Interaction of the chick oviduct progesterone receptor with deoxyribonucleic acid.
The purified DNA binding component (receptor A) of the chick oviduct progesterone receptor has been analyzed for its ability to bind to the cloned ovalbumin gene and to plasmid DNA of various structural compositions. The rapid equilibrium filter adsorption assay of Riggs et al. [Riggs, A. D., Suzuki, H., & Bourgeois, S. (1970) J. Mol. Biol. 48, 67] has been used to demonstrate high affinity binding of the protein to DNA (Kdiss = 10(-10) M at 50 mM KCl, pH 7.2). Studies of association rates are consistent with equilibrium measurements (t 1/2 = 40-80 min). Association of purified receptor with DNA and the kinetics of the interaction have been verified independently by velocity sedimentation techniques. Direct binding assays were performed with the ovalbumin structural gene (cDNA), the entire natural ovalbumin gene containing seven intervening sequences, and various ovalbumin gene fragments coding for the 5' end of the nuclear precursor RNA, intron-exon junctions, and the 3'-noncoding region of the gene. No DNA-sequence specificity was identified for the binding of the receptor protein to any region of ovalbumin gene DNA. In contrast, the structural integrity of the DNA template greatly affected receptor binding. The poorest affinity was to supercoiled DNA and to blunt end, linear duplex gene fragments. The receptor bound saturably to DNA containing limited nicks but became nonsaturable as nicks were increased. Binding of the protein to double-stranded DNA increased susceptibility of the DNA to digestion by the enzyme S1, a single strand specific nuclease. On the basis of preferential receptor binding to single-stranded DNA, a possible mechanism involving DNA helix destabilization is discussed.
D000818:Animals; D002645:Chickens; D004247:DNA / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism; D005260:Female; D007700:Kinetics; D009994:Osmolar Concentration; D010057:Oviducts / Q000378:metabolism*; D011374:Progesterone / Q000378:metabolism; D011485:Protein Binding; D011980:Receptors, Progesterone / Q000378:metabolism*; D013698:Templates, Genetic
D003854:Deoxyribonucleotides; D011980:Receptors, Progesterone; D011374:Progesterone; D004247:DNA