The pathway of male sexual development in mammals is initiated by SRY, a gene on the short arm of the Y chromosome. Its expression in the differentiating gonadal ridge directs testicular morphogenesis, characterized by elaboration of Müllerian inhibiting substance (MIS) and testosterone. SRY and MIS each belong to conserved gene families that function in the control of growth and differentiation. Structural and biochemical studies of the DNA binding domain of SRY (the HMG box) revealed a protein-DNA interaction consisting of partial side chain intercalation into a widened minor groove. Functional studies of SRY in a cell line from embryonic gonadal ridge demonstrated activation of a gene-regulatory pathway leading to expression of MIS. SRY molecules containing mutations associated with human sex reversal have altered structural interactions with DNA and failed to induce transcription of MIS.

SRY, a putative transcription factor encoded by the sex-determining region of the human Y chromosome, regulates a genetic switch in male development. Impairment of this switch leads to intersex abnormalities of the newborn and is observed in association with mutations in the SRY DNAbinding domain [the high-mobility-group (1MG) box]. Here we show that the SRY EMG box exhibits a novel mechanism of DNA recognition: partial intercalation of a nonpolar side chain in the DNA minor groove. Base stacking (but not base pairing) is interrupted at the site of insertion. Sequence specificity reflects topological requirements of partial intercalation rather than direct readout of base-specific functional groups. Our results predict that the SRY HMG box inserts an a-helix into a widened minor groove at the center ofa sharp DNA bend. A similar mechanism may underlie binding of SRY and homologous EMG proteins to four-way junctions (Holliday intermediates) and other noncanonical DNA structures.Protein-DNA recognition is mediated by direct or indirect readout of bases in a DNA sequence (1). Direct readout consists of specific chemical interactions between protein and nucleic-acid bases (2). Indirect readout, inferred from the crystal structure of the trp repressor (TrpR)-operator complex, refers to recognition of sequence-dependent variations in the structure or deformability of the DNA backbone (3). Here we describe a third mechanism of protein-DNA recognition, partial intercalation ofa nonpolar protein side chain through the DNA minor groove. Our studies focus on SRY, a putative transcription factor that initiates male development in mammalian embryogenesis (4). SRY contains a high-mobility-group (HMG) box, a newly recognized motif (5, 6) also implicated in recognition of noncanonical DNA structures (7). A combination of biochemical and 'H NMR methods is used to define a contact between the SRY HMG box and a bent DNA site. This contact generates rules of specificity based on topological requirements of partial intercalation. MATERIALS AND METHODSExperimental design is based on identification ofhigh-affinity SRY target sites in promoters of sex-specific genes (8). The SRY HMG box (designated SRY-p; 85 residues) was expressed in Escherichia coli and purified as described (8). Oligonucleotides used in gel mobility-shift assays were obtained from Oligos, Etc. (Wilsonville, OR). 32P-labeled duplex DNA and SRY-p were incubated at 20°C in 10 mM potassium phosphate, pH 7.4/50 mM KCl/2 mM MgCl2/0.5 mM dithiothreitol/5% glycerol (vol/vol). Each reaction mix-The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.ture contained a variant 15-bp duplex (the test site) and 36-bp duplex containing a native target sequence (the reference site); complexes were resolved by polyacrylamide gel electrophoresis as described in Fig. 1. Quantitation was obtained with a digital fluorescence sca...

Formation of male-specific structures and regression of female primordia are regulated in early male embryogenesis by SRY, a singe-copy gene on the Y chromosome. Assignment of SRY as the testis-determining factor in eutherian mammals is supported by molecular analysis of cytogenetic sex reversal (i.e., XX males and XY females) and by complementary studies of transgenic murine models. Here we characterize the putative DNA-binding domain of SRY, which contains a conserved sequence motif shared by highmobility group nuclear proteins and a newly recognized class of transcription factors. The SRY DNA-binding domain specifically recognizes with nanomolar affinity proximal upstream elements (designated SRYe) in the promoters of the sex-specific genes encoding P450 aromatase and Muflerian inhibiting substance (MIS). P450 aromatase catalyzes the conversion of testosterone to estradiol, and in the male embryo its expression is down-regulated. Conversely, MIS is expressed in the male embryo to induce testicular differentiation and regression of female reproductive ducts. SRYe-binding activity is observed in nuclear extracts obtained from embryonic urogenital ridge immediately preceding morphologic testicular differentiation.