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LA Wyss, A Nilforoushan, F Eichenseher, U Suter, N Blatter, A Marx and SJ Sturla
Abstract
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 investigated newly synthesized base-modified benzimidazole-derived 2´-deoxynucleoside-5´-O-triphosphates in their action on DNA polymerases when performing translesion DNA synthesis past the pro-mutagenic DNA adduct O(6)-benzylguanine (O(6)-BnG). We found that a mutated form of KlenTaq DNA polymerase, i.e. KTqM747K catalyzed O(6)-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(6)-BnG than G, and 5-fold more efficient than natural dTMP misincorporation in adduct bypass. Furthermore, the nucleotide analog BenziTP is required for full-length product formation in O(6)-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.
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Concepts
DNA polymerase I, Reverse transcriptase, DNA polymerase III holoenzyme, Base pair, DNA replication, Polymerase, DNA polymerase, DNA
MeSH headings
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