In vivo mutational characterization of DndE involved in DNA phosphorothioate modification.

Chongde Lai,Lianrong Wang,Teng Huang,Shuangju Wu,Xi Chen,Xiaolin Wu,Zixin Deng,Meijia Gu,Chao Chen,Xiaolin Xiong,Shi Chen,Shuang-Yong Xu

doi:10.1371/journal.pone.0107981

Abstract

DNA phosphorothioate (PT) modification is a recently identified epigenetic modification that occurs in the sugar-phosphate backbone of prokaryotic DNA. Previous studies have demonstrated that DNA PT modification is governed by the five DndABCDE proteins in a sequence-selective and R P stereo-specific manner. Bacteria may have acquired this physiological modification along with dndFGH as a restriction-modification system. However, little is known about the biological function of Dnd proteins, especially the smallest protein, DndE, in the PT modification pathway. DndE was reported to be a DNA-binding protein with a preference for nicked dsDNA in vitro; the binding of DndE to DNA occurs via six positively charged lysine residues on its surface. The substitution of these key lysine residues significantly decreased the DNA binding affinities of DndE proteins to undetectable levels. In this study, we conducted site-directed mutagenesis of dndE on a plasmid and measured DNA PT modifications under physiological conditions by mass spectrometry. We observed distinctive differences from the in vitro binding assays. Several mutants with lysine residues mutated to alanine decreased the total frequency of PT modifications, but none of the mutants completely eliminated PT modification. Our results suggest that the nicked dsDNA-binding capacity of DndE may not be crucial for PT modification and/or that DndE may have other biological functions in addition to binding to dsDNA.

Highlights

Phosphorothioate linkage was originally developed as an artificial means to stabilize oligonucleotides against nuclease degradation [1]
DNA PT modification was first discovered in Streptomyces lividans, and it was later found to be widespread across a range of species due to the horizontal transfer of the dnd genes [3,5,6,7]
To evaluate the influence of the positively charged lysine residues on DNA PT modification under physiological conditions, mutations were made in pJTU1238 to generate a set of plasmids, pWHU668 to pWHU674, carrying the dnd gene cluster and encoding DndE derivatives (Table 1)

Summary

Introduction

Phosphorothioate linkage was originally developed as an artificial means to stabilize oligonucleotides against nuclease degradation [1]. PT linkages are susceptible to Tris peroxide, which accumulates on the anode during conventional or pulsed-field gel electrophoresis, resulting in DNA degradation [3,4]. DNA PT modification was first discovered in Streptomyces lividans, and it was later found to be widespread across a range of species due to the horizontal transfer of the dnd genes [3,5,6,7]. A three-gene cluster, dndFGH, which is located adjacent to the dndBCDE in approximately 86 bacterial species constitutes a restrictionmodification system to restrict foreign DNA. This system displays similarities to methylation-based R-M systems [9,10,11,12]

Methods

Results

Conclusion