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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.)

DNA phosphorothioate (PT) modifications, with one non-bridging phosphate oxygen replaced with sulfur, are widely but sporadically distributed in prokaryotic genomes. Short consensus sequences surround the modified linkage in each strain, although each site is only partially modified. The mechanism that maintains this low-frequency modification status is still unknown. In Salmonella enterica serovar Cerro 87, PT modification is mediated by a four-gene cluster called dptBCDE. Here, we found that deletion of dptB led to a significant increase in intracellular PT modification level. In this deletion, transcription of downstream genes was elevated during rapid cell growth. Restoration of dptB on a plasmid restored wild-type levels of expression of downstream genes and PT modification. In vitro, DptB directly protected two separate sequences within the dpt promoter region from DNase I cleavage. Each protected sequence contained a direct repeat (DR). Mutagenesis assays of the DRs demonstrated that each DR was essential for DptB binding. The observation of two shifted species by gel-shift analysis suggests dimer conformation of DptB protein. These DRs are conserved among the promoter regions of dptB homologs, suggesting that this regulatory mechanism is widespread. These findings demonstrate that PT modification is regulated at least in part at the transcriptional level., (© 2015 John Wiley & Sons Ltd.)

A wide variety of prokaryotes possess DNA modifications consisting of sequence-specific phosphorothioates (PT) inserted by members of a five-gene cluster. Recent genome mapping studies revealed two unusual features of PT modifications: short consensus sequences and partial modification of a specific genomic site in a population of bacteria. To better understand the mechanism of target selection of PT modifications that underlies these features, we characterized the substrate recognition of the PT-modifying enzymes termed DptC, D and E in a cell extract system from Salmonella. The results revealed that double-stranded oligodeoxynucleotides underwent de novo PT modification in vitro, with the same modification pattern as in vivo, i. e., GpsAAC/GpsTTC motif. Unexpectedly, in these in vitro analyses we observed no significant effect on PT modification by sequences flanking GAAC/GTTC motif, while PT also occurred in the GAAC/GTTC motif that could not be modified in vivo. Hemi-PT DNA also served as substrate of the PT-modifying enzymes, but not single-stranded DNA. The PT-modifying enzymes were then found to function as a large protein complex, with all of three subunits in tetrameric conformations. This study provided the first demonstration of in vitro DNA PT modification by PT-modifying enzymes that function as a large protein complex.

Prokaryotes protect their genomes from foreign DNA with a diversity of defence mechanisms, including a widespread restriction-modification (R-M) system involving phosphorothioate (PT) modification of the DNA backbone. Unlike classical R-M systems, highly partial PT modification of consensus motifs in bacterial genomes suggests an unusual mechanism of PT-dependent restriction. In Salmonella enterica, PT modification is mediated by four genes dptB-E, while restriction involves additional three genes dptF-H. Here, we performed a series of studies to characterize the PT-dependent restriction, and found that it presented several features distinct with traditional R-M systems. The presence of restriction genes in a PT-deficient mutant was not lethal, but instead resulted in several pathological phenotypes. Subsequent transcriptional profiling revealed the expression of > 600 genes was affected by restriction enzymes in cells lacking PT, including induction of bacteriophage, SOS response and DNA repair-related genes. These transcriptional responses are consistent with the observation that restriction enzymes caused extensive DNA cleavage in the absence of PT modifications in vivo. However, overexpression of restriction genes was lethal to the host in spite of the presence PT modifications. These results point to an unusual mechanism of PT-dependent DNA cleavage by restriction enzymes in the face of partial PT modification., (© 2014 John Wiley & Sons Ltd.)