Journal: The Journal of Biological Chemistry
Article Title: Spatial organization of DNA sequences directs the assembly of bacterial chromatin by a nucleoid-associated protein
Figure Lengend Snippet: Probing of H-NS-DNA complexes with restriction endonucleases and DNase I footprinting. In A , B , and C , the left panels show agarose gels with digests; the right panels show the change of the relative intensity of specific restriction bands (indicated with black dotted rectangles on the left panel ) in each digestion reaction. Shown is probing with BamHI-HindIII ( A ), BamHI-EcoRI ( B ), and ApaLI ( C ). When the restriction sites are inside the inserted fragment (HindIII or EcoRI sites inside the UPPU, PUUP, or UPUP DNA) the highest protection rate is observed for the UPPU construct (steepest decay of band intensity). When the restriction sites are in the plasmid backbone (ApaLI restriction sites), the highest protection is observed for the PUUP construct and the weakest for UPPU DNA. D , DNase I footprinting of supercoiled UPPU and PUUP constructs at different H-NS concentrations (0–150 n m ). H-NS binding sites 1 and 2 are indicated on the sequencing gel image ( left panel ) by black vertical lines . H-NS induced hypersensitivity ( Position 1 ) and protection in H-NS binding site 2 ( Position 2 ) in UPPU (marked by dotted rectangles ). The graphs on the right show the relative intensity of the Position 1 and Position 2 signals at various H-NS concentrations.
Article Snippet: Probing with restriction enzymes The cleavage reactions of the H-NS nucleoprotein complexes were carried out for 5 min with BamHI-EcoRI and BamHI-HindIII and for 10 min with ApaLI at 37 °C in the same standard New England BioLabs CutSmart buffer.
Techniques: Footprinting, Construct, Plasmid Preparation, Binding Assay, Sequencing