DNA-BINDING PROPERTIES OF A CHEMICALLY SYNTHESIZED DNA-BINDING DOMAINOF HRFX1

RFX DNA binding domain (DBD) is a novel highly conserved motif belongi ng to a large number of dimeric DNA binding proteins which have divers e regulatory functions in eukaryotic organisms, ranging from yeasts to human, To characterize this novel motif, solid phase synthesis of a 7 6mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the; RFX family, has been optimized to yield large quantities (similar to 90 mg) of pure compound, Preliminary two- dimensional H-1 NMR experiments suggested the presence of helical regi ons in this sequence in agreement with previously reported secondary s tructure predictions. In gel mobility shift assays, this synthetic pep tide was shown to bind in a cooperative manner the 23mer duplex oligod eoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoic hiometry of this complex was reduced to 1:1 by decreasing the length o f the DNA sequence to a 13mer oligonucleotide containing a single half -site, Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip, Using this method an association constant (K-a = 4 x 10( 5)/M/s), a dissociation constant (K-D = 6 x 10(-2)/s) and an equilibri um dissociation constant (K-D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide, In the presen ce of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degrees C, illustrating stabilization of the double-stranded co nformation induced by the peptide.