Fig. 3: Structural basis for the DNA binding of SspECTD.
From: Nicking mechanism underlying the DNA phosphorothioate-sensing antiphage defense by SspE
a DNA binding assessment by EMSA. Different concentrations of SspE and its mutants were incubated with 200 ng of linearized pUC19 DNA in 20 mM Tris-HCl (pH 8.0), 100 mM NaCl, and 5% glycerol for 15 min at 30 °C before loading onto a 1% agarose gel. The intensity of the shifted DNA band is expressed as the percentage bound. A nonlinear regression analysis was applied to the datasets using GraphPad Prism software (version 6) to obtain the best-fit curve. Values represent the mean of three biological replicates, and error bars represent standard deviations. b Surface representation of the CTD of SspE colored according to sequence conservation. c Electrostatic surface representation of the CTD of SspE with negative potential shown in red and positive potential shown in blue. d EMSA analysis of the binding of SspECTD and its mutants, shown schematically, with increasing concentrations of EcoRI-linearized pUC19 DNA. All results are representative of three independent experiments. e Model for the binding of SspECTD to a 10-bp DNA fragment (PDB: 5J3G). The conserved positively charged residues, R404 and R408, mutated in this study are represented as sticks. The HNH motif is shown in red. f Plaque assays of S. lividans HXY6 expressing SspABCD-SspE and SspABCD-SspER404S/R408S using 2 μL of each serial tenfold dilution (10-1–10-6) of phage JXY1 at 28 °C. g EMSAs of SspE and SspER100A binding to PT-modified or unmodified pUC19 DNA substrates in the presence of GTP/GMPPCP. The results are representative of two independent experiments. Source data are provided as a Source Data file.
