Probing of DNA-binding sites of Escherichia coli RecA protein utilizing 1-anilinonaphthalene-8-sulfonic acid.
RecA protein of Escherichia coli plays an essential role in homologous recombination of DNA strands. To analyze the interaction of RecA with single-stranded DNA (ssDNA), we performed a fluorescence competition assay employing 1-anilinonaphthalene-8-sulfonic acid (ANS) as an extrinsic fluorescent probe. ANS bound to RecA at three sites, leading to enhancement of ANS fluorescence. Addition of synthetic polynucleotides to the RecA-ANS complex in the absence of a nucleotide quenched the ANS fluorescence, indicating displacement of ANS molecules by ssDNA. Less effective quenching by poly(dA) suggests that the nucleoprotein filament on poly(dA) may differ from those on poly(dT) and poly(dC). A titration experiment with poly(dT) and poly(dA) showed clear stoichiometric binding of 3.5 nucleotides per protein. The site size for poly(dC) was 7.0, which could be explained by the formation of a double helix of poly(dC). ATP and other nucleotides also displaced the ANS. To identify ANS-binding sites, ANS was incorporated into RecA by UV irradiation, and fluorescent peptides were isolated from the proteolytic digest. Sequence analysis suggested that ANS binds to or near the ATP-binding region. These results suggest that the fluorescence quenching and photoincorporation assay using ANS may be useful for the analysis of the interaction of a protein and its ligand.
Masui
R
R
Department of Biology, Graduate School of Science, Osaka University, Japan.
Kuramitsu
S
S
D000227:Adenine Nucleotides / Q000737:chemistry; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000378:metabolism*; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*; D012492:Salts / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D014466:Ultraviolet Rays; D014508:Urea / Q000737:chemistry
D000227:Adenine Nucleotides; D000817:Anilino Naphthalenesulfonates; D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D012492:Salts; C027132:1-anilino-8-naphthalenesulfonate; D014508:Urea; D011938:Rec A Recombinases