2 MUTATIONS IN THE HMG-BOX WITH VERY DIFFERENT STRUCTURAL CONSEQUENCES PROVIDE INSIGHTS INTO THE NATURE OF BINDING TO 4-WAY JUNCTION DNA

Mutation of the highly conserved tryptophan residue in the A-domain HM G-box of HMG1 largely, but not completely, destroys the protein tertia ry structure and abolishes its supercoiling ability, but does not abol ish structure-specific DNA binding to four-way junctions. Circular dic hroism shows that the protein has some residual alpha-helix (<10%) and does not re-fold in the presence of DNA. Structure-specific DNA bindi ng might therefore be a property of some primary structure element, fo r example the N-terminal extended strand, which even in the unfolded p rotein would he held in a restricted conformation by two, largely tran s, X-Pro peptide bonds. However, mutation of P5 or P8 of the A-domain to alanine does not abolish the formation of the (first) complex in a gel retardation assay, which probably arises from binding to the junct ion cross-over, although the P8 mutation does affect the formation of higher complexes which may arise from binding to the junction arms. Si nce mutation of pg in the W49R mutant has no effect on structure-speci fic junction binding, we propose that some some residual alpha-helix i n the protein might be involved, implicating this element in the inter actions of HMG-boxes generally with DNA.