Site-Specific Synthesis of Oligonucleotides Containing 6-Oxo-M(1)dG, the Genomic Metabolite of M(1)dG, and Liquid Chromatography-Tandem Mass Spectrometry Analysis of Its In Vitro Bypass by Human Polymerase iota

The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M(1)dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M(1)dG in genomic DNA is enzymatically oxidized to 6-oxo-M(1)dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M(1)dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M(1)dG lesion on the activity of human polymerase iota (hPol iota). 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 iota primarily incorporates deoxycytidine triphosphate (dCTP) and thymidine triphosphate (dTTP) across from 6-oxo-M(1)dG 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-M(1)dG blocks further replication by the enzyme.