Unscheduled m6A Deposition in RNA via m6ATP Incorporation by DNA Polymerases

N⁶-methyladenosine (m⁶A) is the most abundant modification of mRNA and plays a crucial role in mediating cellular functions, and it is associated with cancer and neurodegenerative diseases. Studies have shown that m⁶A is predominantly deposited on its consensus motif by the m⁶A writer proteins RNA methyltransferase METLL3/METLL14. However, it was found that nonconventional m⁶A deposition by other alternative pathways may also exist and can modulate epitranscriptomic regulation in cells. Thus, understanding the molecular mechanisms underlying nonconventional m⁶A deposition outside the canonical motifs will provide novel insights into the full scope of the functional impact of m⁶A. In this study, we discovered that m⁶ATP was efficiently incorporated by the repairing DNA polymerases pol β and pol η through RNA gap-filling synthesis on an RNA-DNA hybrid. Steady-state kinetics results showed that m⁶ATP was incorporated into RNA by the DNA polymerases with a comparable efficiency to ATP. AlphaFold3-assisted molecular dynamics simulations further elucidated the structural basis for the DNA polymerases to incorporate m⁶ATP into the RNA substrates by showing that the enzymes employed the unique base-stacking mechanism to govern the distance between the 3'-OH group of the 3'-terminus nucleotide of the primer and the 5'-α-phosphate of m⁶ATP to perform their catalysis. Furthermore, we detected a significant amount of m⁶ATP in human cells. We showed that the m⁶ATP level was associated with that of the oxidative stress biomarker 8-oxoGTP in cells, suggesting that unscheduled m⁶A deposition on RNA can be mediated by m⁶ATP incorporation that is associated with cellular oxidative stress. Our study sheds light on the unscheduled m⁶A deposition as a potential alternative mechanism for altering epitranscriptomic modifications.