1. DndEi Exhibits Helicase Activity Essential for DNA Phosphorothioate Modification and ATPase Activity Strongly Stimulated by DNA Substrate with a GAAC/GTTC Motif.
- Author
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Zheng T, Jiang P, Cao B, Cheng Q, Kong L, Zheng X, Hu Q, and You D
- Subjects
- Adenosine Triphosphatases chemistry, Adenosine Triphosphatases genetics, Adenosine Triphosphatases metabolism, Amino Acid Substitution, Bacterial Proteins chemistry, Bacterial Proteins genetics, Catalytic Domain, Computational Biology, DEAD-box RNA Helicases chemistry, DEAD-box RNA Helicases genetics, DNA chemistry, Databases, Genetic, Gene Deletion, Mutation, Nucleotide Motifs, Peptide Fragments genetics, Peptide Fragments metabolism, Phylogeny, Protein Interaction Domains and Motifs, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins metabolism, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Riemerella metabolism, Substrate Specificity, Adenosine Triphosphate metabolism, Bacterial Proteins metabolism, DEAD-box RNA Helicases metabolism, DNA metabolism, Phosphorothioate Oligonucleotides metabolism, Riemerella enzymology
- 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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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