8-Oxoguanine Affects DNA Backbone Conformation in the EcoRI Recognition Site and Inhibits Its Cleavage by the Enzyme.

Joanna J Hoppins,Dmitry O Zharkov,Alexey S Kiryutin,Serge L Smirnov,Stanislav O Fedechkin,David R Gruber,Darya V Petrova,Alexander V Popov,Heather L Miears,Alexandra V Yurkovskaya,Rustem D Kasymov,Anton V Endutkin,Jacob A Brockerman,Shuang-Yong Xu

doi:10.1371/journal.pone.0164424

Abstract

8-oxoguanine is one of the most abundant and impactful oxidative DNA lesions. However, the reasons underlying its effects, especially those not directly explained by the altered base pairing ability, are poorly understood. We report the effect of the lesion on the action of EcoRI, a widely used restriction endonuclease. Introduction of 8-oxoguanine inside, or adjacent to, the GAATTC recognition site embedded within the Drew—Dickerson dodecamer sequence notably reduced the EcoRI activity. Solution NMR revealed that 8-oxoguanine in the DNA duplex causes substantial alterations in the sugar—phosphate backbone conformation, inducing a BI→BII transition. Moreover, molecular dynamics of the complex suggested that 8-oxoguanine, although does not disrupt the sequence-specific contacts formed by the enzyme with DNA, shifts the distribution of BI/BII backbone conformers. Based on our data, we propose that the disruption of enzymatic cleavage can be linked with the altered backbone conformation and dynamics in the free oxidized DNA substrate and, possibly, at the protein—DNA interface.

Highlights

8-oxoguanine is a naturally abundant base lesion with a well characterized mutagenic potential [1]
We report that the activity of restriction endonuclease EcoRI is inhibited by oxoGplaced within, or adjacent to, the recognition site despite the apparent conservation of the protein—DNA interface
In an attempt to follow possible biological consequences of oxoG in DNA, we have compared the activity of EcoRI restriction endonuclease on its natural substrate and in several contexts containing oxoG

Summary

Introduction

8-oxoguanine (oxoG) is a naturally abundant base lesion with a well characterized mutagenic potential [1]. Transcription factors AP-1 and Sp1, methyl-CpG-binding protein MeCP2, and telomere protecting proteins TERF1 and TERF2 poorly bind oxoG-containing recognition sequences [15,16,17], whereas binding of NFκB to its promoter element may either decrease or increase depending on oxoG location in the sequence [18]. This interference of oxoG with normal protein—DNA interactions may potentially have biological consequences such as improper regulation of gene expression, continuing survival of the invasive foreign DNA with a damaged restriction site in a bacterial cell, etc. Our solution NMR analysis of oxoG-containing DNA substrates provides a possible structural explanation (backbone alteration) of this effect

Materials and Methods

Results

Discussion