The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [3H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a 'swivelase' role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, D-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.     

Regulation of the genes for E. coli DNA gyrase: Homeostatic control of DNA supercoiling

DNA gyrase is the bacterial enzyme responsible for converting circular DNA to a negatively supercoiled form. We show that the synthesis of DNA gyrase is itself controlled by DNA supercoiling; synthesis is highest when the DNA template is relaxed. The rates of synthesis in vivo of both the A and B subunits of DNA gyase are increased up to 10-fold by treatments that block DNA gyrase activity and decrease the supercoiling of intracellular DNA. Similarly, efficient synthesis of both gyrase subunits in a cell-free S-30 extract depends on keeping the closed circular DNA template in a relaxed conformation. The results suggest that DNA supercoiling in E. coli is controlled by a homeostatic mechanism. Synthesis of the RecA protein and several other proteins is also increased by treatments that relax intracellular DNA.     

Glutamate racemase is an endogenous DNA gyrase inhibitor

Almost all bacteria possess glutamate racemase to synthesize d-glutamate as an essential component of peptidoglycans in the cell walls. The enforced production of glutamate racemase, however, resulted in suppression of cell proliferation. In the Escherichia coli JM109/pGR3 clone, the overproducer of glutamate racemase, the copy number (i.e. replication efficiency) of plasmid DNA declined dramatically, whereas the E. coli WM335 mutant that is defective in the gene of glutamate racemase showed little genetic competency. The comparatively low and high activities for DNA supercoiling were contained in the E. coli JM109/pGR3 and WM335 cells, respectively. Furthermore, we found that the DNA gyrase of E. coli was modulated by the glutamate racemase of E. coli in the presence of UDP-N-acetylmuramyl-l-alanine, which is a peptidoglycan precursor and functions as an absolute activator for the racemase. This is the first finding of the enzyme protein participating in both d-amino acid metabolism and DNA processing.     

The effect of bacterial DNA gyrase inhibitors on DNA synthesis in mammalian mitochondria

Using isolated rat liver mitochondria, which have previously been shown to carry out true replicative DNA synthesis, we have obtained results which are in accord with the presence and functioning of a DNA gyrase in this organelle. The effects of the Escherichia coli DNA gyrase inhibitors, novobiocin, coumermycin, nalidixic acid and oxolinic acid, upon mtDNA replication suggest the involvement of the putative mitochondrial enzyme in various aspects of this process. First, the preferential inhibition of [³H]dATP incorporation into highly supercoiled DNA together with the appearance of labeled, relaxed DNA are consistent with the involvement of a gyrase in the process of generating negative supercoils in mature mtDNA. Second, the overall depression of incorporation of labeled dATP into mtDNA, including the reduction of radioactivity incorporated into replicative intermediates, suggests a ‘swivelase’ role for the putative gyrase, and this hypothesis is further supported by results obtained on sucrose gradient centrifugation of heat-denatured, d-loop mtDNA. Here, the synthesis of the completed clean circles is inhibited while 9 S initiator strand synthesis is not, suggesting that chain elongation is blocked by the gyrase inhibitors.     

Mu-like prophage strong gyrase site sequences: analysis of properties required for promoting efficient mu DNA replication

The bacteriophage Mu genome contains a centrally located strong gyrase site (SGS) that is required for efficient prophage replication. To aid in studying the unusual properties of the SGS, we sought other gyrase sites that might be able to substitute for the SGS in Mu replication. Five candidate sites were obtained by PCR from Mu-like prophage sequences present in Escherichia coli O157:H7 Sakai, Haemophilus influenzae Rd, Salmonella enterica serovar Typhi CT18, and two strains of Neisseria meningitidis. Each of the sites was used to replace the natural Mu SGS to form recombinant prophages, and the effects on Mu replication and host lysis were determined. The site from the E. coli prophage supported markedly enhanced replication and host lysis over that observed with a Mu derivative lacking the SGS, those from the N. meningitidis prophages allowed a small enhancement, and the sites from the Haemophilus and Salmonella prophages gave none. Each of the candidate sites was cleaved specifically by E. coli DNA gyrase both in vitro and in vivo. Supercoiling assays performed in vitro, with the five sites or the Mu SGS individually cloned into a pUC19 reporter plasmid, showed that the Mu SGS and the E. coli or N. meningitidis sequences allowed an enhancement of processive, gyrase-dependent supercoiling, whereas the H. influenzae or Salmonella serovar Typhi sequences did not. While consistent with a requirement for enhanced processivity of supercoiling for a site to function in Mu replication, these data suggest that other factors are also important. The relevance of these observations to an understanding of the function of the SGS is discussed.     

Interactions of subunits of DNA gyrase

Interaction of DNA gyrase A- and B-subunits during the process of DNA supercoiling was studied. For this purpose a E. coli Cour-1 mutant resistant to coumermycin and containing a mutation in the B-subunit of DNA gyrase was isolated and the influence of the DNA gyrase A-subunit specific inhibitor-nalidixic acid-on DNA supercoiling by wild-type and mutant enzymes was investigated. It turned out that the enzyme from the Cour-1 mutant strain was more sensitive to nalidixic acid than the DNA gyrase from the wild-type strain. Hence, the mutation affecting the B-subunit is capable to change A-subunit properties. That makes it possible to draw the conclusion about a close structural interaction of DNA gyrase subunits during DNA supercoiling.     

RNase HI overproduction is required for efficient full-length RNA synthesis in the absence of topoisomerase I in Escherichia coli

It has long been known that Escherichia coli cells deprived of topoisomerase I (topA null mutants) do not grow. Because mutations reducing DNA gyrase activity and, as a consequence, negative supercoiling, occur to compensate for the loss of topA function, it has been assumed that excessive negative supercoiling is somehow involved in the growth inhibition of topA null mutants. However, how excess negative supercoiling inhibits growth is still unknown. We have previously shown that the overproduction of RNase HI, an enzyme that degrades the RNA portion of an R-loop, can partially compensate for the growth defects because of the absence of topoisomerase I. In this article, we have studied the effects of gyrase reactivation on the physiology of actively growing topA null cells. We found that growth immediately and almost completely ceases upon gyrase reactivation, unless RNase HI is overproduced. Northern blot analysis shows that the cells have a significantly reduced ability to accumulate full-length mRNAs when RNase HI is not overproduced. Interestingly, similar phenotypes, although less severe, are also seen when bacterial cells lacking RNase HI activity are grown and treated in the same way. All together, our results suggest that excess negative supercoiling promotes the formation of R-loops, which, in turn, inhibit RNA synthesis.     

Plasmid DNA supercoiling and survival in long-term cultures of Escherichia coli: role of NaCl

The relationship between the survival of Escherichia coli during long-term starvation in rich medium and the supercoiling of a reporter plasmid (pBR322) has been studied. In aerated continuously shaken cultures, E. coli lost the ability to form colonies earlier in rich NaCl-free Luria-Bertani medium than in NaCl-containing medium, and the negative supercoiling of plasmid pBR322 declined more rapidly in the absence of NaCl. Addition of NaCl at the 24th hour restored both viability and negative supercoiling in proportion to the concentration of added NaCl. Addition of ofloxacin, a quinolone inhibitor of gyrase, abolished rescue by added NaCl in proportion to the ofloxacin added. This observation raises the possibility that cells had the ability to recover plasmid supercoiling even if nutrients were not available and could survive during long-term starvation in a manner linked, at least in part, to the topological state of DNA and gyrase activity.     

Staphylococcus aureus gyrase-quinolone-DNA ternary complexes fail to arrest replication fork progression in vitro. Effects of salt on the DNA binding mode and the catalytic activity of S. aureus gyrase

Type II topoisomerases bind to DNA at the catalytic domain across the DNA gate. DNA gyrases also bind to DNA at the non-homologous C-terminal domain of the GyrA subunit, which causes the wrapping of DNA about itself. This unique mode of DNA binding allows gyrases to introduce the negative supercoils into DNA molecules. We have investigated the biochemical characteristics of Staphylococcus aureus (S. aureus) gyrase. S. aureus gyrase is known to require high concentrations of potassium glutamate (K-Glu) for its supercoiling activity. However, high concentrations of K-Glu are not required for its relaxation and decatenation activities. This is due to the requirement of high concentrations of K-Glu for S. aureus gyrase-mediated wrapping of DNA. These results suggest that S. aureus gyrase can bind to DNA at the catalytic domain independent of K-Glu concentration, but high concentrations of K-Glu are required for the binding of the C-terminal domain of GyrA to DNA and the wrapping of DNA. Thus, salt modulates the DNA binding mode and the catalytic activity of S. aureus gyrase. Quinolone drugs can stimulate the formation of covalent S. aureus gyrase-DNA complexes, but high concentrations of K-Glu inhibit the formation of S. aureus gyrase-quinolone-DNA ternary complexes. In the absence of K-Glu, ternary complexes formed with S. aureus gyrase cannot arrest replication fork progression in vitro, demonstrating that the formation of a wrapped ternary complex is required for replication fork arrest by a S. aureus gyrase-quinolone-DNA ternary complex.     

Mechanisms for defining supercoiling set point of DNA gyrase orthologs: I. A nonconserved acidic C-terminal tail modulates Escherichia coli gyrase activity

DNA topoisomerases manage chromosome supercoiling and organization in all cells. Gyrase, a prokaryotic type IIA topoisomerase, consumes ATP to introduce negative supercoils through a strand passage mechanism. All type IIA topoisomerases employ a similar set of catalytic domains for function; however, the activity and specificity of gyrase are augmented by a specialized DNA binding and wrapping element, termed the C-terminal domain (CTD), which is appended to its GyrA subunit. We have discovered that a nonconserved, acidic tail at the extreme C terminus of the Escherichia coli GyrA CTD has a dramatic and unexpected impact on gyrase function. Removal of the CTD tail enables GyrA to introduce writhe into DNA in the absence of GyrB, an activity exhibited by other GyrA orthologs, but not by wild-type E. coli GyrA. Strikingly, a "tail-less" gyrase holoenzyme is markedly impaired for DNA supercoiling capacity, but displays normal ATPase function. Our findings reveal that the E. coli GyrA tail regulates DNA wrapping by the CTD to increase the coupling efficiency between ATP turnover and supercoiling, demonstrating that CTD functions can be fine-tuned to control gyrase activity in a highly sophisticated manner.     

An Escherichia coli DNA topoisomerase I mutant has a compensatory mutation that alters two residues between functional domains of the DNA gyrase A protein.

Nucleotide sequence analysis revealed that the compensatory gyrA mutation in Escherichia coli DM750 affects DNA supercoiling by interchanging the identities of Ala-569 and Thr-586 in the DNA gyrase A subunit. These residues flank Arg-571, a site for trypsin cleavage that splits gyrase A protein between DNA breakage-reunion and DNA-binding domains. The putative interdomain locations of the DM750 mutation and that of E. coli DM800 (in gyrase B protein) suggests that these compensatory mutations may reduce DNA supercoiling activity by altering allosteric interactions in the gyrase complex.     

A superhelical spiral in the Escherichia coli DNA gyrase A C-terminal domain imparts unidirectional supercoiling bias

DNA gyrase is unique among type II topoisomerases in that its DNA supercoiling activity is unidirectional. The C-terminal domain of the gyrase A subunit (GyrA-CTD) is required for this supercoiling bias. We report here the x-ray structure of the Escherichia coli GyrA-CTD (Protein Data Bank code 1ZI0). The E. coli GyrA-CTD adopts a circular-shaped beta-pinwheel fold first seen in the Borrelia burgdorferi GyrA-CTD. However, whereas the B. burgdorferi GyrA-CTD is flat, the E. coli GyrA-CTD is spiral. DNA relaxation assays reveal that the E. coli GyrA-CTD wraps DNA inducing substantial (+) superhelicity, while the B. burgdorferi GyrA-CTD introduces a more modest (+) superhelicity. The observation of a superhelical spiral in the present structure and that of the Bacillus stearothermophilus ParC-CTD structure suggests unexpected similarities in substrate selectivity between gyrase and Topo IV enzymes. We propose a model wherein the right-handed ((+) solenoidal) wrapping of DNA around the E. coli GyrA-CTD enforces unidirectional (-) DNA supercoiling.     

Dissection of the nucleotide cycle of B. subtilis DNA gyrase and its modulation by DNA

DNA topoisomerases catalyze the inter-conversion of different topological forms of DNA. While all type II DNA topoisomerases relax supercoiled DNA, DNA gyrase is the only enyzme that introduces negative supercoils into DNA at the expense of ATP hydrolysis. We present here a biophysical characterization of the nucleotide cycle of DNA gyrase from Bacillus subtilis, both in the absence and presence of DNA. B. subtilis DNA gyrase is highly homologous to its well-studied Escherichia coli counterpart, but exhibits unique mechanistic features. The active heterotetramer of B. subtilis DNA gyrase is formed by mixing the GyrA and GyrB subunits. GyrB undergoes nucleotide-induced dimerization and is an ATP-operated clamp. The intrinsic ATPase activity of gyrase is stimulated tenfold in the presence of plasmid DNA. However, in contrast to the E. coli homolog, the rate-limiting step in the nucleotide cycle of B. subtilis GyrB is ATP hydrolysis, not product dissociation or an associated conformational change. Furthermore, there is no cooperativity between the two DNA and ATP binding sites in B. subtilis DNA gyrase. Nevertheless, the enzyme is as efficient in negative supercoiling as the E. coli DNA gyrase. Our results provide evidence that the evolutionary goal of efficient DNA supercoiling can be realized by similar architecture, but differences in the underlying mechanism. The basic mechanistic features are conserved among DNA gyrases, but the kinetics of individual steps can vary significantly even between closely related enzymes. This suggests that each topoisomerase represents a different solution to the complex reaction sequence in DNA supercoiling.     

Peptides based on CcdB protein as novel inhibitors of bacterial topoisomerases

The ccd toxin-antitoxin system of the F plasmid encodes CcdB, a protein that poisons the essential Escherichia coli DNA gyrase, unique type IIA topoisomerase able to introduce negative supercoils into DNA. Based on CcdB structure, a series of linear peptide analogues were obtained by the solid-phase methodology. One of these peptides (CcdBET2) displayed inhibition of the supercoiling activity of bacterial DNA gyrase with a concentration required for complete inhibition (IC(100)=10 microM) lower than the wild type CcdB. For Topo IV, a second type IIA bacterial topoisomerase, CcdBET2 was better inhibited the relaxation activity with an IC(100) of 5 microM (wt CcdB>10 microM). The replacement of Gly, present in the three C-terminal amino acid residues, by Glu, abolished the capacity to inhibit the gyrase but not the Topo IV activities. These findings demonstrate that the mechanism by which CcdBET2 inhibits DNA gyrase is different of the mechanism by which inhibits Topo IV. Therefore, CcdBET2 is a new type II topoisomerase inhibitor with specificity for Topo IV.     

nifH promoter activity is regulated by DNA supercoiling in Sinorhizobium meliloti

In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown ceils, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoiling-dependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.