Issue 9, 2017

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.

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

Supplementary files

Article information

Article type
Edge Article
Submitted
24 May 2017
Accepted
05 Jul 2017
First published
06 Jul 2017
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2017,8, 6380-6388

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

S. Wang, Y. Song, Y. Wang, X. Li, B. Fu, Y. Liu, J. Wang, L. Wei, T. Tian and X. Zhou, Chem. Sci., 2017, 8, 6380 DOI: 10.1039/C7SC02340E

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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