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.     

Prophage induction by DNA topoisomerase II poisons and reactive-oxygen species: role of DNA breaks.

Various compounds were evaluated for their ability to induce prophage lambda in the Escherichia coli WP2s(lambda) microscreen assay. The inability of a DNA gyrase subunit B inhibitor (novobiocin) to induce prophage indicated that inhibition of the gyrase's ATPase was insufficient to elicit the SOS response. In contrast, poisons of DNA gyrase subunit A (nalidixic acid and oxolinic acid) were the most potent inducers of prophage among the agents examined here. This suggested that inhibition of the ligation function of subunit A, which also has a DNA nicking activity, likely resulted in DNA breaks that were available (as single-stranded DNA) to act as strong SOS-inducing signals, leading to prophage induction. Agents that both intercalated and produced reactive-oxygen species (the mammalian DNA topoisomerase II poisons, adriamycin, ellipticine, and m-AMSA) were the next most potent inducers of prophage. Agents that produced reactive-oxygen species only (hydrogen peroxide and paraquat) were less potent than adriamycin and ellipticine but more potent than m-AMSA. Agents that intercalated but did not generate reactive-oxygen species (actinomycin D) or that did neither (teniposide) were unable to induce prophage, suggesting that intercalation alone may be insufficient to induce prophage. These results illustrate the variety of mechanisms (and the relative effectiveness of these mechanisms) by which agents can induce prophage. Nonetheless, these agents may induce prophage by producing essentially the same type of DNA damage, i.e., DNA strand breaks. The potent genotoxicity of the DNA gyrase subunit A poisons illustrates the genotoxic consequences of perturbing an important DNA-protein complex such as that formed by DNA and DNA topoisomerase.     

Increased expression of biodegradative threonine dehydratase of Escherichia coli by DNA gyrase inhibitors

The synthesis of inducible biodegradative threonine dehydratase of Escherichia coli increased several-fold in the presence of the DNA gyrase inhibitors, nalidixic acid and coumermycin. Temperature-sensitive gyrB mutants expressed higher levels of dehydratase as compared to an isogenic gyrB+ strain. Immunoblotting experiments showed increased synthesis of the dehydratase protein in the presence of gyrase inhibitors; addition of rifampicin and chloramphenicol to cells actively synthesizing enzyme preventing new enzyme production. Increased expression of dehydratase by gyrase inhibitors was accompanied by relaxation of supercoiled DNA.     

Recombination between chloroplast DNAs does not occur in sexual crosses of Oenothera

Summary Induction of the SOS genes is required for efficient repair of damaged DNA in Escherichia coli. SOS induction by nalidixic acid or oxolinic acid, two inhibitors of DNA gyrase, requires the RecBC enzyme of E. coli. We report here that the nuclease activity of RecBC enzyme is not needed for SOS induction by these agents. We suggest that the unwinding activity of RecBC enzyme produces single-stranded DNA which activates the RecA protein to stimulate LexA repressor cleavage and SOS induction.     

DNA gyrase is a host factor required for transposition of Tn5

We show that DNA gyrase is required for transposition of Tn5. Coumermycin, a potent inhibitor of DNA gyrase subunit B, inhibits transposition in a wild-type strain, but has no effect on strains carry ing a coumermycin-resistant allele in gyrB. In addition, strains containing a thermolabile subunit A of gyrase (gyrA43) are defective for transposition at a nonpermissive temperature. The requirement for gyrase is due to a requirement for supercoiled DNA. We showed this by introducing into the gyrA43 strain a deletion of the gene encoding topoisomerase I. The introduction of the second mutation caused an increase in the superhelical density of DNA as well as an increase in the transposition frequency. This also implies that if the DNA is supercoiled there is no further requirement for gyrase. Experiments with coumermycin support this, because the drug does not inhibit transposition if the recipient DNA remains supercoiled. This indicates that if the DNA acting as recipient of the transposon is deficient in supercoils, it will be a poor substrate for transposition. We also describe a system in which a gene on a multicopy plasmid can be efficiently introduced into the Escherichia coli chromosome.     

SOS independent survival against mitomycin C induced lethality in a rifampicin-nalidixic acid-resistant mutant of Escherichia coli

A combination of specific rifampicin-resistant (rpoB87) and nalidixic acid-resistant (gyrA87) mutations results in a marked increase in the survival of Escherichia coli against mitomycin C-induced lethality in mutants defective for SOS induction and excision repair. Although the response does not seem to be obligatorily dependent upon the RecA protein, the efficiency is markedly increased in its presence, even in a conventionally inactive form. This response is not elicited against lethality due to ultraviolet radiation or N-methyl-N' -nitro-N-nitrosoguanidine exposure. The combination of rpoB87 and gyrA87 mutations also greatly alleviates post-mitomycin C degradation of DNA under SOS non-inducible conditions. It is proposed that the rpoB subunit of RNA polymerase and gyrA subunit of DNA gyrase could participate in the repair of certain types of DNA damage, such as cross-links, in a mode independent of SOS-regulated excision repair and post-replication repair.     

Characterization of the Nucleoid-associated Protein YejK

Nucleoid-associated proteins play an important role in condensing chromosomal DNA and regulating gene expression. We report here the characterization of the nucleoid-associated protein YejK, which was detected in a yeast two-hybrid screen using the ParE subunit of topoisomerase IV as bait. The purified protein likely exists in a monomer-dimer equilibrium in solution and can form tetramers. Cross-linking of the protein bound to DNA suggests that the active form could be either a dimer or tetramer. YejK, which is present at about 24,000 copies of monomer per mid-log phase cell, binds double-stranded DNA with a site size of 12–14 base pairs/monomer, does not display a significant preference for either bent compared with straight DNA or supercoiled compared with relaxed DNA, and untwists DNA somewhat as it binds. YejK binds RNA, but not single-stranded DNA, with 65% of the avidity with which it binds DNA. However, cells deleted for yejK do not show defects in either RNA or protein synthesis. YejK interacts with all the subunits of both DNA gyrase and topoisomerase IV and has measurable effects on their activities. In the presence of YejK, relaxation of negatively supercoiled DNA by topoisomerase IV becomes distributive, whereas relaxation of positively supercoiled DNA is stimulated. Relaxation of negatively supercoiled DNA by DNA gyrase is inhibited, whereas the extent of supercoiling of relaxed DNA is limited. A YejK-GFP chimera is an effective marker for the nucleoid in live cell imaging.     

Positively supercoiled plasmid DNA is produced by treatment of Escherichia coli with DNA gyrase inhibitors.

A procedure has been developed whereby the relative amounts of the topoisomers of E. coli plasmid can be determined for cells grown under a variety of conditions. Several applications of the procedure are presented. Addition of either novobiocin or oxolinic acid, two inhibitors of DNA gyrase, gives rise to positively supercoiled plasmid. A likely model for the introduction of positive supercoils, involving DNA gyrase itself, is discussed. Oxolinic acid is also shown to induce linearization of plasmid in vivo. Starvation of cells for ATP is shown to cause relaxation of plasmid. The shift of a gyrB temperature-sensitive strain to the restrictive temperature is also shown to cause plasmid relaxation. Finally, it is noted that polyamine starvation of E. coli has no detectable effect on the distribution of topoisomers.     

SOS induction in a subpopulation of structural maintenance of chromosome (Smc) mutant cells in Bacillus subtilis

The structural maintenance of chromosome (Smc) protein is highly conserved and involved in chromosome compaction, cohesion, and other DNA-related processes. In Bacillus subtilis, smc null mutations cause defects in DNA supercoiling, chromosome compaction, and chromosome partitioning. We investigated the effects of smc mutations on global gene expression in B. subtilis using DNA microarrays. We found that an smc null mutation caused partial induction of the SOS response, including induction of the defective prophage PBSX. Analysis of SOS and phage gene expression in single cells indicated that approximately 1% of smc mutants have fully induced SOS and PBSX gene expression while the other 99% of cells appear to have little or no expression. We found that induction of PBSX was not responsible for the chromosome partitioning or compaction defects of smc mutants. Similar inductions of the SOS response and PBSX were observed in cells depleted of topoisomerase I, an enzyme that relaxes negatively supercoiled DNA.     

Probing the two-gate mechanism of DNA gyrase using cysteine cross-linking.

Cross-linking a pair of novel cysteine residues on either side of the bottom dimer interface of DNA gyrase blocks catalytic supercoiling. Limited strand passage is allowed, but release of the transported DNA segment (T segment) via opening of the bottom dimer interface is prevented. In contrast, ATP-independent relaxation of negatively supercoiled DNA is completely abolished, suggesting that T-segment entry via the bottom gate is blocked. These findings support a two-gate model for supercoiling by DNA gyrase and suggest that relaxation by gyrase is the reverse of supercoiling. Cross-linking a truncated version of gyrase (A64(2)B2), which lacks the DNA wrapping domains, does not block ATP-dependent relaxation. This indicates that passage of DNA through the bottom dimer interface is not essential for this reaction. The mechanistic implications of these results are discussed.     

Phenotypes conferred by the Bacillus subtilis recM13 mutation and the din23 fusion.

The din23 fusion encodes a B. subtilis SOS-inducible regulatory region fused to the E. coli lacZ gene (Love et al., 1985). A strain encoding the din23 fusion and a recM13 allele showed low-level constitutive beta-galactosidase expression, was induced for beta-galactosidase production by DNA gyrase inhibitors but not by DNA-damaging agents, and was slightly induced by a variety of agents which do not normally induce the SOS regulon. The din23 fusion itself resulted in high levels of spontaneous prophage induction in wild-type, recM- and recA-hosts, despite the fact that the din23recM13 strain was not induced for beta-galactosidase production by DNA-damaging agents. The results suggest that the recM gene may be involved with the regulation of the RecA protease-mediated SOS response, while the din23 gene may be involved with the regulation of an alternative function which results in the cleavage of prophage repressor.     

Heat-stable and heat-labile thymidylate synthases B of Bacillus subtilis: comparison of the nucleotide and amino acid sequences

Heat treatment of wild-type Escherichia coli cells led to a transient relaxation of negatively supercoiled plasmid DNA and there was no recovery of DNA torsional strain in the DNA in gyrA mutant cells. After heat treatment, DnaK and GroEL proteins were synthesized continuously in the gyrA mutant cells, whereas they were synthesized only transiently in wild-type cells. Thus, change in superhelical density of the DNA correlated with the temperature-induced expression of heat shock proteins. Inhibitors of DNA gyrase (nalidixic acid, novobiocin), an organic solvent (ethanol) and a psychotropic drug (chlorpromazine) all stimulated relaxation of cellular DNA over the same concentration range that induces heat shock proteins. As DNA relaxation was induced by heat treatment or chemicals in an rpoH mutant, the process is not the result of induced synthesis of heat shock proteins.     

Impact of DNA gyrase inhibition by antisense ribozymes on rec A in <Emphasis Type="Italic">E. coli</Emphasis>

The chromosome of E. coli is maintained in a negatively supercoiled state, and supercoiling levels are affected by growth phase and a variety of environmental stimuli. Regulation of DNA supercoiling yields a complex spectrum of effects on the E. coli recA system. Previous studies indicated that inhibition of DNA gyrase by antibiotics that act on the DNA gyrase A subunit results in turning on the recA system. Here we show that antisense ribozymes that act on the DNA gyrase A subunit can also induce recA. We used real time PCR and immunoblot to analyze the impact of DNA gyrase A inhibition by antisense ribozymes on recA expression. When gyrase A was inhibited by the RNase P mediated antisense ribozymes the expression of recA was induced around 130-fold as seen by real time PCR analysis. This suggests that repair pathway is induced by antisense ribozymes against DNA gyrase A and the damage produced by these ribozymes may be similar to that produced by fluroquinolones.     

Coumarin and quinolone action in archaebacteria: evidence for the presence of a DNA gyrase-like enzyme.

The action of novobiocin and coumermycin (two coumarins which interact with the gyrB subunit of eubacterial DNA gyrase) and ciprofloxacin (a fluoroquinolone which interacts with the gyrA subunit of DNA gyrase) was tested on several archaebacteria, including five methanogens, two halobacteria, and a thermoacidophile. Most strains were sensitive to doses of coumarins (0.02 to 10 micrograms/ml) which specifically inhibit DNA gyrase in eubacteria. Ciprofloxacin inhibited growth of the haloalkaliphilic strain Natronobacterium gregoryi and of the methanogen Methanosarcina barkeri. In addition, ciprofloxacin partly relieved the sensitivity to coumarins (and vice versa). Novobiocin inhibited DNA replication in Halobacterium halobium rapidly and specifically. Topological analysis has shown that the 1.7-kilobase plasmid from Halobacterium sp. strain GRB is negatively supercoiled; this plasmid was relaxed after novobiocin treatment. These results support the existence in archaebacteria of a coumarin and quinolone target related to eubacterial DNA gyrase.     

A class of gyrase mutants of Salmonella typhimurium show quinolone-like lethality and require Rec functions for viability

We have identified a new class of DNA gyrase mutants of Salmonella typhimurium that show chronic derepression of the SOS regulon. Thus, these mutants mimic the response of wild-type cells to gyrase inhibitors of the quinolone family. SOS induction by conditional lethal mutations gyrA208 or gyrB652, like that mediated by quinolones, is completely dependent on the function of the recB gene product. Introduction of recA or recB null mutations into these strains exacerbates their temperature-sensitive phenotype and prevents growth at the otherwise permissive temperature of 37°C. Selection of suppressors that concomitantly restore growth at 37°C and SOS induction in a recB^? background yielded mutations that relieve the RecB requirement for homologous recombination; namely, sbcB mutations as well as mutations at a new locus that was named sbcE. Such mutations also restore SOS induction in quinolone-treated gyr⁺recB^? strains. These findings indicate that Rec functions are needed for growth of the gyrase mutants at 37°C and suggest that recombinational repair intermediates constitute the SOS-inducing signal in the mutants as well as in quinolone-treated wild-type bacteria. Unlike quinolones, however, the gyr mutations described in this study do not cause detectable accumulation of ‘cleavable’ gyrase–DNA complexes in plasmid or chromosomal DNA. Yet gyrA208 (the only allele tested) was found to trigger RecB-mediated reckless degradation of chromosomal DNA in recA^? cells at restrictive temperatures. Indirect evidence suggests that double-stranded DNA ends, entry sites for the RecBCD enzyme, are generated in the gyr mutants by the breakage of DNA-replication forks. We discuss how this could occur and how recombinational rescue of collapsed replication forks could account for cell survival (and SOS induction) in the gyr mutants as well as in quinolone-treated bacteria.     

An atypical type II topoisomerase from Mycobacterium smegmatis with positive supercoiling activity

Topoisomerases are essential ubiquitous enzymes, falling into two distinct classes. A number of eubacteria including Escherichia coli, typically contain four topoisomerases, two type I topoisomerases and two type II topoisomerases viz. DNA gyrase and topoisomerase IV. In contrast several other bacterial genomes including mycobacteria, encode for one type I topoisomerase and a DNA gyrase. Here we describe a new type II topoisomerase from Mycobacterium smegmatis which is different from DNA gyrase or topoisomerase IV in its characteristics and origin. The topoisomerase is distinct with respect to domain organization, properties and drug sensitivity. The enzyme catalyses relaxation of negatively supercoiled DNA in an ATP-dependent manner and also introduces positive supercoils to both relaxed and negatively supercoiled substrates. The genes for this additional topoisomerase are not found in other sequenced mycobacterial genomes and may represent a distant lineage.     

Real-time detection of DNA topological changes with a fluorescently labeled cruciform

Topoisomerases are essential cellular enzymes that maintain the appropriate topological status of DNA and are the targets of several antibiotic and chemotherapeutic agents. High-throughput (HT) analysis is desirable to identify new topoisomerase inhibitors, but standard in vitro assays for DNA topology, such as gel electrophoresis, are time-consuming and are not amenable to HT analysis. We have exploited the observation that closed-circular DNA containing an inverted repeat can release the free energy stored in negatively supercoiled DNA by extruding the repeat as a cruciform. We inserted an inverted repeat containing a fluorophore-quencher pair into a plasmid to enable real-time monitoring of plasmid supercoiling by a bacterial topoisomerase, Escherichia coli gyrase. This substrate produces a fluorescent signal caused by the extrusion of the cruciform and separation of the labels as gyrase progressively underwinds the DNA. Subsequent relaxation by a eukaryotic topoisomerase, human topo IIα, causes reintegration of the cruciform and quenching of fluorescence. We used this approach to develop a HT screen for inhibitors of gyrase supercoiling. This work demonstrates that fluorescently labeled cruciforms are useful as general real-time indicators of changes in DNA topology that can be used to monitor the activity of DNA-dependent motor proteins.     

The DNA supercoiling-sensitive expression of the Salmonella typhimurium his operon requires the his attenuator and is modulated by anaerobiosis and by osmolarity

Bacterial cells possess a subset of genes whose expression correlates with changes in DNA supercoiling brought about by anaerobic growth and by growth at high osmolarity. It has been shown previously that expression of the histidine biosynthetic operon of Salmonella typhimurium is derepressed by relaxation of supercoiled DNA. Here, we confirm that a his::MudJ operon fusion in S. typhimurium can be induced by treatment with the DNA gyrase inhibitor novobiocin in a dose-dependent manner, and show that the level of derepression is higher in stationary phase than in mid-exponential phase cultures. Furthermore, expression of his is repressed by anaerobiosis and by osmolarity, two environmental parameters which increase the negative supercoiling of bacterial DNA. Novobiocin induction of his is also repressed by growing the cells either at high osmolarity or anaerobically. Both environmental repression and novobiocin induction of his require the his attenuator. In addition, derepression of his expression by novobiocin and its repression by anaerobiosis or osmolarity are independent of the stringent response gene, relA.