A role of basic residues and the putative intercalating phenylalanine of the HMG-1 box B in DNA supercoiling and binding to four-way DNA junctions

HMG thigh mobility group) 1 is a chromosomal protein with two homologous DN A-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG bo xes, can recognize structural distortion of DNA, such as four-way DNA junct ions (4WJs), that are very likely to have features common to their natural, yet unknown, cellular binding targets. HMG-1 can also bend/loop DNA and in troduce negative supercoils in the presence of topoisomerase I in topologic ally closed DNAs, Results of our gel shift: assays demonstrate that mutatio n of Arg(97) within the extended N-terminal strand of the B domain signific antly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Sever al basic amino acids of the extended N-terminal strand (Lys(96)/Arg(97)) an d helix I (Arg(110)/Lys(114)) Of the B domain participate in DNA binding an d supercoiling, The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoile d DNA and 4WJs. We conclude that the B domain of HMG-1 can tolerate substit utions of a number of amino acid residues without abolishing the structure- specific recognition of 4WJs, whereas mutations of most of these residues s everely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive,