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
posted on 2021-12-03, 21:04authored byPlamen
P. Christov, Robyn Richie-Jannetta, Philip J. Kingsley, Anoop Vemulapalli, Kwangho Kim, Gary A. Sulikowski, Carmelo J. Rizzo, Amit Ketkar, Robert L. Eoff, Carol A. Rouzer, Lawrence J. Marnett
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.