The epsilon subunit of DNA polymerase III Is involved in the nalidixic acid-induced SOS response in Escherichia coli

Quinolone antibacterial drugs such as nalidixic acid target DNA gyrase in Escherichia coli. These inhibitors bind to and stabilize a normally transient covalent protein-DNA intermediate in the gyrase reaction cycle, referred to as the cleavage complex. Stabilization of the cleavage complex is necessary but not sufficient for cell killing--cytotoxicity apparently results from the conversion of cleavage complexes into overt DNA breaks by an as-yet-unknown mechanism(s). Quinolone treatment induces the bacterial SOS response in a RecBC-dependent manner, arguing that cleavage complexes are somehow converted into double-stranded breaks. However, the only proteins known to be required for SOS induction by nalidixic acid are RecA and RecBC. In hopes of identifying additional proteins involved in the cytotoxic response to nalidixic acid, we screened for E. coli mutants specifically deficient in SOS induction upon nalidixic acid treatment by using a dinD::lacZ reporter construct. From a collection of SOS partially constitutive mutants with disruptions of 47 different genes, we found that dnaQ insertion mutants are specifically deficient in the SOS response to nalidixic acid. dnaQ encodes DNA polymerase III epsilon subunit, the proofreading subunit of the replicative polymerase. The deficient response to nalidixic acid was rescued by the presence of the wild-type dnaQ gene, confirming involvement of the epsilon subunit. To further characterize the SOS deficiency of dnaQ mutants, we analyzed the expression of several additional SOS genes in response to nalidixic acid using real-time PCR. A subset of SOS genes lost their response to nalidixic acid in the dnaQ mutant strain, while two tested SOS genes (recA and recN) continued to exhibit induction. These results argue that the replication complex plays a role in modulating the SOS response to nalidixic acid and that the response is more complex than a simple on/off switch.     

Role of recombinational repair in sensitivity to an antitumour agent that inhibits bacteriophage T4 type II DNA topoisomerase

The bacteriophage T4-encoded type II DNA topoisomerase is the major target for the antitumour agent m-AMSA (4-(9-acridinylamino)methanesulphon-m-anisidide) in phage-infected bacterial cells. Inhibition of the purified enzyme by m-AMSA results in formation of a cleavage complex that contains the enzyme covalently attached to DNA on both sides of a double-strand break. In this article, we provide evidence that this cleavage complex is responsible for inhibition of phage growth and that recombinational repair can reduce sensitivity to the antitumour agent, presumably by eliminating the complex (or some derivative thereof). First, topoisomerase-deficient mutants were shown to be resistant to m-AMSA, indicating that m-AMSA inhibits growth by inducing the cleavage complex rather than by inhibiting enzyme activity. Second, mutations in several phage genes that encode recombination proteins (uvsX, uvsY, 46 and 59) increased the sensitivity of phage T4 to m-AMSA, strongly suggesting that recombination participates in the repair of topoisomerase-mediated damage. Third, m-AMSA stimulated recombination in phage-infected bacterial cells, as would be expected from the recombinational repair of DNA damage. Finally, m-AMSA induced the production of cleavage complexes involving the T4 topoisomerase within phage-infected cells.     

Iron‐sulfur cluster carrier proteins involved in the assembly of Escherichia coli NADH:ubiquinone oxidoreductase (complex I)

The NADH:ubiquinone oxidoreductase (respiratory complex I) is the main entry point for electrons into the Escherichia coli aerobic respiratory chain. With its sophisticated setup of 13 different subunits and 10 cofactors, it is anticipated that various chaperones are needed for its proper maturation. However, very little is known about the assembly of E. coli complex I, especially concerning the incorporation of the iron‐sulfur clusters. To identify iron‐sulfur cluster carrier proteins possibly involved in the process, we generated knockout strains of NfuA, BolA, YajL, Mrp, GrxD and IbaG that have been reported either to be involved in the maturation of mitochondrial complex I or to exert influence on the clusters of bacterial complex. We determined the NADH and succinate oxidase activities of membranes from the mutant strains to monitor the specificity of the individual mutations for complex I. The deletion of NfuA, BolA and Mrp led to a decreased stability and partially disturbed assembly of the complex as determined by sucrose gradient centrifugation and native PAGE. EPR spectroscopy of cytoplasmic membranes revealed that the BolA deletion results in the loss of the binuclear Fe/S cluster N1b.     

Isolation of SOS constitutive mutants of Escherichia coli

The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.