THE EFFECT OF FREE DNA ON THE INTERACTIONS OF THE ESTROGEN-RECEPTOR BOUND TO HORMONE, PARTIAL ANTAGONIST OR PURE ANTAGONIST WITH TARGET DNA

Interactions between the lamb uterine estrogen receptor occupied by estradiol, 4-hydroxytamoxifen (a non-steroidal partial estrogen antagonist) or ICI 164,384 (a steroidal pure estrogen antagonist), and the vitellogenin A2 estrogen-response element (vit ERE) were compared using a biotinylated 25-base all-palindromic double-stranded oligonucleotide, containing vit ERE (b-ERE), which allowed isolation of the b-ERE . receptor . [H-3]ligand assembly on streptavidin-Sepharose. The results of saturation analyses of the three receptor . [H-3]ligand complexes by increasing amounts of b-ERE were quite similar for the proportion of complexes able to interact with b-ERE (which varied from 30% to 65% according to experiments) and for the equilibrium dissociation constant [K-d (0 degrees C) approximate to 1.2 nM, assuming that the receptor interacted as a dimer with b-ERE]. With each ligand, receptor binding to ERE did not change the rate of ligand dissociation from the receptor at 20 degrees C. The rate of estrogen receptor dissociation from b-ERE, measured at 20 degrees C in the presence of a given concentration of ERE, did not vary according to the ligand bound to the receptor; however, this dissociation rate increased linearly over the ERE concentration range (0.5-10 mu M). The experimental rate constant (k(_)) of estrogen receptor dissociation from b-ERE appeared to be the sum of the basal dissociation-rate constant (k(-)(o) approximate to 0.011 min(-1)), corresponding to spontaneous dissociation which would occur in the absence of ERE, and of the ERE-induced dissociation-rate constant, proportional to the used concentration of ERE (k(-)(i) approximate to 4500 C(ERE)M(-1) min(-1), where C-ERE is the molar concentration of ERE). Non-target DNA also induced receptor dissociation from b-ERE, but its efficiency was 6-10-fold lower than that of ERE. We conclude that, the two antiestrogens are as efficient as estradiol in promoting estrogen receptor binding to a single vit ERE; the low or nil ability of antiestrogens to induce estrogenic responses is probably not linked with the receptor DNA-binding step; DNA binding does not seem to affect the conformation of the filled hormone-binding site of the receptor at 20 degrees C, interactions of receptor dimers with DNA seems to proceed by direct transfer of receptor dimers between DNA strands.