Functional analysis of DNA bending and unwinding by the high mobility group domain of LEF-1.
LEF-1 (lymphoid enhancer-binding factor 1) is a cell type-specific member of the family of high mobility group (HMG) domain proteins that recognizes a specific nucleotide sequence in the T cell receptor (TCR) alpha enhancer. In this study, we extend the analysis of the DNA-binding properties of LEF-1 and examine their contributions to the regulation of gene expression. We find that LEF-1, like nonspecific HMG-domain proteins, can interact with irregular DNA structures such as four-way junctions, albeit with lower efficiency than with specific duplex DNA. We also show by a phasing analysis that the LEF-induced DNA bend is directed toward the major groove. In addition, we find that the interaction of LEF-1 with a specific binding site in circular DNA changes the linking number of DNA and unwinds the double helix. Finally, we identified two nucleotides in the LEF-1-binding site that are important for protein-induced DNA bending. Mutations of these nucleotides decrease both the extent of DNA bending and the transactivation of the TCR alpha enhancer by LEF-1, suggesting a contribution of protein-induced DNA bending to the function of TCR alpha enhancer.
Giese
K
K
Howard Hughes Medical Institute, Department of Microbiology, University of California, San Francisco 94143-0414, USA.
Pagel
J
J
Grosschedl
R
R
D004278:DNA, Superhelical / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006609:High Mobility Group Proteins / Q000378:metabolism*; D051821:Lymphoid Enhancer-Binding Factor 1; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics; D014157:Transcription Factors / Q000378:metabolism*
D004278:DNA, Superhelical; D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D051821:Lymphoid Enhancer-Binding Factor 1; D016693:Receptors, Antigen, T-Cell, alpha-beta; D014157:Transcription Factors