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Originally published In Press as doi:10.1074/jbc.M112366200 on March 4, 2002
J. Biol. Chem., Vol. 277, Issue 21, 18404-18410, May 24, 2002
Equilibrium Binding Assays Reveal the Elevated Stoichiometry
and Salt Dependence of the Interaction between Full-length Human
Sex-determining Region on the Y Chromosome (SRY) and DNA*
Stephanie
Baud ,
Emmanuel
Margeat§,
Serge
Lumbroso¶,
Françoise
Paris¶ ,
Charles
Sultan¶,
Catherine
Royer, and
Nicolas
Poujol**
From the Centre de Biochimie Structurale, UMR INSERM
554, CNRS 5048, Université Montpellier I, 29 rue de
Navacelles, 34090 Montpellier, France, ¶ INSERM U439, Pathologie
Moléculaire des Récepteurs Nucléaires, 70 rue de
Navacelles et Laboratoire d'Hormonologie, CHU Montpellier, 34295 Cedex
France, and Unité Endocrinologie Pédiatrique,
Hôpital A. de Villeneuve, CHU
Montpellier, 34295 Cedex France
In an effort to better define the molecular
mechanism of the functional specificity of human sex-determining region
on the Y chromosome (SRY), we have carried out equilibrium binding
assays to study the interaction of the full-length bacterial-expressed protein with a DNA response element derived from the CD3 gene enhancer. These assays are based on the observation of the fluorescence anisotropy of a fluorescein moiety covalently bound to the target oligonucleotide. The low anisotropy value due to the fast tumbling of
the free oligonucleotide in solution increases substantially upon
binding the protein to the labeled target DNA. Our results indicate
that the full-length human wild-type SRY (SRYWT)
forms a complex of high stoichiometry with its target DNA. Moreover, we
have demonstrated a strong salt dependence of both the affinity and
specificity of the interaction. We have also addressed the DNA bending
properties of full-length human SRYWT in solution by
fluorescence resonance energy transfer and revealed that maximal bending is achieved with a protein to DNA ratio significantly higher
than the classical 1:1. Oligomerization thus appears, at least in
vitro, to be tightly coupled to SRY-DNA interactions. Alteration of protein-protein interactions observed for the
mutant protein SRYY129N, identified in a patient presenting
with 46,XY sex reversal, suggests that oligomerization may play an
important role in vivo as well.
*
This work was supported in part by the CNRS, INSERM, La
Fondation pour la Recherche Médicale, L'Association pour la
Recherche sur le Cancer, and the Région Languedoc-Roussillon.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Supported by a doctoral grant from the French Ministère de
l'Education, de la Recherche et de la Technologie and a grant from the
Fondation pour la Recherche Médicale.
**
A postdoctoral fellow (INSERM poste d'accueil recherche clinique).
To whom correspondence should be addressed. Present address: Centre
Biologique Médical, Laboratoire d'Analyses Médicales, 16 rue du 8 Mai 1945, Sète, 34200 France. Tel.:
33-467-748-317; Fax: 33-467-181-667; E-mail:
poujol-at-home@wanadoo.fr.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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