Protein footprinting reveals specific binding modes of a high mobility group protein I to DNAs of different conformation.
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.
Frank
O
O
III. Zoologisches Institut-Entwicklungsbiologie, Universität Göttingen, Humboldtallee 34A, 37073 Göttingen, Germany.
Schwanbeck
R
R
Wiśniewski
J R
JR
D000818:Animals; D001665:Binding Sites / Q000502:physiology; D002683:Chironomidae / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D025741:HMGA1a Protein; D006609:High Mobility Group Proteins / Q000737:chemistry*; D006801:Humans; D017665:Hydroxyl Radical / Q000378:metabolism; D019476:Insect Proteins / Q000737:chemistry; D016899:Interferon-beta / Q000235:genetics; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry; D014157:Transcription Factors / Q000737:chemistry*
D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D019476:Insect Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D014157:Transcription Factors; D025741:HMGA1a Protein; D017665:Hydroxyl Radical; D016899:Interferon-beta; D004247:DNA; D010450:Endopeptidases