The m(6)A methylation perturbs the Hoogsteen pairing-guided incorporation of an oxidized nucleotide

Natural nucleic acid bases can form Watson–Crick (WC) or Hoogsteen (HG) base pairs. Importantly, 8-oxo-2′-deoxyguanosine (8-oxo-dG) in DNA or 8-oxo-dG 5′-triphosphate (8-oxo-dGTP) favors a syn conformation because of the steric repulsion between O8 and O4′ of the deoxyribose ring. 8-oxo-dGTP can be incorporated into DNA opposite the templating adenine (A) using HG pairing as the dominant mechanism. Both RNA and DNA can be methylated at the N6 position of A to form N (6)-methyladenine (m(6)A). It has been found that certain viral infections may trigger an increase in the production of both 8-oxo-dGTP and m(6)A. The current study aims to systematically explore the effects of m(6)A methylation on HG base pairs and the consequent nucleotide incorporation. Our thermodynamic melting study shows that the m(6)A·8-oxo-dG is significantly less stable than the A·8-oxo-dG base pair in the paired region of a DNA duplex. Moreover, we have used pre-steady-state kinetics to examine the incorporation of 8-oxo-dGTP opposite m(6)A relative to A by a variety of reverse transcriptase (RT) enzymes and DNA polymerase (DNA pol) enzymes such as the human immunodeficiency virus type 1 (HIV-1) RT and human DNA pol β. The results demonstrate that all of these enzymes incorporate 8-oxo-dGTP less efficiently opposite m(6)A relative to A. Considering the steric bulk of the purine–purine pair between 8-oxo-dG and A, m(6)A methylation may affect the HG pairing to a great extent. Hence, it will be unfavorable to incorporate 8-oxo-dGTP into the growing strand opposite m(6)A. Moreover, the impeded incorporation of 8-oxo-dGTP opposite m(6)A has been extended to determine m(6)A at pre-defined positions in human rRNA. Our study may provide new insights into the roles of m(6)A in reducing the mutagenic potential of cellular 8-oxo-dGTP.