Ogawa H, Ogawa T
Department of Biology, Faculty of Science, Osaka University, Japan.
Adv Biophys. 1990;26:33-49. doi: 10.1016/0065-227x(90)90006-f.
Treatments that damage DNA or inhibit DNA synthesis in E. coli induce the expression of a set of functions called SOS functions that are involved in DNA repair, mutagenesis, arrest of cell division and prophage induction. Induction of SOS functions is triggered by inactivation of the LexA repressor or a phage repressor. Inactivation of these repressors results from their cleavage by the E. coli RecA protein in the presence of single-stranded DNA and a nucleoside triphosphate. We found that these cleavage reactions are controlled by two mechanisms in vitro: one is through the structural change of the RecA protein in the ternary complex, RecA-ssDNA-ATP-gamma-S. The active ternary complex is formed by binding of ATP-gamma-S to a complex of RecA protein and ssDNA. On the other hand, when the RecA protein binds to ATP-gamma-S prior to its binding to ssDNA, the resulting complex has no or only very weak cleavage activity toward the repressor. This structural change is negatively controlled by its C-terminal part. The loss of the 25 amino acid residues from the C-terminal leads the RecA protein to stable binding to dsDNA as well as ssDNA, and the protein takes the activated form for the repressor cleavage constitutively. The other mechanism is through the structural change of the repressor. The cleavage reaction of a phi 80cI repressor is greatly stimulated by the presence of d(G-G), and d(G-G) stimulates the cleavage by binding to the C-terminal half of the phi 80cI repressor. Moreover, the C-terminal fragment of the cleaved products of the 80cI repressor was able to cleave a phi 80cI-lambda chimeric repressor. These results strongly suggested that the active site of the repressor cleavage was located in the C-terminal domain of the repressor and that the C-terminal fragment produced by the cleavage could cleave the repressor.
在大肠杆菌中，破坏DNA或抑制DNA合成的处理会诱导一组称为SOS功能的功能表达，这些功能与DNA修复、诱变、细胞分裂停滞和原噬菌体诱导有关。SOS功能的诱导是由LexA阻遏物或噬菌体阻遏物的失活触发的。这些阻遏物的失活是由于它们在单链DNA和核苷三磷酸存在下被大肠杆菌RecA蛋白切割所致。我们发现，这些切割反应在体外受两种机制控制：一种是通过三元复合物RecA-ssDNA-ATP-γ-S中RecA蛋白的结构变化。活性三元复合物是由ATP-γ-S与RecA蛋白和ssDNA的复合物结合形成的。另一方面，当RecA蛋白在结合ssDNA之前先结合ATP-γ-S时，所形成的复合物对阻遏物没有或只有非常弱的切割活性。这种结构变化受其C末端部分的负调控。从C末端缺失25个氨基酸残基会导致RecA蛋白与dsDNA和ssDNA稳定结合，并且该蛋白持续采取用于切割阻遏物的活化形式。另一种机制是通过阻遏物的结构变化。d(G-G)的存在极大地刺激了φ80cI阻遏物的切割反应，并且d(G-G)通过与φ80cI阻遏物的C末端一半结合来刺激切割。此外，φ80cI阻遏物切割产物的C末端片段能够切割φ80cI-λ嵌合阻遏物。这些结果强烈表明，阻遏物切割的活性位点位于阻遏物的C末端结构域，并且切割产生的C末端片段可以切割阻遏物。
