Aberrant DNA N6‐methyladenine incorporation via adenylate kinase 1 is suppressed by ADAL deaminase‐dependent 2′‐deoxynucleotide pool sanitation

Abstract

AbstractIntracellular decay of N6‐methyladenine (m6A)‐containing RNA potentially induces aberrant N6‐methyl‐2′‐adenine (6mdA) misincorporation into DNA. Biophysically, misincorporated 6mdA may destabilize the DNA duplex in a manner similar to bona fide methylated 6mdA DNA, thereby affecting DNA replication and transcription. Utilizing heavy stable isotope labeling and ultrasensitive UHPLC–MS/MS assay, we demonstrate that intracellular m6A‐RNA decay does not generate free 6mdA species, nor lead to any misincorporated DNA 6mdA in most mammalian cell lines tested, unveiling the existence of a sanitation mechanism that prevents 6mdA misincorporation. Depletion of deaminase ADAL increases the levels of free 6mdA species, concomitant with the presence of DNA‐misincorporated 6mdA resulting from intracellular RNA m6A decay, suggesting that ADAL catabolizes 6mdAMP in vivo. Furthermore, we show that the overexpression of adenylate kinase 1 (AK1) promotes 6mdA misincorporation, while AK1 knockdown diminishes 6mdA incorporation, in ADAL‐deficient cells. We conclude that ADAL together with other factors (such as MTH1) contributes to 2′‐deoxynucleotide pool sanitation in most cells but compromised sanitation (e.g., in NIH3T3 cells) and increased AK1 expression may facilitate aberrant 6mdA incorporation. This sanitation mechanism may provide a framework for the maintenance of the epigenetic 6mdA landscape.