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Specific Incorporation of an Artificial Nucleotide Opposite a Mutagenic DNA Adduct by a DNA Polymerase

Specific Incorporation of an Artificial Nucleotide Opposite a Mutagenic DNA Adduct by a DNA Polymerase

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WYSS, Laura A., Arman NIFOROUSHAN, Fritz EICHENSEHER, Ursina SUTER, Nina BLATTER, Andreas MARX, Shana J. STURLA, 2015. Specific Incorporation of an Artificial Nucleotide Opposite a Mutagenic DNA Adduct by a DNA Polymerase. In: Journal of the American Chemical Society : JACS. 137(1), pp. 30-33. ISSN 0002-7863. eISSN 1520-5126. Available under: doi: 10.1021/ja5100542

@article{Wyss2015Speci-30771, title={Specific Incorporation of an Artificial Nucleotide Opposite a Mutagenic DNA Adduct by a DNA Polymerase}, year={2015}, doi={10.1021/ja5100542}, number={1}, volume={137}, issn={0002-7863}, journal={Journal of the American Chemical Society : JACS}, pages={30--33}, author={Wyss, Laura A. and Niforoushan, Arman and Eichenseher, Fritz and Suter, Ursina and Blatter, Nina and Marx, Andreas and Sturla, Shana J.} }

2015-04-17T06:29:39Z Sturla, Shana J. eng Wyss, Laura A. Blatter, Nina Blatter, Nina Eichenseher, Fritz 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 O<sup>6</sup>-benzylguanine (O<sup>6</sup>-BnG). We found that a mutated form of KlenTaq DNA polymerase, i.e., KTqM747K, catalyzed O<sup>6</sup>-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 O<sup>6</sup>-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 O<sup>6</sup>-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. Eichenseher, Fritz Suter, Ursina Sturla, Shana J. Marx, Andreas Niforoushan, Arman 2015-04-17T06:29:39Z Niforoushan, Arman Specific Incorporation of an Artificial Nucleotide Opposite a Mutagenic DNA Adduct by a DNA Polymerase 2015 Wyss, Laura A. Marx, Andreas Suter, Ursina

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