Banding patterns of pHZ2031 respectively linearized using four different but unique restriction enzymes (EcoRV, EcoRI, XhoI and NdeI) before treatment with activated Tris-buffer

<p><b>Copyright information:</b></p><p>Taken from "DNA modification by sulfur: analysis of the sequence recognition specificity surrounding the modification sites"</p><p></p><p>Nucleic Acids Research 2007;35(9):2944-2954.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1888814.</p><p>© 2007 The Author(s)</p> Two fragments adding up to the size of linearized pHZ2031 (5.4 kb) were always seen at their expected positions after cleavage at a common modification site, as exemplified by cleavage at specific modification site 10 (), indicated as two white asterisks in each respective gel panel. (EcoRV: 4182 and 1231; EcoRI: 3027 and 2386; XhoI: 3976 and 1437; NdeI: 4687 and 726)

A Novel Target of IscS in <em>Escherichia coli:</em> Participating in DNA Phosphorothioation

<div><p>Many bacterial species modify their DNA with the addition of sulfur to phosphate groups, a modification known as DNA phosphorothioation. DndA is known to act as a cysteine desulfurase, catalyzing a key biochemical step in phosphorothioation. However, bioinformatic analysis revealed that 19 out of the 31 known <em>dnd</em> gene clusters, contain only four genes (<em>dndB-E</em>), lacking a key cysteine desulfurase corresponding gene. There are multiple cysteine desulfurase genes in <em>Escherichia coli</em>, but which one of them participates into DNA phosphorothioation is unknown. Here, by employing heterologous expression of the <em>Salmonella enterica dnd</em> gene cluster named <em>dptBCDE</em> in three <em>E. coli</em> mutants, each of which lacked a different cysteine desulfurase gene, we show that IscS is the only cysteine desulfurase that collaborates with <em>dpt</em>B-E, resulting in DNA phosphorothioation. Using a bacterial two-hybrid system, protein interactions between IscS and DptC, and IscS and DptE were identified. Our findings revealed IscS as a key participant in DNA phosphorothioation and lay the basis for in-depth analysis of the DNA phosphorothioation biochemical pathway.</p> </div

Plasmids that are used in this study.

<p>Plasmids that are used in this study.</p

Strains that are used in this study.

<p>Strains that are used in this study.</p

Protein interactions between IscS and Dpt proteins.

<p>A.The bar graph shows protein interactions that enable the <i>E. coli</i> cells to survive on medium containing 3AT (3-amino-1,2,4-triazole). F, pBT-LGF2; P, pTRG-Gal11P; S, pBT-IscS; B, pTRG-DptB; C, pTRG-DptC; D, pTRG-DptD; E, pTRG-DptE; G, pTRG only. F and P were co-expressed as positive control; S and G were co-expressed as negative control. <i>E. coli</i> can grow on 3-AT selective screening medium only when there is a binding interaction between the fusion proteins expressed from the bait and target plasmids. B. Dual selection plate containing 3-amino-1,2,4-triazole and streptomycin. F+P, LGF2+GallP (growth, positive control); S+B, IscS+DptB (no growth, no interaction); S+C, IscS+DptC (growth indicating protein interaction); S+D, IscS+DptD (no growth, no interaction); S+E, IscS+DptE (growth indicating protein interaction); S+G, IscS+pTRG (no growth, negative control). C. Interactions between IscS and DptC as well as IscS and DptE confirmed by pull-down experiments. Left panel: IscS (N terminus Strep tagged) extraction was mixed with _GSTDptC or _GSTDptE extraction and then purified by Streptactin affinity purification. Western blot was done using antibody against GST. Right panel: the mixture was purified by GST affinity purification. Western blotting was done using antibody against StreptagII.</p

IscS might participate DNA phosphorothioation directly.

<p>Ethidium bromide-stained agarose gels. TAE (top gel), samples run in normal TAE buffer; PAA (bottom gel), samples run in TAE containing PAA. Expression of <i>S. enterica dptB-E</i> resulted in DNA S-modification and a fluorescent smear in all samples, except for <i>E. coli ΔiscS</i>. <i>IscS</i> was therefore the only gene that was required for DNA S-modification among the tested deletions.</p

Specific activities of WT and point mutants of DndA measured using in vitro cysteine desulfurase activity assay.

<p>Assays were performed for five times, and the average values of specific activities along with standard deviations of the measurements were shown.</p

Crystal structure of DndA from <i>Streptomyces lividans</i>.

<p>(<b>A</b>) <b>Overall structure of the DndA dimer.</b> The structure is viewed perpendicular to the two-fold axis of the dimer. The two protomers are shown in magenta and green, respectively. Their bound PLP cofactors are presented as sticks, with carbon atoms yellow, nitrogen atoms blue, oxygen atoms red, and phosphorus atoms orange. (<b>B</b>) <b>Structure of a protomer of DndA.</b> α helices are shown in cyan, β sheets are shown in magenta, and loops are shown in pink. PLP and its covalently linked Lys200 of DndA, as well as the catalytic Cys327 (mutated to serine in our study), are shown in stick representation.</p

The binding site of PLP on DndA.

<p>(<b>A</b>) <b>PLP is located in a deep surface pocket on DndA.</b> The two protomers of DndA are shown in surface representation, with only one PLP shown in stick representation. The protomer of DndA harboring this PLP is colored in light grey, whereas the other protomer is colored in dark grey. Blue, red, yellow, and orange represent nitrogen, oxygen, carbon, and phosphorus atoms, respectively. (<b>B</b>) <b>The interaction interface between PLP and DndA.</b> DndA is shown in grey, with carbon atoms of its side chains and PLP shown in green. Blue, red, and orange represent nitrogen, oxygen, and phosphorus atoms, respectively. Hydrogen bonds are represented by magenta dashed lines. The orange circle indicates the presumable location of the carboxylate group of the L-cysteine substrate.</p

Structural comparison of DndA with related cysteine desulfurases/selenocysteine lyase.

<p>(<b>A</b>) Structural superimposition of DndA (red), IscS (green, PDB code 1P3W), NifS (cyan, PDB code 1ECX), CsdB (magenta, PDB code 1C0N), and SufS (blue, PDB code 1T3I). Their bound PLP's are shown as sticks. (<b>B</b>) In DndA, the active site Cys327 is located on a β strand, and its distance from PLP is ∼16 Å. In IscS (<b>C</b>) and NifS (<b>D</b>), the active site cysteines are located on relatively long loops, and are not visible in the crystal structure. Visible residues closest to the catalytic cysteines on the primary sequence are no less than 9 Å from PLP. In CsdB (<b>E</b>) and SufS (<b>F</b>), the active site cysteines are located on relatively short loops, and are ∼7 Å from PLP.</p