THE SRY CANTILEVER MOTIF DISCRIMINATES BETWEEN SEQUENCE-SPECIFIC AND STRUCTURE-SPECIFIC DNA RECOGNITION - ALANINE MUTAGENESIS OF AN HMG BOX

The high-mobility-group (HMG) box defines a DNA-bending motif conserve d among architectural transcription factors. A ''hydrophobic wedge'' a t the protein surface provides a mechanism of DNA bending: disruption of base stacking by insertion of a sidechain ''cantilever.'' First des cribed in the mammalian testis-determining factor SRY, the cantilever motif consists of adjacent aromatic and nonpolar sidechains at the cru x of the HMG box (residues 12 and 13). Here, the role of these side ch ains in DNA recognition is investigated by alanine mutagenesis. F12A a nd I13A substitutions in the SRY HMG box each permit native folding an d thermal stability (as monitored by circular dichroism and H-1-NMR) b ut eliminate sequence-specific DNA-binding activity (as detected by ge l-mobility shift). On binding to the sharp angles of a four-way DNA ju nction (4WJ), however, the substitutions each promote formation of a h igh-molecular-weight aggregate, presumably by DNA-dependent oligomeriz ation. The substitutions have opposite effects on initial binding to t he 4WJ: whereas such binding is attenuated ten-fold by F12A, it is enh anced by I13A. A foreshortened ''alanine cantilever'', not observed am ong specific HMG boxes, occurs in a non-specific domain (HMG-1A) and m ay enhance architecture-selective DNA recognition.