High affinity binding of estrogen receptor to recombinant plasmids containing (dA-dC)n.(dG-dT)n sequences.
Estrogen receptor is a gene regulatory protein that is present in a subset of breast tumors and in normal sex accessory tissues of vertebrates. The receptor protein forms a high affinity complex with estradiol and mediates its action. The mechanism of action of estrogen receptor (ER) involves its binding to specific DNA sequences in the genome and the stimulation of the expression of estrogen-responsive genes. In order to understand the molecular characteristics of ER-DNA interactions, we studied the relative binding affinity of rabbit uterine ER to recombinant plasmids: pDHf2 with 23-base pair (dA-dC)n.(dG-dT)n insert; pDHf14 with 60-base pair (dA-dC)n.(dG-dT)n insert; pDHg16 with 23-base pair (dG-dC)n.(dG-dC)n insert; and the parental plasmid pDPL6 without any insert. We conducted this study by using a DNA-cellulose elution assay in which the receptor was bound to DNA-cellulose and then eluted with different concentrations of plasmid DNAs in 10 mM Tris.HCl and 1 mM dithiothreitol, pH 7.4. The efficacy of a plasmid to bind to ER was determined from the concentration of the plasmid DNA required to elute 50% of the receptor from DNA-cellulose. The 50% elution concentrations were as follows: pDHf2, 3.2 micrograms/ml; pDHf14, 0.85 micrograms/ml; pDHg16, 200 micrograms/ml; and pDPL6, 340 micrograms/ml. Linearization of pDHf14 with HindIII reduced its binding efficacy only slightly, with a 50% elution concentration of the linear plasmid at 3.2 micrograms/ml. In contrast, the linear polynucleotide, poly(deoxyadenylate-deoxycytidylate).poly(deoxyguanylate-deoxythymidylat e [poly(dA-dC).poly(dG-dT)] and calf thymus DNA were 1650 and 880-fold less efficient, respectively, than pDHf14 in eluting ER from DNA-cellulose. These results show that the presence of short stretches of (dA-dC)n.(dG-dT)n sequences has a dramatic effect in increasing the binding affinity of ER to DNA. Since these sequences are reported to be conformationally ultrapolymorphic, a possible explanation for this increased binding might be the assumption of a conformational state of the inserted sequence to a form that has high affinity for ER.
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Department of Environmental and Community Medicine, University of Medicine and Dentistry, New Jersey-Robert Wood Johnson Medical School, Piscataway 08854.
Thomas
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D000818:Animals; D002482:Cellulose / Q000031:analogs & derivatives; D002499:Centrifugation, Density Gradient; D004247:DNA / Q000031:analogs & derivatives; D004251:DNA Transposable Elements; D004274:DNA, Recombinant / Q000378:metabolism*; D005260:Female; D066298:In Vitro Techniques; D009690:Nucleic Acid Conformation; D010957:Plasmids; D011089:Polydeoxyribonucleotides / Q000378:metabolism*; D011817:Rabbits; D011960:Receptors, Estrogen / Q000378:metabolism*; D014599:Uterus / Q000378:metabolism
D004251:DNA Transposable Elements; D004274:DNA, Recombinant; C033190:DNA-cellulose; D011089:Polydeoxyribonucleotides; D011960:Receptors, Estrogen; C044165:poly(d(G-T).d(C-A)); D002482:Cellulose; D004247:DNA