1. Site-Specific Synthesis of Oligonucleotides Containing 6-Oxo-M1dG, the Genomic Metabolite of M1dG, and Liquid Chromatography–Tandem Mass Spectrometry Analysis of Its In Vitro Bypass by Human Polymerase ι
- Author
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Philip J. Kingsley, Carmelo J. Rizzo, Plamen P. Christov, Kwangho Kim, Anoop Vemulapalli, Robert L. Eoff, Carol A. Rouzer, Amit Ketkar, Lawrence J. Marnett, Gary A. Sulikowski, and Robyn Richie-Jannetta
- Subjects
chemistry.chemical_classification ,biology ,Deoxyguanosine triphosphate ,Deoxycytidine triphosphate ,Oligonucleotide ,General Medicine ,Toxicology ,chemistry.chemical_compound ,chemistry ,Biochemistry ,Deoxyadenosine triphosphate ,biology.protein ,heterocyclic compounds ,Nucleotide ,Polymerase ,DNA ,Thymidine triphosphate - Abstract
The lipid peroxidation product malondialdehyde and the DNA peroxidation product base-propenal react with dG to generate the exocyclic adduct, M1dG. This mutagenic lesion has been found in human genomic and mitochondrial DNA. M1dG in genomic DNA is enzymatically oxidized to 6-oxo-M1dG, a lesion of currently unknown mutagenic potential. Here, we report the synthesis of an oligonucleotide containing 6-oxo-M1dG and the results of extension experiments aimed at determining the effect of the 6-oxo-M1dG 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-M1dG 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-M1dG blocks further replication by the enzyme.
- Published
- 2021