Structure-specific binding of the two tandem HMG boxes of HMG1 to four-wayjunction DNA is mediated by the A domain

We have investigated the nature of the "structure-specific" binding of the tandem A and B HMG boxes of high mobility group protein 1 (HMG1) to four-wa y junction DNA. AB didomain binding favours the open, planar form of the ju nction, as shown by reaction with potassium permanganate. Site-directed cle avage of the DNA by a 1,10-phenanthroline-copper moiety attached to unique natural or engineered cysteine residues in the A or B domain shows that the two Linked HMG boxes are not functionally equivalent in four-way junction binding. The A domain of the didomain binds to the centre of the junction, mediating structure-specific binding; the concave surface of the domain int eracts with the widened minor groove at the centre, contacting one of the f our strands of the junction, and the short arm comprising helices I and II and the connecting loop protrudes into the central hole. The B domain makes contacts along one of the arms, presumably stabilising the binding of the didomain through additional non-sequence-specific interactions. The isolate d B domain can, however, bind to the centre of the junction. The preferenti al binding of the A domain of the AB didomain to the centre correlates with our previous finding of a higher preference of the isolated A domain than of the B domain for this structurally distinct DNA ligand. It is probably a t least partly due to the higher positive surface potential in the DNA-bind ing region of the A domain (in particular to an array of positively charged side-chains suitably positioned to interact with the negatively charged ph osphates surrounding the central hole of the junction) and partly to differ ences in residues corresponding to those that intercalate between bases in other HMG box/DNA complexes. (C) 1999 Academic Press.