Sassanfar M, Roberts J W
Section of Biochemistry, Molecular and Cell Biology, Cornell University, Ithaca, NY 14853.
J Mol Biol. 1990 Mar 5;212(1):79-96. doi: 10.1016/0022-2836(90)90306-7.
The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.
大肠杆菌的SOS基因包括许多DNA修复基因，它们可被DNA损伤诱导。虽然诱导过程中的核心生化事件，即通过单链DNA与ATP结合激活RecA蛋白以促进LexA阻遏物的裂解已为人所知，但DNA损伤后引发这种激活的细胞事件尚未得到很好的理解。我们在此提供证据表明，典型诱变剂紫外线损伤后的主要诱导途径需要活跃的复制叉；这一结果支持了这样的模型，即DNA复制会留下间隙，在损伤诱导的损伤处延伸停止，从而提供激活RecA蛋白的单链DNA。为了定量检测诱导信号的直接产物——激活的RecA蛋白，我们设计了一种测定完整LexA阻遏物消失速率的方法。利用这种方法，我们研究了诱导过程的早期阶段。DNA损伤后几分钟内即可检测到LexA的裂解，且其发生在蛋白质合成不存在的情况下。通过追踪LexA在细胞中的重新积累，我们检测到了DNA修复和诱导信号的消失。利用这种方法，我们测量了野生型和各种突变细胞中LexA的含量，表征了各种诱导处理后诱导信号产生的动力学和条件，最后，证明了紫外线诱导SOS时对DNA复制的需求。
