Photocross-links between single-stranded DNA and Escherichia coli RecA protein map to loops L1 (amino acid residues 157-164) and L2 (amino acid residues 195-209).
To function as a repair and recombination protein, RecA has to be assembled as an active filament on single-stranded DNA in the presence of ATP or its analogs. We have identified amino acids in the primary DNA binding site of RecA that interact with single-stranded DNA by photocross-linking. A nucleoprotein complex consisting of RecA protein bound to a monosubstituted oligonucleotide bearing a 5-iododeoxyuracil cross-linking moiety was irradiated with long wavelength ultraviolet radiation to effect cross-linking with RecA protein. Subsequent trypsin digestion, followed by purification and peptide sequencing, revealed the cross-linking of two independent peptides, amino acid residues 153-169 and 199-216. Met164 from loop L1 and Phe203 from loop L2 were determined to be the exact points of cross-linking. Thus, our data confirm and extend predictions about the DNA binding domain of RecA protein based on the molecular structure of RecA (Story, R. M., Weber, I. T., and Steitz, T. A. (1992) Nature 355, 318-325).
Genetics and Biochemistry Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1810, USA.
V A
VA
Malkov
R D
RD
Camerini-Otero
D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010777:Photochemistry; D017433:Protein Structure, Secondary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism* / Q000528:radiation effects; D014357:Trypsin; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011938:Rec A Recombinases; D014357:Trypsin