Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-Methyladenine in Human Cells

DNA N-6-methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope -labeled deoxynucleoside [N-15(5)]-2'-deoxyadenosine ([N-15(5)]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [N-15(5)]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine N-15, and further utilizes the purine salvage pathway to generate mainly both [N-15(4)]-dA and [N-15(4)]-2'-deoxyguanosine ([N-15(4)]-dG) in mammalian genomes. However, [N-15(5)]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [N-15(4)]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.