Nucleic Acids Research, Vol 26, Issue 9 2143-2149, Copyright © 1998 by Oxford University Press
F Cornille, P Emery, W Schuler, C Lenoir, B Mach, BP Roques and W Reith
The RFX DNA binding domain (DBD) is a novel highly conserved motif
belonging to a large number of dimeric DNA binding proteins which have
diverse regulatory functions in eukaryotic organisms, ranging from yeasts
to human. To characterize this novel motif, solid phase synthesis of a
76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a
prototypical member of the RFX family, has been optimized to yield large
quantities (approximately 90 mg) of pure compound. Preliminary
two-dimensional1H NMR experiments suggested the presence of helical regions
in this sequence in agreement with previously reported secondary structure
predictions. In gel mobility shift assays, this synthetic peptide was shown
to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide
corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to
an inverse repeat present in the 23mer. The stoichiometry of this complex
was reduced to 1:1 by decreasing the length of 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 equilibrium dissociation constant (K D = 153 nM) were
determined for binding of hRFX1/DBD to the double- stranded 13mer
oligonucleotide. In the presence of hRFX1/DBD the melting temperature of
the 13mer DNA was increased by 16 degreesC, illustrating stabilization of
the double-stranded conformation induced by the peptide.
ARTICLES
DNA binding properties of a chemically synthesized DNA binding domain of hRFX1
Departement de Pharmacochimie Moleculaire et Structurale, INSERM U266, CNRS URA D1500, UFR des Sciences Pharmaceutiques et Biologiques, 4 Avenue de l'Observatoire, 75270 Paris Cedex 06, France. cornille@pharmacie.univ-paris5.fr
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