Masui R, Kuramitsu S
Department of Biology, Graduate School of Science, Osaka University, Japan.
Biochemistry. 1998 Sep 1;37(35):12133-43. doi: 10.1021/bi980541p.
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.
大肠杆菌的RecA蛋白在DNA链的同源重组中起着至关重要的作用。为了分析RecA与单链DNA（ssDNA）的相互作用，我们进行了一项荧光竞争测定，采用1-苯胺基萘-8-磺酸（ANS）作为外在荧光探针。ANS在三个位点与RecA结合，导致ANS荧光增强。在没有核苷酸的情况下，向RecA-ANS复合物中添加合成多核苷酸会淬灭ANS荧光，这表明ssDNA取代了ANS分子。聚（dA）的淬灭效果较差，表明聚（dA）上的核蛋白细丝可能与聚（dT）和聚（dC）上的不同。用聚（dT）和聚（dA）进行的滴定实验表明，每个蛋白质有3.5个核苷酸的明显化学计量结合。聚（dC）的位点大小为7.0，这可以通过聚（dC）双螺旋的形成来解释。ATP和其他核苷酸也能取代ANS。为了确定ANS结合位点，通过紫外线照射将ANS掺入RecA中，并从蛋白水解消化物中分离出荧光肽。序列分析表明，ANS结合在ATP结合区域或其附近。这些结果表明，使用ANS的荧光淬灭和光掺入测定法可能有助于分析蛋白质与其配体的相互作用。
