Two mutations of basic residues within the N-terminus of HMG-1 B domain with different effects on DNA supercoiling and binding to bent DNA

High mobility group (HMG) 1 protein and its two homologous DNA-binding doma ins, A and B ("HMG-boxes"), can bend and supercoil DNA in the presence of t opoisomerase I, as well as recognize differently bent and distorted DNA str uctures, including four-way DNA junctions, supercoiled DNA and DNA modified with anticancer drug cisplatin. Here we show that the lysine-rich part of the linker region between A and B domains of HMG-1, the (TKKKFKD91)-T-85 se quence that is attached to the N-terminus of the B domain within HMG-1, is a prerequisite for a preferential binding of the B domain to supercoiled DN A. The above sequence is also essential for a high-affinity binding of the B domain to DNA containing a site-specific major 1,2-d(GpG) intrastrand DNA adduct of cisplatin. Mutation of Arg(97), but not Lys(90) [Lys(90) forms a specific cross-link with platinum(II) in major groove of cisplatin-modifie d DNA; Kane, S. A., and Lippard, S. J. (1996) Biochemistry 35, 2180-2188], to alanine significantly (>40-fold) reduces affinity of the B domain to cis platin-modified DNA, inhibits the ability of the B domain to bend (ligase-m ediated circularization) or supercoil DNA, and results in a loss of the pre ferential binding of the B domain to supercoiled DNA without affecting the structural-specificity of the HMG-box for four-way DNA junctions. Some of t he reported activities of the B domain are enhanced when the B domain is co valently linked to the A domain. We propose that binding of the A/B linker region within the major DNA groove helps the two HMG-1 domains to anchor to the minor DNA groove to facilitate their DNA binding and ether activities.