Site-Specific Synthesis of Oligonucleotides Containing 6-Oxo-MdG, the Genomic Metabolite of MdG, and Liquid Chromatography-Tandem Mass Spectrometry Analysis of Its In Vitro Bypass by Human Polymerase ι.

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, MdG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. MdG in genomic DNA is enzymatically oxidized to 6-oxo-MdG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-MdG and the results of extension experiments aimed at determining the effect of the 6-oxo-MdG lesion on the activity of human polymerase iota (hPol ι). For this purpose, a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to obtain reliable quantitative data on the utilization of poorly incorporated nucleotides. Results demonstrate that hPol ι primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-MdG with approximately equal efficiency, whereas deoxyadenosine triphosphate (dATP) and deoxyguanosine triphosphate (dGTP) are poor substrates. Following the incorporation of a single nucleotide opposite the lesion, 6-oxo-MdG blocks further replication by the enzyme.