Specific photocrosslinking of DNA-protein complexes: identification of contacts between integration host factor and its target DNA.
Azide moieties have been specifically placed in the backbone of DNA by chemical coupling between azidophenacyl bromide and uniquely positioned phosphorothioate residues. The derivatized DNA forms specific complexes with a DNA-binding protein and, following irradiation with 302-nm light, makes specific crosslinks to the protein. Isolation of this covalent complex, followed by tryptic digestion and Edman degradation of the resulting crosslinked peptide, identifies the portion of the protein that is near the derivatized segment of the target DNA. We use this method to probe the interaction between a specific DNA sequence and integration host factor (IHF) protein. A single IHF heterodimer is known to contact > 25 bp of DNA and thereby introduce a sharp bend. Two segments of a typical IHF site were derivatized with aryl azide. Although the segments were separated by only 5 bp, they crosslinked to different subunits of IHF. The locations of the crosslinks support our current view for the way IHF protein binds to and bends its specific targets.
Yang
S W
SW
Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, MD 20892-4034.
Nash
H A
HA
D000595:Amino Acid Sequence; D001386:Azides; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry*; D034982:Integration Host Factors; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D013873:Thionucleotides; D014357:Trypsin
D001386:Azides; D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; D034982:Integration Host Factors; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D013873:Thionucleotides; D004247:DNA; D014357:Trypsin