CONTACTS BETWEEN STEROID-HORMONE RECEPTORS AND THYMINES IN DNA - AN INTERFERENCE METHOD

Understanding the mechanisms by which regulatory proteins recognize genetic information stored in DNA relies on the availability of methods to analyze their interaction with individual nucleotides and their reactive groups. Here we describe the use of KMnO4 to analyze the contacts between steroid hormone receptors and thymines within a hormone responsive element. Although several pyrimidine residues are highly conserved among different receptor binding sites their participation in sequence recognition has not been directly studied. Using an interference procedure based on selective modification of the thy mine ring by KMnO4, we detect intimate contacts between the glucocorticoid or progesterone receptors and three thymine residues within the promoter distal receptor binding site of the mouse mammary tumor virus (-190/-160). A comparison of binding data obtained with oligonucleotides containing desoxyuridine, bromodesoxyuridine, cytosine, or 5′-methylcytosine instead of thymines demonstrates that the methyl group of those three thymines contributes to the free energy of binding. This simple method could be of general utility for the study of sequence-specific protein-DNA interactions.