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.
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 CD3epsilon 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 (SRY(WT)) 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 SRY(WT) 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 SRY(Y129N), identified in a patient presenting with 46,XY sex reversal, suggests that oligomerization may play an important role in vivo as well.
Baud
Stephanie
S
Centre de Biochimie Structurale, UMR INSERM 554, CNRS 5048, Université Montpellier I, 29 rue de Navacelles, 34090 Montpellier, France.
Margeat
Emmanuel
E
Lumbroso
Serge
S
Paris
Françoise
F
Sultan
Charles
C
Royer
Catherine
C
Poujol
Nicolas
N
D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004735:Energy Transfer; D005453:Fluorescence; D006801:Humans; D009687:Nuclear Proteins; D011485:Protein Binding; D051876:Sex-Determining Region Y Protein; D012965:Sodium Chloride / Q000737:chemistry*; D014157:Transcription Factors; D014998:Y Chromosome
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C497745:SRY protein, human; D051876:Sex-Determining Region Y Protein; D014157:Transcription Factors; D012965:Sodium Chloride; D004247:DNA