Footprinting protein–DNA complexes using the hydroxyl radical

Abstract

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.

The multifunctional histone-like protein Lsr2 protects mycobacteria against reactive oxygen intermediates

“…Lsr2 appears to use a different mechanism to protect mycobacteria against oxidative stress that requires direct protein-DNA binding. Many DNA binding proteins locally protect DNA from ROI in vitro, indeed this phenomenon is the basis for DNA foot printing studies (30); thus, one could question the physiological relevance of the in vitro protection which we observed. However, Lsr2 differs from classical DNA binding proteins in its ability to bind long DNA sequences with relative nonspecificity, to oligomerize, and to regulate expression of a large number of genes (11,15).…”

Binding of the RamR Repressor to Wild-Type and Mutated Promoters of the ramA Gene Involved in Efflux-Mediated Multidrug Resistance in Salmonella enterica Serovar Typhimurium

“…To better decipher at the molecular level the interaction of RamR with P ramA , the 97-bp DNA fragment described above was physically mapped using high-resolution hydroxyl radical footprinting (HRF) (7). Footprinting experiments were performed on both the top strand (i.e., the ramA coding strand) and the bottom strand of the DNA fragment.…”

“…Hydroxyl radical footprinting (HRF), rather than DNase I footprinting, was chosen because it is not limited by the steric hindrance associated with the DNase I and DNA-binding proteins because of the small size of the diffusing chemical nuclease (hydroxyl radicals). It can therefore provide high-resolution footprinting of DNAprotein complexes and structural detail for them (7). The two complementary oligonucleotides corresponding to the 97-bp fragment of the ramR-ramA intergenic region were synthesized (Sigma-Aldrich) and separately 5= labeled with [␥-32 P]ATP (Perkin-Elmer, Villebon-sur-Yvette, France) at 30 Ci per 10-l reaction mixture using the T4 polynucleotide kinase (New England BioLabs, Ipswich, MA).…”

Genetic and biochemical characteristics of the histone-like protein DR0199 in Deinococcus radiodurans

“…radiodurans DR0199 protein appeared to utilize a different mechanism to protect D. radiodurans against DNA damage stresses that require direct protein-DNA binding. Many DNA-binding proteins locally protect DNA from reactive oxygen species in vitro, and this phenomenon is indeed the basis for DNA footprinting studies (Jain & Tullius, 2008). Protection may also be related to alterations in DNA conformation.…”