DNA Footprinting: Detecting DNA Protein Interactions DNA footprinting is another method that allows you to determine if a DNA binding protein interacts with a specific region of DNA. In a footprinting assay you can identify regions of the genome that are protected from nucleases by a DNA binding protein. The ability of a DNA binding protein to protect DNA from a nucleus gives us information regarding the binding of the protein of interest to the DNA sequence. Control: The first step is to label naked DNA (grey lines) with radioactive phosphate (green ball) at the 5` end. The next step is to lightly digest the DNA fragments with a nuclease that recognizes double stranded DNA. The concentration of nuclease in the reaction mixture is very low and the reaction is carried out for a very short period of time. This ensures that each fragment is digested at only one position. Since the nuclease lacks sequence specificity, it will digest each fragment at a random location. The nuclease will break the phosphodiester bond that exists between adjacent nucleotides. If you digest a large enough population of DNA fragments you will generate a collection of broken fragments that differ from each other by a single nucleotide. You can separate these fragments on an agarose gel, transfer them to a charged nitrocellulose filter, and visualize the fragments on X-ray film (the radioactive nucleotides will emit radiation that is captured by the film). You can see a ladder of DNA fragments: see red box.

DNA Footprinting: Detecting DNA Protein Interactions DNA footprinting is another method that allows you to determine if a DNA binding protein interacts with a specific region of DNA. In a footprinting assay you can identify regions of the genome that are protected from nucleases by a DNA binding protein. The ability of a DNA binding protein to protect DNA from a nucleus gives us information regarding the binding of the protein of interest to the DNA sequence. Control: The first step is to label naked DNA (grey lines) with radioactive phosphate (green ball) at the 5` end. The next step is to lightly digest the DNA fragments with a nuclease that recognizes double stranded DNA. The concentration of nuclease in the reaction mixture is very low and the reaction is carried out for a very short period of time. This ensures that each fragment is digested at only one position. Since the nuclease lacks sequence specificity, it will digest each fragment at a random location. The nuclease will break the phosphodiester bond that exists between adjacent nucleotides. If you digest a large enough population of DNA fragments you will generate a collection of broken fragments that differ from each other by a single nucleotide. You can separate these fragments on an agarose gel, transfer them to a charged nitrocellulose filter, and visualize the fragments on X-ray film (the radioactive nucleotides will emit radiation that is captured by the film). You can see a ladder of DNA fragments: see red box.

DNA Footprinting: Detecting DNA-Protein Interactions Experiment: In a separate experiment the naked DNA (which has been radiolabeled) is mixed with a purified DNA binding protein. After a short incubation the reaction is treated with a cross-linking reagent to stabilize DNA-protein interactions (if any exist). The reaction is then washed with several salt solutions to remove excess unbound protein. Just like the control experiment, the DNA fragments are lightly digested with a nuclease that recognizes double stranded DNA. The nuclease will digest the naked DNA but will be unable to digest the regions that is protected by the DNA binding protein – this is because the DNA binding protein is covering the DNA strands and preventing the nuclease from having access to the double helix. After the reaction is completed, the DNA binding proteins are removed with a reverse cross-linking reagent. The remaining DNA fragments are loaded onto an agarose gel, transferred to a charged nitrocellulose filter, and exposed to X-ray film. You will see most of the bands on the film (like the control). However, there will be a spot on where DNA fragments are missing. These represent the regions that could not be digested by the nuclease – they were protected by the DNA binding protein (blue boxes). Remember the undigested fragments (left image, purple arrow) are very large and will run near the top of the gel as the full-length fragment

DNA Footprinting: Detecting DNA-Protein Interactions Experiment: In a separate experiment the naked DNA (which has been radiolabeled) is mixed with a purified DNA binding protein. After a short incubation the reaction is treated with a cross-linking reagent to stabilize DNA-protein interactions (if any exist). The reaction is then washed with several salt solutions to remove excess unbound protein. Just like the control experiment, the DNA fragments are lightly digested with a nuclease that recognizes double stranded DNA. The nuclease will digest the naked DNA but will be unable to digest the regions that is protected by the DNA binding protein – this is because the DNA binding protein is covering the DNA strands and preventing the nuclease from having access to the double helix. After the reaction is completed, the DNA binding proteins are removed with a reverse cross-linking reagent. The remaining DNA fragments are loaded onto an agarose gel, transferred to a charged nitrocellulose filter, and exposed to X-ray film. You will see most of the bands on the film (like the control). However, there will be a spot on where DNA fragments are missing. These represent the regions that could not be digested by the nuclease – they were protected by the DNA binding protein (blue boxes). Remember the undigested fragments are very large and will run near the top of the gel as the full-length fragment (left image, green arrow).