The DNA-binding site of the RecA protein. Photochemical cross-linking of Tyr103 to single-stranded DNA.
To investigate the DNA-binding site in the Escherichia coli RecA protein, RecA was covalently cross-linked to oligodeoxythymidine [p(dT)14] by irradiation with ultraviolet light. We identified the site of cross-linking of the protein when the RecA.p(dT)14 complex was formed in the absence of nucleotide cofactor as well as in the presence of adenosine 5'-[gamma-thio]triphosphate. When RecA.p(dT)14 complex formed without nucleotide cofactor was irradiated with ultraviolet light, a cross-linked peptide was found after digestion with Achromobactor lyticus protease I. Amino acid composition of the peptide was determined. The results indicated that the site of cross-linking was in the region spanning amino acid residues 89-106. Further digestion of the cross-linked fragment with Staphylococcus aureus V8 protease indicated that Tyr103 was the site of cross-linking. When the complex formed with adenosine 5'-[gamma-thio]triphosphate was irradiated with ultraviolet light, two cross-linked sites were detected, which were in the region of residues 89-106 and residues 178-183. These regions are far from the two disordered loops in the crystal structure, which were suggested to be DNA-binding sites by Story et al. [Story, R. M., Weber, T. W. & Steitz, T. A. (1992) Nature 355, 318-325].
Morimatsu
K
K
Department of Molecular Protozoology, Osaka University, Japan.
Horii
T
T
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D004277:DNA, Single-Stranded / Q000378:metabolism* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010777:Photochemistry; D011938:Rec A Recombinases / Q000378:metabolism* / Q000528:radiation effects; D014443:Tyrosine / Q000378:metabolism*; D014466:Ultraviolet Rays
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011938:Rec A Recombinases