Characterization of Hoxd1 protein-DNA-binding specificity using affinity chromatography and random DNA oligomer
1Laboratory of Molecular Biology, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Canada.
Abstract:
1. Hoxd1 is member of the labial subfamily of Hox genes that has a conserved 60 amino acid homeodomain region. The homeodomain is an important determining factor in the binding of the protein to specific DNA sequence(s). DNA-binding specificity for the Hoxd1 protein has not been determined previously. 2. We have employed a rapid affinity chromatography method to determine optimal DNA binding sequences for the 109 amino acid Hoxd1 peptide, comprising the homeodomain and the entire carboxy terminal region of the Hoxd1 protein. 3. Labial Hox proteins have intrinsically weak DNA-binding activity that has been attributed to the nonbasic residues at positions 2 and 3 in the N-terminal arm of the homeodomain. The presence of the Hoxd1 carboxy terminal region negated the influence of the nonbasic residues and facilitated Hoxd1 DNA-binding specificity. 4. DNA sequences bound to the Hoxd1 peptide-affinity column were separated from a random pool of oligonucleotide sequences by gradient elution and enriched by polymerase chain reaction. Preferred sequences were identified on 5' and 3' of a TAAT core, extending the binding site to T/AT/gTAATTGTA. 5. Stability and specificity of optimal DNA-binding sequence for Hoxd1 homeodomain were determined by equilibrium and kinetic studies. Dissociation coefficient constant (KD) was estimated to be 8.6 x 10(-9) M and the DNA-Hoxd1 homeodomain complex has a half life (t(1/2)) of 12.7 min. 6. A molecular model of Hoxd1 homeodomain-DNA interaction based on the X-ray coordinates of Antennapedia homeodomain-DNA complex has revealed novel interactions of key Hoxd1 residues at the protein-DNA interface.
