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

Michal Štros,Eva Muselı́Ková

doi:10.1074/jbc.m007167200

Abstract

HMG (high mobility group) 1 is a chromosomal protein with two homologous DNA-binding domains, the HMG boxes A and B. HMG-1, like its individual HMG boxes, can recognize structural distortion of DNA, such as four-way DNA junctions (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 introduce negative supercoils in the presence of topoisomerase I in topologically closed DNAs. Results of our gel shift assays demonstrate that mutation of Arg(97) within the extended N-terminal strand of the B domain significantly (>50-fold) decreases affinity of the HMG box for 4WJs and alters the mode of binding without changing the structural specificity for 4WJs. Several basic amino acids of the extended N-terminal strand (Lys(96)/Arg(97)) and helix I (Arg(110)/Lys(114)) of the B domain participate in DNA binding and supercoiling. The putative intercalating hydrophobic Phe(103) of helix I is important for DNA supercoiling but dispensable for binding to supercoiled DNA and 4WJs. We conclude that the B domain of HMG-1 can tolerate substitutions of a number of amino acid residues without abolishing the structure-specific recognition of 4WJs, whereas mutations of most of these residues severely impair the topoisomerase I-mediated DNA supercoiling and change the sign of supercoiling from negative to positive.

Highlights

HMG proteins 1 and 2 are relatively abundant and highly conserved chromatin-associated proteins that are present in all vertebrate cell nuclei [1, 2]
The NMR structures of the HMG box domains of HMG-1 [5, 6], HMG-D [7, 25], NHP6A [26], LEF-1 [27] and SRY complexed to DNA [28], mutational analyses, and a “domain swap” (12, 29 –32; reviewed in Ref. 4) indicated that DNA binding/bending occurs through the concave surface of the L-shaped HMG box containing a cluster of conserved hydrophobic amino acids
Strategy of the Mutational Analysis and Preparation of B Domain and Mutants—The aim of this paper was to carry out mutational analysis of the central domain of the chromosomal protein HMG-1 to identify amino acid residues involved in structure-specific binding to 4WJs or DNA unwinding in the presence of topoisomerase I

Summary

Introduction

HMG (high mobility group) proteins 1 and 2 are relatively abundant and highly conserved chromatin-associated proteins that are present in all vertebrate cell nuclei [1, 2]. In addition to the ability of HMG-1/2 to bind non-B-DNA structures, the proteins can induce (in the presence of topoisomerase I) negative supercoils in topologically closed domains of DNA, stabilize DNA loops in complex nucleoprotein structures, and facilitate binding of certain sequence-specific proteins to their target DNA (11, 16, 19 –24). The NMR structures of the HMG box domains of HMG-1 [5, 6], HMG-D [7, 25], NHP6A [26], LEF-1 [27] and SRY complexed to DNA [28], mutational analyses, and a “domain swap” (12, 29 –32; reviewed in Ref. 4) indicated that DNA binding/bending occurs through the concave surface of the L-shaped HMG box containing a cluster of conserved hydrophobic amino acids (hydrophobic core). We have introduced mutations into the B domain of HMG-1 to assess the functional importance of a number of highly conserved residues, including the putative intercalating hydrophobic Phe103 of helix I, for the ability of the HMG-1 box domain to introduce supercoils in topologically closed DNA (in the presence of topoisomerase I) and to bind

Objectives

Results

Conclusion