title: In vivo mutational characterization of DndE involved in DNA phosphorothioate modification.
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 RP 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.
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China; College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang, China.
authors.forename: Chongde
authors.initials: C
authors.lastname: Lai
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China; Taihe Hospital, Hubei University of Medicine, Shiyan, Hubei, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wu
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Chao
authors.initials: C
authors.lastname: Chen
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Teng
authors.initials: T
authors.lastname: Huang
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Xiong
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Shuangju
authors.initials: S
authors.lastname: Wu
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Meijia
authors.initials: M
authors.lastname: Gu
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Zixin
authors.initials: Z
authors.lastname: Deng
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Xi
authors.initials: X
authors.lastname: Chen
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Shi
authors.initials: S
authors.lastname: Chen
authors.affiliation: Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, and School of Pharmaceutical Sciences, Wuhan University, Wuhan, China.
authors.forename: Lianrong
authors.initials: L
authors.lastname: Wang
mesh_terms: D000409:Alanine / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics* / Q000378:metabolism; D044127:Epigenesis, Genetic; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000378:metabolism; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D019779:Salmonella enterica / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D010710:Phosphates; D054735:Phosphorothioate Oligonucleotides; D004247:DNA; D008239:Lysine; D000409:Alanine; C035638:thiophosphoric acid