Rare nucleobase DNA N6-methyladenine (6mA) is recently found in high eukaryotes (1, 2). It is of intensive interest to seek conclusive evidences to support the presence of post-replicative 6mA modification in mammal (3). Here we exploited two stable isotope-labeling strategies for indicating either nucleobase adenine (by ([15N5]-dA) (4) or N6-methyl group (by [13CD3]-L-methionine) to trace methylase-generated genomic 6mA in cells. Theoretically, methylating actions on DNA adenine base should yield genomic 6mA in a form bearing a stable isotope label. Astonishingly, by both labeling strategies, the detected 6mA is present exclusively in the non-labeled form in embryonic stem cells of human and mouse. We also found that non-labeled genomic 6mA increased with extracellular N6-methyladenine base-containing nucleosides and RNA/DNA fragments. By the use of either of [15N5]-6mA deoxynucleoside and [CD3]-m6A nucleoside as a stable-isotope tracer, we observed genomic DNA 6mA predominantly in the stable isotope-labeled form, supporting DNA polymerase-dependent incorporation of 6mA. Instead of methylases, we identified DNA polymerase l as one of major polymerases responsible for accumulation of 6mA in G1 phase of ES cells. Noteworthy, these incorporated DNA 6mA can't be eliminated by demethylase alkbh1 and by the depletion of Mettl4. Collectively, we show and prove a new source of DNA N6-methyladenine base in mammal, implicating the complexity of DNA N6-methyladenine in eukaryotes.

LC-MS method for accurate identification of DNA polymerase-mediated DNA N6-methyladenine incorporation

ACS Spring Meeting 2021

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.