The m6A methylation perturbs the Hoogsteen pairing-guided incorporation of an oxidized nucleotide

Abstract

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 N⁶-methyladenine (m⁶A). It has been found that certain viral infections may trigger an increase in the production of both 8-oxo-dGTP and m⁶A. The current study aims to systematically explore the effects of m⁶A methylation on HG base pairs and the consequent nucleotide incorporation. Our thermodynamic melting study shows that the m⁶A·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 m⁶A 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 m⁶A relative to A. Considering the steric bulk of the purine–purine pair between 8-oxo-dG and A, m⁶A 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 m⁶A. Moreover, the impeded incorporation of 8-oxo-dGTP opposite m⁶A has been extended to determine m⁶A at pre-defined positions in human rRNA. Our study may provide new insights into the roles of m⁶A in reducing the mutagenic potential of cellular 8-oxo-dGTP.