Incorporation of oxidized guanine nucleoside 5'-triphosphates in DNA with DNA polymerases and preparation of single-lesion carrying DNA.
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 (dN (ox)TP) including imidazolone associated with oxazolone (dIzTP/dZTP), dehydroguanidinohydantoin (dOGhTP), and oxaluric acid (dOxaTP). We showed that the single-nucleotide incorporation of these dN (ox)TP 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.
Mourgues
Sophie
S
Laboratoire de Chimie de Coordination du CNRS, 205 Route de Narbonne, Toulouse Cedex 4, France.
Trzcionka
Jérôme
J
Vasseur
Jean-Jacques
JJ
Pratviel
Geneviève
G
Meunier
Bernard
B
D004247:DNA / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D010084:Oxidation-Reduction
D006160:Guanosine Triphosphate; D004247:DNA; D004259:DNA-Directed DNA Polymerase