AN SRY MUTATION CAUSING HUMAN SEX REVERSAL RESOLVES A GENERAL MECHANISM OF STRUCTURE-SPECIFIC DNA RECOGNITION - APPLICATION TO THE 4-WAY DNA JUNCTION

SRY, a genetic ''master switch'' for male development in mammals, exhibits two biochemical activities: sequence-specific recognition of duplex DNA and sequence-independent binding to the sharp angles of four-way DNA junctions. Here, we distinguish between these activities by analysis of a mutant SRY associated with human sex reversal (46, XY female with pure gonadal dysgenesis). The substitution (I68T in human SRY) alters a nonpolar side chain in the minor-groove DNA recognition or-helix of the HMG box [Haqq, C. M., King, C.-Y., Ukiyama, E., Haqq, T. N., Falsalfi, S., Donahoe, P. K., and Weiss, M. A. (1994) Science 266, 1494-1500]. The native (but not mutant) side chain inserts between specific base pairs in duplex DNA, interrupting base stacking at a site of induced DNA bending. Isotope-aided H-1-NMR spectroscopy demonstrates that analogous side-chain insertion occurs on binding of SRY to a four-way junction, establishing a shared mechanism of sequence- and structure-specific DNA binding. Although the mutant DNA-binding domain exhibits >50-fold reduction in sequence-specific DNA recognition, near wild-type affinity for four-way junctions is retained. Our results (i) identify a shared SRY-DNA contact at a site of either induced or intrinsic DNA bending, (ii) demonstrate that this contact is not required to bind an intrinsically bent DNA target, and (iii) rationalize patterns of sequence conservation or diversity among HMG boxes. Clinical association of the I68T mutation with human sex reversal supports the hypothesis that specific DNA recognition by SRY is required for male sex determination.