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.