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Incorporation of Oxidized Guanine Nucleoside 5′-Triphosphates in DNA with DNA Polymerases and Preparation of Single-Lesion Carrying DNA

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journal contribution
posted on 22.04.2008, 00:00 authored by Sophie Mourgues, Jérôme Trzcionka, Jean-Jacques Vasseur, Geneviève Pratviel, Bernard Meunier
We investigated the incorporation of oxidatively modified guanine residues in DNA using three DNA polymerases, Escherichia coli Kf exo+, Kf exo−, and Taq DNA polymerase. We prepared nucleoside 5′-triphosphates with modified bases (dNoxTP) including imidazolone associated with oxazolone (dIzTP/dZTP), dehydroguanidinohydantoin (dOGhTP), and oxaluric acid (dOxaTP). We showed that the single-nucleotide incorporation of these dNoxTP at the 3′-end of a primer DNA strand was possible opposite C or G for dIzTP/dZTP, opposite C for dOGhTP using the Klenow fragment, and opposite C for dOxaTP using Taq. The efficiency of these misincorporations was compared to that of the nucleoside 5′-triphosphate modified with the mutagenic guanine lesion 8-oxo-G opposite A or C as well as to that of the natural dNTPs. The reaction was found not competitive. However, the ability of Kf exo− to further copy the whole template DNA strand from the primer carrying one modified residue at the 3′-end proved to be easy and rapid. The two-step polymerization process consisting of the single-nucleotide extension followed by the full extension of a primer afforded a method for the preparation of tailored double-stranded DNA oligonucleotides carrying a single modified base at a precise site on any sequence. This very rapid method allowed the incorporation of unique residues in DNA that were not available before due to their unstable character.