Sites of reaction of Escherichia coli DNA gyrase on pBR322 in vivo as revealed by oxolinic acid-induced plasmid linearization

pBR322 DNA, linearized by lysis of an oxolinic acid-treated culture of Escherichia coli strain DK6recA- (pBR322) with sodium dodecyl sulfate, was purified, treated with DNA polymerase in the presence of the four deoxynucleoside triphosphates, and ligated to DNA linkers containing the XhoI recognition sequence. Most of the drug-resistant colonies resulting from transformation of E. coli with this material bore plasmids that appeared by restriction enzyme analysis to differ from pBR322 only by the introduction of an XhoI site. The XhoI sites in plasmids from 93 transformants were distributed unevenly around the pBR322 map. Maxam-Gilbert DNA sequence analysis of 36 of these plasmids, labeled at the 5' termini of the XhoI sites, revealed that 29 of them contained, in addition to the XhoI linker, a duplication of four base-pairs of the pBR322 sequence surrounding the linker. Therefore, oxolinic acid-induced linearization must have resulted in 5'-terminal extensions of four bases, the configuration known to result from oxolinic acid-induced DNA cleavage by DNA gyrase in vitro. The sequence data thus allowed the determination of the precise point at which linearization occurred, apparently by the abortion of a gyrase-DNA covalent intermediate that existed in vivo. When the 19 different sites of the 29 plasmids were compared, the following set of rules could be derived: (formula; see text) where N is any nucleotide, R is a purine, and Y is a pyrimidine. Cleavage occurred at the line between the eighth and ninth positions from the left. The parenthetical G and T were preferred secondarily to T and G, respectively, whereas T and G in the 13th position from the left were equally preferred. Several of these rules are similar to those proposed previously based on several in vitro gyrase cleavage sites. Some of our rules show dyad symmetry around the axis midway between the cleavage points in the two strands, while others are distinctly asymmetric.     

DNA gyrase complex with DNA: determinants for site-specific DNA breakage.

DNA gyrase catalyses DNA supercoiling by making a transient double-stranded DNA break within its 120-150 bp binding site on DNA. Addition of the inhibitor oxolinic acid to the reaction followed by detergent traps a covalent enzyme-DNA intermediate inducing sequence-specific DNA cleavage and revealing potential sites of gyrase action on DNA. We have used site-directed mutagenesis to examine the interaction of Escherichia coli gyrase with its major cleavage site in plasmid pBR322. Point mutations have been identified within a short region encompassing the site of DNA scission that reduce or abolish gyrase cleavage in vitro. Mapping of gyrase cleavage sites in vivo reveals that the pBR322 site has the same structure as seen in vitro and is similarly sensitive to specific point changes. The mutagenesis results demonstrate conclusively that a major determinant for gyrase cleavage resides at the break site itself and agree broadly with consensus sequence studies. The gyrase cleavage sequence alone is not a good substrate, however, and requires one or other arm of flanking DNA for efficient DNA breakage. These results are discussed in relation to the mechanism and structure of the gyrase complex.     

Sequence specificity of Bacillus subtilis DNA gyrase in vivo

Linearization of pBG0 (a hydrid between Escherichia coli plasmid pBR322 and Staphylococcus aureus plasmid pUB110) was performed by lysis of the oxolinic acid treated Bacillus subtilis protoplasts with sodium dodecyl sulfate. This plasmid DNA linearization was used both for a detailed mapping of DNA gyrase cleavage sites of various strength and for the nucleotide sequence determinations at the points of gyrase-mediated scission by introducing the XhoI linker DNA. A total of 40 plasmids carrying inserted XhoI linker were sequenced by labeling 3' termini of XhoI sites; 38 of them were found to contain a duplication of four base-pairs of the plasmid sequence flanking the linker, which were characteristic of the oxolinic acid-induced DNA cleavage by E. coli DNA gyrase in vitro and in vivo. The relative strength of these sequenced sites was established by comparing their positions to the sites mapped on the appropriate plasmid genome. This allowed us to propose a consensus sequence of B. subtilis DNA gyrase in vivo cleavage site:GNAT GATCATNC% MathType!MTEF!2!1!+-% feaafeart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXafv3ySLgzGmvETj2BSbqefm0B1jxALjhiov2D% aebbfv3ySLgzGueE0jxyaibaiiYdd9qrFfea0dXdf9vqai-hEir8Ve% ea0de9qq-hbrpepeea0db9q8as0-LqLs-Jirpepeea0-as0Fb9pgea% 0lrP0xe9Fve9Fve9qapdbaqaaeGacaGaaiaabeqaamaabaabcaGcba% GaaeikaiaabsfacaqGPaGaaeiiaiaabccacaqGGaGaaeiiaiaabcca% caqGOaGaae4raiaabMcacaqGGaGaaeiiaiaabccacaqGGaGaaeiiai% aabccacaqGGaGaaeiiaiaabccacaqGGaGaaeiiaiaabccacaqGGaGa% aeiiaiaabccacaqGOaGaaeyqaiaabMcaaaa!4E92!\[{\rm{(T) (G) (A)}}\]where N is any nucleotide. The bases in parentheses were preferred secondarily. The involvement of DNA gyrase in illegitimate recombination events in Bacillus subtilis is discussed.     

The role of DNA-gyrase from Bacillus subtilis during intermolecular irregular recombination]

The location of oxolinis acid-induced gyrase cleavage sites on pBR322 and pUB110 plasmid DNA in Bacillus subtilis cells has been studied and established. The treated Bacillus subtilis protoplasts were used in the study. Coordinates of the gyrase cleavage sites were compared to the location of the illegitimate recombination sites precisely mapped on the plasmid genomes. The obtained data indicate involvement of the DNA gyrase in formation of a fraction of recombinants in Bacillus subtilis.     

Formation of lambda transducing phage in vitro: involvement of DNA gyrase

We found that transducing phages carrying the gal or bio regions of the Escherichia coli genome were formed during in vitro packaging of endogenous lambda DNA. Structural analysis of the transducing phage genomes indicated that they were formed by abnormal excision of lambda prophage. Formation of transducing phages was stimulated by oxolinic acid, an inhibitor of DNA gyrase, implying that DNA gyrase participates in the abnormal excision of lambda prophage. When pBR322 DNA was added to the reaction mixture, transducing phages into which pBR322 had been inserted were produced at a high frequency. This reaction was also stimulated by oxolinic acid. Sequence analyses revealed that pBR322 is inserted into the sites of abnormal excision of the prophage. These results show that transducing phages can be formed by DNA gyrase-dependent illegitimate recombination in an in vitro system and that secondary recombination takes place frequently at the site where the first recombination occurs.     

Norfloxacin-induced DNA gyrase cleavage complexes block Escherichia coli replication forks, causing double-stranded breaks in vivo

Antibacterial quinolones inhibit type II DNA topoisomerases by stabilizing covalent topoisomerase-DNA cleavage complexes, which are apparently transformed into double-stranded breaks by cellular processes such as replication. We used plasmid pBR322 and two-dimensional agarose gel electrophoresis to examine the collision of replication forks with quinolone-induced gyrase-DNA cleavage complexes in Escherichia coli. Restriction endonuclease-digested DNA exhibited a bubble arc with discrete spots, indicating that replication forks had been stalled. The most prominent spot depended upon the strong gyrase binding site of pBR322, providing direct evidence that quinolone-induced cleavage complexes block bacterial replication forks in vivo. We differentiated between stalled forks that do or do not contain bound cleavage complex by extracting DNA under different conditions. Resealing conditions allow gyrase to efficiently reseal the transient breaks within cleavage complexes, while cleavage conditions cause the latent breaks to be revealed. These experiments showed that some stalled forks did not contain a cleavage complex, implying that gyrase had dissociated in vivo and yet the fork had not restarted at the time of DNA isolation. Additionally, some branched plasmid DNA isolated under resealing conditions nonetheless contained broken DNA ends. We discuss a model for the creation of double-stranded breaks by an indirect mechanism after quinolone treatment.     

The cleavage of DNA at phosphorothioate internucleotidic linkages by DNA gyrase.

We have constructed a plasmid which contains 22 copies of a 147 bp DNA fragment which contains the major DNA gyrase cleavage site from plasmid pBR322 (located at base-pair 990). We have found that this fragment is efficiently bound and cleaved by gyrase. The selectivity for the sequence corresponding to position 990 in pBR322 is maintained even when this site is located only 15 bp from one end of the 147 bp fragment. A strategy for the specific incorporation of a single thiophosphoryl linkage into the 147 bp fragment has been developed, and gyrase has been shown to catalyse efficient cleavage of fragments bearing phosphorothioate linkages at the gyrase cleavage site in one or both strands.     

Mechanism of illegitimate recombination: common sites for recombination and cleavage mediated by E. coli DNA gyrase

Summary Illegitimate recombination dependent on DNA gyrase in a cell-free system has previously been described. We have now mapped DNA gyrase cleavage sites in the vicinity of known recombination sites in pBR322. Among five recombination sites examined, three were found to coincide with a DNA gyrase cleavage site. This result suggests that the cleavage of DNA by DNA gyrase has a central role in the recombination process.     

In the presence of subunit A inhibitors DNA gyrase cleaves DNA fragments as short as 20 bp at specific sites.

A key step in the supercoiling reaction is the DNA gyrase-mediated cleavage and religation step of double-stranded DNA. Footprinting studies suggest that the DNA gyrase binding site is 100-150 bp long and that the DNA is wrapped around the enzyme with the cleavage site located near the center of the fragment. Subunit A inhibitors interrupt this cleavage and resealing cycle and result in cleavage occurring at preferred sites. We have been able to show that even a 30 bp DNA fragment containing a 20 bp preferred cleavage sequence from the pBR322 plasmid was a substrate for the DNA gyrase-mediated cleavage reaction in the presence of inhibitors. This DNA fragment was cleaved, although with reduced efficiency, at the same sites as a 122 bp DNA fragment. A 20 bp DNA fragment was cleaved with low efficiency at one of these sites and a 10 bp DNA fragment was no longer a substrate. We therefore propose that subunit A inhibitors interact with DNA at inhibitor-specific positions, thus determining cleavage sites by forming ternary complexes between DNA, inhibitors and DNA gyrase.     

A biochemical analysis of the interaction of DNA gyrase with the bacteriophage Mu, pSC101 and pBR322 strong gyrase sites: the role of DNA sequence in modulating gyrase supercoiling and biological activity

Replication of bacteriophage Mu DNA, a process requiring efficient synapsis of the prophage ends, takes place within the confines of the Escherichia coli nucleoid. Critical to ensuring rapid synapsis is the function of the SGS, a strong gyrase site, located at the centre of the Mu genome. Replacement of the SGS by the strong gyrase sites from pSC101 or pBR322 fails to support efficient prophage replication. To probe the unique SGS properties we undertook a biochemical analysis of the interaction of DNA gyrase with the Mu SGS, pSC101 and pBR322 sites. In binding and cleavage assays the order of efficacy was pSC101 > Mu SGS > pBR322. However, in supercoiling assays the Mu SGS (cloned into pUC19) exhibited a strong enhancement of gyrase-catalysed supercoiling over pUC19 alone; the pSC101 site showed none and the pBR322 site gave a moderate improvement. Most striking was the Mu SGS-dependent increase in processivity of the gyrase reaction. This highly processive supercoiling coupled with efficient binding may account for the unique biological properties of the SGS. The results emphasize the importance of the DNA substrate as an active component in modulating the gyrase supercoiling reaction, and in determining the biological roles of specialized gyrase sites.     

Evidence for a conformational change in the DNA gyrase-DNA complex from hydroxyl radical footprinting.

We have used the technique of hydroxyl radical footprinting to probe the complex between DNA gyrase and a 198 bp DNA fragment containing the preferred gyrase cleavage site from plasmid pBR322. We find that gyrase protects 128 bp from the hydroxyl radical with the central 13 bp (adjacent to the gyrase cleavage site) being most strongly protected. Flanking the central region are arms showing periodic protection from the reagent suggesting a helical repeat of 10.6 bp, consistent with the DNA being wrapped upon the enzyme surface. The presence of 5'-adenylyl-beta,gamma-imidodiphosphate or a quinolone drug causes alteration of the protection pattern consistent with a conformational change in the complex involving one arm of the wrapped DNA. The significance of these results for the mechanism of DNA supercoiling by gyrase is discussed.     

Replacement of the Bacteriophage Mu Strong Gyrase Site and Effect on Mu DNA Replication

The bacteriophage Mu strong gyrase site (SGS) is required for efficient replicative transposition and functions by promoting the synapsis of prophage termini. To look for other sites which could substitute for the SGS in promoting Mu replication, we have replaced the SGS in the middle of the Mu genome with fragments of DNA from various sources. A central fragment from the transposing virus D108 allowed efficient Mu replication and was shown to contain a strong gyrase site. However, neither the strong gyrase site from the plasmid pSC101 nor the major gyrase site from pBR322 could promote efficient Mu replication, even though the pSC101 site is a stronger gyrase site than the Mu SGS as assayed by cleavage in the presence of gyrase and the quinolone enoxacin. To look for SGS-like sites in the Escherichia coli chromosome which might be involved in organizing nucleoid structure, fragments of E. coli chromosomal DNA were substituted for the SGS: first, repeat sequences associated with gyrase binding (bacterial interspersed mosaic elements), and, second, random fragments of the entire chromosome. No fragments were found that could replace the SGS in promoting efficient Mu replication. These results demonstrate that the gyrase sites from the transposing phages possess unusual properties and emphasize the need to determine the basis of these properties.     

Genetic analysis of the strong gyrase site (SGS) of bacteriophage Mu: localization of determinants required for promoting Mu replication

The Mu strong gyrase site (SGS), located in the centre of the Mu genome, is required for efficient Mu replication, as it promotes synapsis of the prophage termini. Other gyrase sites tested, even very strong ones, were unable to substitute for the SGS in Mu replication. To determine the features required for its unique properties, a deletion analysis was performed on the SGS. For this analysis, we defined the 20 bp centred on the midpoint of the 4 bp staggered cleavage made by gyrase to be the 'core' and the flanking sequences to be the 'arms'. The deletion analysis showed that (i) approximately 40 bp of the right arm is required, in addition to core sequences, for both efficient Mu replication and gyrase cleavage; and (ii) the left arm was not required for efficient Mu replication, although it was required for efficient gyrase cleavage. These observations implicated the right arm as the unique feature of the SGS. The second observation showed that strong gyrase cleavage and Mu replication could be dissociated and suggested that even weak gyrase sites, if supplied with the right arm of the SGS, could promote Mu replication. Hybrid sites were constructed with gyrase sites that could not support efficient Mu replication. The SGS right arm was used to replace one arm of the strong pSC101 gyrase site or the weaker pBR322 site. The pSC101 hybrid site allowed efficient Mu replication, whereas the pBR322 hybrid site allowed substantial, but reduced, replication. Hence, it appears that optimal Mu replication requires a central strong gyrase site with the properties imparted by the right arm sequences. Possible roles for the SGS right arm in Mu replication are addressed.     

Effect of nalidixic acid and novobiocin on pBR322 genetic expression in Escherichia coli minicells.

The effects of two deoxyribonucleic acid (DNA) gyrase inhibitors, nalidixic acid and novobiocin, on the gene expression of plasmid pBR322 in Escherichia coli minicells were studied. Quantitative estimates of the synthesis of pBR322-coded polypeptides in novobiocin-treated minicells showed that the synthesis of a polypeptide of molecular weight of 34,000 (the tetracycline resistance protein) was reduced to 11 to 20% of control levels, whereas the amount of a polypeptide of 30,500 (the beta-lactamase precursor) was increased to as much as 200%. Nalidixic acid affected the synthesis of the tetracycline resistance protein similarly to novobiocin, although to a lesser extent. The effects of nalidixic acid were not observed in a nalidixic-resistant mutant; those induced by novobiocin were only partially suppressed in a novobiocin-resistant mutant. The synthesis of one of the inducible tetracycline-resistant proteins (34,000) coded by plasmid pSC101 was also reduced in nalidixic acid- and novobiocin-treated minicells. These results suggest that the gyrase inhibitors modified the interaction of ribonucleic acid polymerase with some promoters, either by decreasing the supercoiling density of plasmid DNA or by altering the association constant of the gyrase to specific DNA sites.     

DNA gyrase can cleave short DNA fragments in the presence of quinolone drugs.

We have analysed the DNA cleavage reaction of DNA gyrase using oligonucleotides annealed to a single-stranded M13 derivative containing a preferred gyrase cleavage site. We find that gyrase can cleave duplexes down to approximately 20 bp in size in the presence of the quinolone drugs ciprofloxacin and oxolinic acid. Ciprofloxacin shows a variation in its site specificity with an apparent preference for G bases adjacent to the cleavage sites, whereas oxolinic acid stimulates cleavage predominantly at the previously determined site. With either drug, cleavage will not occur within 6 bases from the end of a DNA duplex or a nick. We suggest that cleavage site specificity with short DNA duplexes is determined by drug-DNA interactions whereas with longer fragments the positioning effect of the DNA wrap around gyrase prescribes the site of cleavage.     

DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid.

Treatments in vivo of Escherichia coli with oxolinic acid, a potent inhibitor of DNA gyrase and DNA synthesis, lead to DNA cleavage when extracted chromosomes are incubated with sodium dodecyl sulfate. This DNA breakage has properties similar to those obtained in vitro with DNA gyrase reaction mixtures designed to assay production of supertwists: it is oxolinic acid-dependent, sodium dodecyl sulfate-activated, and at saturating drug concentrations produces double-strand DNA cleavage with a concommitant tight association of protein and DNA. In addition, identical treatments performed on a nalA mutant strain exhibit no DNA cleavage. Thus the DNA cleavage sites probably correspond to chromosomal DNA gyrase sites. Sedimentation measurements of the DNA cleavage products indicate that there are approximately 45 DNA breaks per chromosome. This value is similar to the number of domains of supercoiling found in isolated Escherichia coli chromosomes, suggesting one gyrase site per domain. At low oxolinic acid concentrations single-strand cleavages predominate after sodium dodecyl sulfate treatment, and the inhibition of DNA synthesis parallels the number of sites that obtain a single-strand scission. Double-strand breaks arise from the accumulation of single-strand cleavages in accordance with a model where each cleavage site contains two independent drug targets, one on each DNA strand. Since the nicking-closing subunit of gyrase is the target of oxolinic acid in vitro, we suggest that each gyrase site contains two nicking-closing subunits, one on each DNA strand, and that DNA synthesis requires both to be functional.