Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors.

Abstract

The amalian sex-determining gene SRY (sex-determining region on Y chromosome) encodes a member of the high mobility group (HMG) . In contrast, proteins with a single HMG domain, including LEF-1 (6, 7), TCF-1 (8), and the human sex-determining factor SRY (14,15), were found to interact with DNA in a sequence-specific manner. DNA binding by these proteins was found to be characterized by two unusual features. (i) HMG-domain proteins contact DNA primarily through the minor groove of the double helix (20)(21)(22). (ii) DNA binding by LEF-1 and SRY was shown to be accompanied by very sharp bends in the DNA helix (21, 23).Comparison of the amino acid sequences of the HMG domains associated with the h-and mSRY proteins indicated a 70%o identity, which represents a significantly lower evolutionary conservation than the typically >90%o sequence identity observed with mammalian homologs of other DNA binding domains (24-27). Thus, the inability of the hSRY gene to induce sex reversal in female mouse embryos could, in principle, be explained by amino acid changes that have resulted in different DNA binding properties of the h-and mSRY HMG domains. Alternatively, the species specificity of SRY function could be accounted for by interactions of hand mSRY with different and/or possibly species-specific regulatory proteins. Here, we compared DNA binding by hand mSRY and found differences in the specificity of sequence recognition and DNA contacts. These differences may contribute to the species-specific function of this regulatory protein. MATERIALS AND METHODS Plasmid Construction and Expression of h-and mSRY11MG-Domain Peptides. Two overlapping oligonucleotides, together containing the coding region for the hSRY HMGdomain peptide [amino acids 5-92 (25)], were annealed, extended with DNA polymerase, and ligated into plasmid pGEX-3X (Pharmacia). The peptide contained two additional amino acid residues at the N terminus (GI) and three additional amino acid residues at the C terminus (NSS), due to the position of the translational stop codon in pGEX-3X. The mSRY HMG-domain peptide was as described (21). The glutathionine S-transferase-SRY HMG fusion proteins were expressed and purified as described (20).Electrophorefic Mobility Shift Assay and Methylation Interference Analysis. DNA binding reactions and analysis of the protein-DNA complexes were essentially as described (6). For methylation interference analysis, double-stranded TCR-11 oligonucleotide (Fig. 1) was labeled either at the 5' end of the coding or noncoding strand and methylated with dimethyl sulfate (28). DNA binding reactions were performed using 50 fmol of the single end-labeled probe, 500 ng of sonicated salmon sperm DNA, and 20 ng of recombinant mor hSRY HMG-domain peptide.Circular Permutation Analysis and Determination of the Bending Ange. Circularly permuted DNA fragments containing the SRY binding site were generated as described (21). Bending angles were estimated by taking the ratio of the Abbreviations: HMG, high mobility group; h, human; m, muri...

A novel SRY missense mutation affecting nuclear import in a 46,XY female patient with bilateral gonadoblastoma

“…6 In 10 -15% of XY patients with male-to-female sex reversal (46,XY DSD), inactivating mutations in SRY have been identified, with the majority residing in the HMG domain, and affecting the binding and bending of DNA. 7,8 At the Nand C-terminal ends of this domain, two nuclear localization signals (NLSs) are present. The biological effect of a number of the reported mutations can be explained by the fact that they reside in one of these NLSs, thereby disrupting nuclear import.…”

Mouse μ Opioid Receptor Distal Promoter Transcriptional Regulation by SOX Proteins

“…7A). It was previously reported that SOX proteins achieve DNA sequence specificity through subtle preferences for flanking nucleotides and that this is likely to be dictated by signature amino acids in their HMG domains (43,44). For example, the optimal SOX9 binding sequence, AGAACAATGG, contained a core DNA binding element AACAAT, flanked by 5Ј-AG and 3Ј-GG nucleotides.…”

Molecular mechanisms involved in mammalian primary sex determination

“…The HMG domain is highly conserved among species, and consists of a 79 amino-acid polypeptide. This domain comprises three a-helices that can form a L-signature shape and interacts with the consensus sequence (A/T)ACAA(T/A) in the minor groove of DNA with a significantly high affinity, and causes an approximate 658-808 bend at the binding site (Giese et al 1994, Chew et al 2005, Palasingam et al 2009, Jauch et al 2012. Apart from the HMG domain, the regions outside the HMG domain of SRY are poorly conserved among mammals without any identified structural/functional domain.…”