HIGH-AFFINITY INTERACTION OF DIPTERAN HIGH-MOBILITY GROUP (HMG) PROTEINS-1 WITH DNA IS MODULATED BY COOH-TERMINAL REGIONS FLANKING THE HMG BOX DOMAIN

The cells of the dipteran insects Chironomus and Drosophila contain hi gh mobility group (HMG) 1 proteins that are homologous to the HMG1 pro tein of mammals but comprise one instead of two DNA-binding HMG boxes. Mobility shift assays have revealed that Chironomus cHMG1a and cHMG1b bind double strand and four-way junction DNA in a similar way at appa rent dissociation constants in the range of 7.5-20 x 10(-9) M. Both pr oteins are monomeric and highly asymmetric molecules in solution. cHMG 1a and cHMG1b exhibit Stokes' radii of 2.4 and 2.3 nm, respectively, a nd both show a frictional ratio of 1.5. Despite these similarities in their hydrodynamic properties, the binding site of cHMG1a on DNA is si milar to 1.5 of the size found for the cHMG1b. Enzymatically and chemi cally prepared peptides of cHMG1a as well as bacterially expressed cHM G1a with terminal deletions and point substitutions showed that sequen ces flanking the folded domain that constitutes the HMG box are essent ial for the interaction of the HMG box with DNA. In particular, change s in the number of positive and negative charges, respectively, within basic and acidic domains modulated the DNA binding affinity of the cH MG1a protein. The alteration of fluorescence of the Trp residues sugge st that this modulation is due to interaction of the acidic domain wit h the positively charged HMG box.