Protein Footprinting Reveals Specific Binding Modes of a High Mobility Group Protein I to DNAs of Different Conformation

Abstract

The high mobility group proteins I and Y (HMGI/Y) are abundant components of chromatin. They are thought to derepress chromatin, affect the assembly and activity of the transcriptional machinery, and associate with constitutive heterochromatin during mitosis. HMGI/Y protein molecules contain three potential DNA-binding motifs (AT-hooks), but the extent of contacts between DNA and the entire protein has not been determined. We have used a protein-footprinting procedure to map regions of the Chironomus HMGI protein molecule that are involved in contacts with DNA. We find that in the presence of double-stranded DNA all AT-hook motifs are protected against hydroxyl radical proteolysis. In contrast, only two motifs were protected in the presence of four-way junction DNA. Large regions that flank the AT-hook motifs were found to be strongly protected against proteolysis in complexes with interferon-beta promoter DNA, suggesting amino acid residues outside the AT-hooks considerably contribute to DNA binding.