Comparison of the DNA binding characteristics of the related zinc finger proteins WT1 and EGR1.
The interactions of the related zinc finger proteins WT1 and EGR1 with DNA have been investigated using a quantitative binding assay. A recombinant peptide containing the four zinc fingers of WT1 binds to the dodecamer DNA sequence GCG-TGG-GCG-TGT with an apparent dissociation constant (Kd) of (1.14 +/- 0.09) x 10(-9) M under conditions of 0.1 M KCl, pH 7.5, at 22 degrees C. Under the same conditions, a recombinant peptide containing the three zinc fingers of EGR1 binds to the dodecamer sequence, the first nine bases comprising the EGR consensus binding site, with an apparent Kd of (3.55 +/- 0.24) x 10(-9) M. The nature of the equilibrium binding of each peptide to DNA was investigated as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1 with DNA is an entropy-driven process, while the formation of the EGR1-DNA complex is favored by enthalpy and entropy. The DNA binding activities of both proteins have broad pH optima centered at pH 8.0. The binding of both proteins to DNA shows similar sensitivity to ionic strength, with approximately 7.7 +/- 0.8 ion pairs formed in the EGR1-DNA complex and 9.2 +/- 1.8 ion pairs formed in the WT1-DNA complex. Results of measuring the effects of point mutations in the DNA binding site on the affinity of WT1 and EGR1 indicates a significant difference in the optimal binding sites: for EGR1, the highest affinity binding site has the sequence GNG-(T/G)GG-G(T/C)G, while for WT1 the highest affinity binding site has the sequence G(T/C)G-(T/G)GG-GAG-(T/C)G(T/C).
Hamilton
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TB
Department of Biochemistry and Microbiology, University of Victoria, British Columbia, Canada.
Borel
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F
Romaniuk
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PJ
D000595:Amino Acid Sequence; D000818:Animals; D000838:Anions; D001482:Base Composition; D001665:Binding Sites / Q000235:genetics; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D051766:Early Growth Response Protein 1; D016162:Genes, Wilms Tumor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D017874:Immediate-Early Proteins; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D013696:Temperature; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D025721:WT1 Proteins; D016335:Zinc Fingers / Q000235:genetics*
D000838:Anions; D002413:Cations, Divalent; D002414:Cations, Monovalent; D004268:DNA-Binding Proteins; C497325:EGR1 protein, human; D051766:Early Growth Response Protein 1; C486109:Egr1 protein, mouse; D017874:Immediate-Early Proteins; D014157:Transcription Factors; D025721:WT1 Proteins; D008274:Magnesium