Footprinting protein-DNA complexes using the hydroxyl radical.
Hydroxyl radical footprinting has been widely used for studying the structure of DNA and DNA-protein complexes. The high reactivity and lack of base specificity of the hydroxyl radical makes it an excellent probe for high-resolution footprinting of DNA-protein complexes; this technique can provide structural detail that is not achievable using DNase I footprinting. Hydroxyl radical footprinting experiments can be carried out using readily available and inexpensive reagents and lab equipment. This method involves using the hydroxyl radical to cleave a nucleic acid molecule that is bound to a protein, followed by separating the cleavage products on a denaturing electrophoresis gel to identify the protein-binding sites on the nucleic acid molecule. We describe a protocol for hydroxyl radical footprinting of DNA-protein complexes, along with a troubleshooting guide, that allows researchers to obtain efficient cleavage of DNA in the presence and absence of proteins. This protocol can be completed in 2 d.
Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, USA.
Swapan S
SS
Jain
Thomas D
TD
Tullius
D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018983:DNA Footprinting / Q000379:methods*; D017665:Hydroxyl Radical; D046911:Macromolecular Substances; D011485:Protein Binding; D023302:Protein Footprinting / Q000379:methods*; D011506:Proteins / Q000737:chemistry / Q000378:metabolism
D046911:Macromolecular Substances; D011506:Proteins; D017665:Hydroxyl Radical; D004247:DNA