title: DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.
abstract: Phosphorothioate (PT) modification of DNA, in which the non-bridging oxygen of the backbone phosphate group is replaced by sulfur, is governed by the DndA-E proteins in prokaryotes. To better understand the biochemical mechanism of PT modification, functional analysis of the recently found PT-modifying enzyme DndEi, which has an additional domain compared with canonical DndE, from Riemerella anatipestifer is performed in this study. The additional domain is identified as a DNA helicase, and functional deletion of this domain in vivo leads to PT modification deficiency, indicating an essential role of helicase activity in PT modification. Subsequent analysis reveals that the additional domain has an ATPase activity. Intriguingly, the ATPase activity is strongly stimulated by DNA substrate containing a GAAC/GTTC motif (i.e. the motif at which PT modifications occur in R. anatipestifer) when the additional domain and the other domain (homologous to canonical DndE) are co-expressed as a full-length DndEi. These results reveal that PT modification is a biochemical process with DNA strand separation and intense ATP hydrolysis.
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030.
authors.forename: Tao
authors.initials: T
authors.lastname: Zheng
authors.affiliation: the Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Shanghai 200240, and.
authors.forename: Pan
authors.initials: P
authors.lastname: Jiang
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030.
authors.forename: Bo
authors.initials: B
authors.lastname: Cao
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030.
authors.forename: Qiuxiang
authors.initials: Q
authors.lastname: Cheng
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030.
authors.forename: Lingxin
authors.initials: L
authors.lastname: Kong
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Zheng
authors.affiliation: the Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences, Shanghai 200240, and huqinghai@caas.cn.
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Hu
authors.affiliation: From the State Key Laboratory of Microbial Metabolism, and School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai 200030, the Joint International Research Laboratory of Metabolic and Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, China dlyou@sjtu.edu.cn.
authors.forename: Delin
authors.initials: D
authors.lastname: You
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D019295:Computational Biology; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D030541:Databases, Genetic; D017353:Gene Deletion; D009154:Mutation; D059372:Nucleotide Motifs; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D054735:Phosphorothioate Oligonucleotides / Q000378:metabolism*; D010802:Phylogeny; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D060494:Riemerella / Q000201:enzymology* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D010446:Peptide Fragments; D054735:Phosphorothioate Oligonucleotides; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases; D053487:DEAD-box RNA Helicases
keywords: ATPase; DNA enzyme; DNA helicase; bacteria; microbiology