Learn about how to assay DNA-protein interactions.
Assaying DNAProtein Interactions
There are four important techniques for assaying DNA-protein interactions. The first two techniques (Filter Binding and Gel Mobility Shift) determine if your DNA is interacting with protein. The other two techniques (DNase Footprinting and DMS Footprinting) indicate where the target site of interaction between protein and DNA lies on the DNA.
Nitrocellulose membrane filters are commonly used to filter-sterilize solutions. Nitrocellulose filters can bind DNA, but only under certain conditions. Singlestranded DNA binds readily to nitrocellulose, but double stranded DNA by itself does not. On the other hand protein does bind, and if a protein is bound to double-stranded DNA the protein-DNA complex will bind. Please refer to Nitrocellulose Filter Binding Assay article for more information.
Gel Mobility Shift
In order to measure DNA-protein interactions this technique relies on the simple fact that a small DNA has a much higher mobility in gel electrophoresis than the same DNA does when it is bound to a protein. We therefore can label a sort double stranded DNA fragment, then mix it with a protein, and electrophorese the complex. Then we subject the gel in autoradiography to detect the labeled species. The small size of the DNA is important in this assay. If a whole gene were used its mobility would already be very low, and the mobility shift caused by protein binding would be difficult to detect. Therefore we must know approximately where the protein binds to the DNA so we can prepare a short restriction fragment, or even a synthetic double-stranded oligonucleotide that will contain the target site for the protein, but little else.
Once we know that a protein binds to a given DNA we can use footprinting to find out where the target site lies on the DNA. Dnase footprinting relies on the fact that a protein, by binding to DNA, covers the binding site and so protects it from attack by Dnase. In this sense it leaves a "footprint" on the DNA. The first step in a footprinting experiment is to end-label the DNA. We can label either strand, but only one per experiment. Next, we bind the protein to the DNA. Then we treat the DNA-protein complex with Dnase I under mild conditions (very little Dnase), so that an average of only one cut occurs per DNA molecule. Next, we remove the protein from the DNA, separate the DNA strands, and electrophorese the resulting fragments on a high resolution polyacylamide gel alongside size markers. Fragments will arise from the other end of the DNA as well, but we will not detect them because they are unlabeled. We always include a control with DNA alone (no protein), and usually use more than one protein concentration so we can see by the gradual disappearance of the bands in the footprint region that protection of the DNA depends on the concentration of the added protein. The footprint represents the region of DNA protected by the protein, and therefore tells us where the protein binds.
Dnase footprinting gives a good idea of the location of the binding site for the protein, but Dnase is a macromolecule and is therefore a rather blunt instrument for probing the fine details of the binding site. Which means, that there may be gaps in the interaction between protein and DNA that Dnase would not fit into and therefore would not detect. Moreover, DNA binding proteins frequently perturb the DNA within the binding region, distorting the double helix. These perturbations are interesting, but are not generally detected by Dnase footprinting because the protein keeps the Dnase away. To do more detailed footprinting, we need a smaller molecule that can fit into the nooks and crannies of the DNA-protein complex and reveal more of the subtleties of the interaction. A favorite tool for this job is the methylating agent dimethylsulfate (DMS).
With DMS footprinting we start off in the same way as in Dnase footprinting, by end-labeling the DNA and binding the protein. Then we methylate the DNA-protein complex with DMS, using a mild treatment so that on average only one methylation even occurs per DNA molecule. Next, we dislodge the protein, and treat the DNA with a reagent that removes methylated purines, creating apurinic sites (deoxyriboses without bases) on the DNA. Then we break the DNA at these apurinic sites. These reactions are the same as the Maxam-Gilbert DNA sequencing reactions. Finally we electrophorese the DNA fragments and autoradiograph the gel to detect the labeled DNA bands. Each band ends next to a nucleotide that was methylated and thus unprotected by the protein.
See the DNA Molecule in 3-Dimensions