The ε Subunit of DNA Polymerase III Is Involved in the Nalidixic Acid-Induced SOS Response in Escherichia coli

Type II DNA topoisomerases are essential enzymes involved in processes such as DNA replication, chromosome segregation, and transcription (7, 26, 55). Many clinically important antibacterial and antitumor agents target type II topoisomerases by altering the equilibrium of the topoisomerase reaction cycle to stabilize the normally transient intermediate called the cleavage complex (9, 11). Antibacterial quinolones such as nalidixic acid target DNA gyrase (and topoisomerase IV in certain bacteria) by stabilizing the cleavage complex, which consists of the topoisomerase linked via phosphotyrosine bonds to both 5′ ends of a staggered double-stranded DNA break (DSB). While the cleavage complex contains a latent DSB, this break is bridged by the covalently attached enzyme, and intact DNA is reconstituted when the enzyme completes the reaction cycle and dissociates. A variety of results argue that cleavage complex formation is necessary but not sufficient for cytotoxicity (reviewed in reference 11). Some cellular processing event apparently converts a subset of the cleavage complexes into cytotoxic lesions, because inhibition of RNA or protein synthesis can mitigate quinolone sensitivity (3, 10, 11, 28). However, the detailed mechanism(s) of cytotoxicity remains unclear. A number of observations argue that quinolone treatment leads to frank DSBs that are likely involved in cytotoxicity. First, treatment of cells with bactericidal concentrations of a quinolone causes free rotation of chromosomal DNA in the presence of ethidium bromide, which is characteristic of broken DNA ends (3). Second, mutational inactivation of proteins involved in DSB repair (RecA and RecBC) leads to drug hypersensitivity (23, 30). Finally, nalidixic acid induces the bacterial SOS response in a manner that is dependent on the RecBC enzyme, which requires double-strand ends for activity (31, 36). SOS is the bacterial DNA damage response, in which more than 20 genes are upregulated after DNA damage (13, 54). Notably, mutations in recA, recB, or recC eliminate the nalidixic acid-induced SOS response and cause hypersensitivity to nalidixic acid, implicating drug-induced DSBs in both the SOS response and cytotoxicity. DNA replication plays an important role in the cytotoxicity of antitumor agents that target type II topoisomerase in eukaryotes. Thus, eukaryotic cells are more sensitive to these inhibitors during S phase than during G1, and the DNA polymerase inhibitor aphidicolin abrogates sensitivity to topoisomerase inhibitors (8, 17, 57). There are conflicting reports on whether DNA replication is important in the bacterial response to quinolones. In favor of a role for DNA replication, Gudas and Pardee (14) found that the SOS response to nalidixic acid was blocked after temperature shift-up of a temperature-sensitive dnaA mutant. Furthermore, we recently found that replication forks are blocked in vivo at the sites of quinolone-stabilized cleavage complexes on replicating plasmids (41). The blocked forks were sometimes broken in vivo, raising the possibility that replication-dependent breaks could be a cytotoxic lesion. On the other hand, two reports argue against a role for DNA replication in quinolone action. Sassanfar and Roberts (43) reported that SOS was not prevented after a temperature shift of a dnaC(Ts) uvrB mutant. In a direct measure of quinolone cytotoxicity, Zhao et al. (58) found that temperature shift-up of a dnaB(Ts) mutant does not protect against killing by nalidixic acid or ciprofloxacin (a fluoroquinolone). In summary, while it is clear that quinolones effectively block DNA replication, the physiological consequences of this blocked replication are still uncertain. The only genes known to be required for the SOS response to nalidixic acid are recA and recBC (13, 54). In a prior study, we used an SOS reporter system to screen for Escherichia coli mutants specifically deficient in SOS induction upon nalidixic acid treatment, hoping to find additional genes involved in this process. However, all 18 mutants with this phenotype had insertions in recB or recC (37). Using the same SOS reporter construct, we also isolated a collection of mutants constitutive for the SOS response (39). Compared to a lexA mutant, all other insertion mutants were only partially constitutive for SOS. It is possible that a partially constitutive mutant would also be defective for SOS induction after nalidixic acid—such a mutant would probably have been missed in the Newmark et al. (37) screen for induction defects because it would be expressing the reporter construct regardless of the presence of nalidixic acid. We therefore examined the SOS response of each mutant in this collection, looking for mutants that are specifically defective in response to nalidixic acid. We found insertions in one gene, dnaQ, that cause this phenotype. The dnaQ gene encodes the ɛ subunit of DNA polymerase III, arguing that this subunit in particular, and the replication complex in general, is important in the nalidixic acid-induced SOS response. We also found that the dnaQ insertions are defective for induction of only a subset of SOS genes, implying greater complexity in the SOS response.