Specific Photocrosslinking of DNA-Protein Complexes: Identification of Contacts Between Integration Host Factor and Its Target DNA

• Published in: Proceedings of the National Academy of Sciences - PNAS 1994-12, Vol.91 (25), p.12183-12187
• Main Authors: Yang, Shu-Wei, Nash, Howard A.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Azides; Bacterial Proteins - chemistry; Bacterial Proteins - isolation & purification; Bacterial Proteins - metabolism; Base Sequence; Binding Sites; Biochemistry; Bromides; Cross-Linking Reagents - chemistry; Crosslinking; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - isolation & purification; DNA - metabolism; DNA probes; DNA-Binding Proteins - chemistry; Integration Host Factors; Irradiation; Models, Molecular; Molecular Sequence Data; Nucleic Acid Conformation; Oligodeoxyribonucleotides - chemistry; Oligonucleotides; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Protein Structure, Secondary; Proteins; Thionucleotides; Trypsin
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.91.25.12183

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.