Fig. 1. Genomic incorporation of DNA N⁶-methyladenine and the contribution of DNA polymerase λ.

a Flow diagram of tracing DNA 6mA in mES cells by [¹⁵N₅]-dA or [¹³CD₃]-L-methionine. (b, c) UHPLC-MS/MS chromatograms (b) and quantification (c) of unlabeled dA, [¹⁵N₄]-dA, and [¹⁵N₅]-dA in the genome of mES cells. (d, e) UHPLC-MS/MS chromatograms (d) and quantification (e) of unlabeled 6mA, [¹⁵N₄]-6mA, and [¹⁵N₅]-6mA in the genome of mES cells. Note: [¹⁵N₅]-dA was used as an initiation tracer and would be converted into [¹⁵N₄]-dA in genomic DNA.⁹ The distinct cell cycle phases are indicated in (b–e). (f, g) UHPLC-MS/MS quantification of [¹³CD₃]-5mC (f) and [¹³CD₃]-6mA (g) in the genomes of non-synchronized and G1 phase ES cells. [¹³CD₃]-L-methionine was used for tracing stable isotope-labeled methyl group. h UHPLC-MS/MS quantification of genomic 6mA levels in pol λ^+/+ and pol λ^−/− mES cells. The unsorted cells at G1 phase were directly obtained by L-mimosine treatment. The sorted cells at sub G1 and G1 phases were obtained by flow cytometry sorting of the L-mimosine-treated cells. i UHPLC-MS/MS quantification of the labeled genomic 6mA ([CD₃]-6mA) levels in pol λ^+/+ and pol λ^−/− mES cells. The cells were treated with [CD₃]-m⁶A alone or co-treated with [CD₃]-m⁶A and L-mimosine. ND, not detected.