyss, Andreas Marx, Nina Blatter |
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| Rok vydání: | 2014 |
| Předmět: | |
| Zdroj: | Journal of the American Chemical Society. 137:30-33 |
| ISSN: | 1520-5126 0002-7863 |
| DOI: | 10.1021/ja5100542 |
| Popis: | The ability to detect DNA modification sites at single base resolution could significantly advance studies regarding DNA adduct levels, which are extremely difficult to determine. Artificial nucleotides that are specifically incorporated opposite a modified DNA site offer a potential strategy for detection of such sites by DNA polymerase-based systems. Here we investigate the action of newly synthesized base-modified benzimidazole-derived 2′-deoxynucleoside-5′-O-triphosphates on DNA polymerases when performing translesion DNA synthesis past the pro-mutagenic DNA adduct O6-benzylguanine (O6-BnG). We found that a mutated form of KlenTaq DNA polymerase, i.e., KTqM747K, catalyzed O6-BnG adduct-specific processing of the artificial BenziTP in favor of the natural dNTPs. Steady-state kinetic parameters revealed that KTqM747K catalysis of BenziTP is 25-fold more efficient for template O6-BnG than G, and 5-fold more efficient than natural dTMP misincorporation in adduct bypass. Furthermore, the nucleotide analogue BenziTP is required for full-length product formation in O6-BnG bypass, as without BenziTP the polymerase stalls at the adduct site. By combining the KTqM747K polymerase and BenziTP, a first round of DNA synthesis enabled subsequent amplification of Benzi-containing DNA. These results advance the development of technologies for detecting DNA adducts. |
| Databáze: | OpenAIRE |
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