Photochemical cross-linking of the Escherichia coli single-stranded DNA-binding protein to oligodeoxynucleotides. Identification of phenylalanine 60 as the site of cross-linking.
The single-stranded DNA-binding proteins from bacteriophage T4, F plasmid, Escherichia coli, and calf thymus can all be covalently cross-linked in vitro to thymine oligonucleotides by irradiating the respective protein-oligonucleotide complexes with ultraviolet light. More extensive studies on the E. coli single-stranded DNA-binding protein (SSB) indicate that this reaction is dependent upon both the length of the oligonucleotide and the dose of ultraviolet irradiation. Using anion-exchange and reverse-phase ion-pairing high-performance liquid chromatography we have isolated a specific cross-linked tryptic peptide comprising residues 57-62 of the SSB protein with the sequence valine-valine-leucine-phenylalanine-glycine-lysine. Solid-phase sequence analysis of the covalent [32P] p(dT)8-peptide complex indicates that phenylalanine 60 is the site of cross-linking. This amino acid is located within the general region of SSB (residues 1-115) that has previously been shown to contain the DNA-binding site (Williams, K. R., Spicer, E. K., LoPresti, M. B., Guggenheimer, R. A., and Chase, J. W. (1983) J. Biol. Chem. 258, 3346-3355). The high-performance liquid chromatography purification procedure we have devised to isolate cross-linked peptide-oligonucleotide complexes should be of general applicability and should facilitate future structure/function studies on other nucleic acid-binding proteins.
D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000528:radiation effects*; D009838:Oligodeoxyribonucleotides / Q000378:metabolism*; D009841:Oligonucleotides / Q000378:metabolism*; D010649:Phenylalanine; D010957:Plasmids; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013604:T-Phages / Q000378:metabolism; D014466:Ultraviolet Rays
D000596:Amino Acids; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D010649:Phenylalanine