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

Fisher, L.M.; Barot, H A; Cullen, Martin E.

doi:10.1002/j.1460-2075.1986.tb04375.x

Cited by 57 publications

(44 citation statements)

References 35 publications

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“…Propagation of the plasmids was in E. coli DH5ao [F-endA1 hsdR17 (rK-MK+) supE44 thi-J DNA sequencing. DNA sequences were determined both by the chain termination method (27) and by the chemical modification method (19) as previously described (6,30).…”

Section: Methodsmentioning

confidence: 99%

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins

1992

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We have determined the nucleotide sequence of a 5.3-kb segment of the Staphylococcus aureus chromosome that includes the gyrA and gyrB genes coding for both subunits of DNA gyrase, the enzyme that catalyzes ATP-dependent DNA supercoiling. The gene order at this locus, dnoA-dnaN-recF-gyrB-gyrA, is similar to that found in the BaciUus subtilis replication origin region. S. aureus recF, gyrB, and gyrA genes are closely spaced, occupy the same reading frame, and may be coordinately expressed. The S. aureus gyrB and gyrA genes encode 640-and 889-residue proteins, respectively, that share strong homology with other bacterial gyrase subunits, notably those from B. subtilis. These results are discussed in regard to the mechanism of DNA gyrase and its role as a target for the 4-quinolones and other antistaphylococcal agents.DNA gyrase, bacterial topoisomerase II, catalyzes ATPdependent DNA supercoiling and is essential for bacterial growth, being involved in DNA replication, transcription, and recombination (9, 37). The Escherichia coli enzyme consists of two A and two B subunits, the 97-and 90-kDa products of the gyrA (48 min) and gyrB genes (82 min), respectively (37). The subunits provide different functions in the DNA strand passage process by which gyrase mediates topological changes in DNA (20). The GyrA subunits form a transient double-strand break in DNA by linking to each complementary DNA strand via . A second DNA duplex is then passed through the gyrase-DNA gate in a process coupled to ATP binding and hydrolysis by the B subunits (20). Several lines of evidence show that the E. coli gyrase subunits are each organized into domains with discrete enzymatic functions (26), as first suggested by studies of a fragment of the gyrase B protein (7).Two classes of drug act by targeting DNA gyrase. Antibacterial 4-quinolones, such as ciprofloxacin, interrupt DNA breakage and resealing by the A subunits (8), whereas the coumarin drugs novobiocin and coumermycin interfere with ATP utilization by the B proteins (10, 32). Gyrase inhibitors have been widely used, particularly against Staphylococcus aureus, a gram-positive organism for which few clinically effective agents are available (13). Both ciprofloxacin and novobiocin are active against S. aureus, including methicillin-resistant strains (11,13,25,36), although resistance to fluoroquinolones has rapidly emerged (16,28,29 (2), and Neisseria gonorrhoeae (31), and from Pseudomonas putida (24). MATERIALS AND METHODSBacterial strains, plasmids, reagents. Cloning of the gyrBgyrA locus from S. aureus 81231 as overlapping inserts contained in plasmids pRH1, pRH2, and pRH3 has been described previously (14). Propagation of the plasmids was in E. coli DH5ao [F-endA1 hsdR17 (rK-MK+) supE44 thi-J recAI gyrA96 rell ]. E. coli XL-1 Blue {endAl hsdRJ7 (rK-MK+) supE44 thi-J X-recAl gyrA96 reLAl (lac) [F' proAB lacIVAMI5 TnlO(Tetr)

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Section: Methodsmentioning

confidence: 99%

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins

1992

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“…The 5'-end-labeling, using T4 polynucleotide kinase, was carried out as described previously (6 (29). Cells were grown in 10 liters of L broth to an optical density at 600 nm of -1.5, pelleted, washed with 50 mM Tris hydrochloride, pH 8.0-10% glycerol-1 mM dithiothreitol, and stored at -70°C as described previously (19).…”

Section: Methodsmentioning

confidence: 99%

“…Addition of detergent to gyrase-DNA complexes formed in the presence of oxolinic acid results in site-specific double-stranded DNA breakage and covalent attachment of the gyrase A subunits to each 5'-phosphate end via tyrosine (Tyr)-122 (6,7,9,15,22,31). Thus, the A subunits appear to promote DNA breakage and reunion during catalysis, a process interrupted by quinolone inhibitors.…”

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confidence: 99%

Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones

Cullen

Wyke

Kuroda

et al. 1989

Antimicrob Agents Chemother

193

166

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Nalidixic acid, enoxacin, and other antibacterial 4-quinolones inhibit DNA gyrase activity by interrupting DNA breakage and reunion by A subunits of the A2B2 gyrase complex. Despite their clinical importance, the mode of quinolone action and mechanisms of resistance are poorly understood at the molecular level. Using a DNA fragment enrichment procedure, we isolated the gyrA gene from a uropathogenic Escherichia coli strain that encodes a gyrase A protein cross-resistant to a variety of quinolones. When complemented with gyrase B subunit, the purified A protein reconstituted DNA supercoiling activity 100-fold more resistant to inhibition by enoxacin than the susceptible enzyme and failed to mediate quinolone-dependent DNA cleavage. Nucleotide sequence analysis revealed that the gene differed at 58 nucleotide positions compared with the K-12 gyrA sequence. The 875-amino-acid residue-resistant gyrase A protein differed at three positions from its wild-type E. coli K-12 counterpart: tryptophan, glutamate, and serine replaced serine, aspartate, and alanine residues at positions 83, 678, and 828, respectively. By genetic analysis of chimeric gyrA genes in a gyrA(Ts) background, we showed that the Ser-83-*Trp mutation in the gyrase A protein was solely responsible for high-level bacterial resistance to nalidixic acid and fluoroquinolones.Bacterial DNA is maintained in a negatively supercoiled state by DNA gyrase, an ATP-dependent type II DNA topoisomerase (10). Gyrase is essential for cell viability, being implicated in a range of DNA transactions, including DNA replication and recombination, and in the control of gene expression (8,35). Gyrase catalyzes DNA supercoiling by an interesting and unusual mechanism. It passes a duplex DNA segment through a transient double-stranded DNA break made within a 120-to 150-base-pair (bp) loop of DNA wrapped on the surface of the tetrameric A2B2 gyrase complex. DNA strand passage is the salient mechanistic feature that allows DNA supercoiling and ATP-independent DNA relaxation by gyrase and also accounts for its catenation or decatenation and DNA unknotting activities (3,16,18).Antibacterial quinolones such as oxolinic acid inhibit DNA supercoiling by gyrase in vitro and rapidly arrest DNA replication in vivo (2, 9, 12, 28). Addition of detergent to gyrase-DNA complexes formed in the presence of oxolinic acid results in site-specific double-stranded DNA breakage and covalent attachment of the gyrase A subunits to each 5'-phosphate end via tyrosine 7,9,15,22,31). Thus, the A subunits appear to promote DNA breakage and reunion during catalysis, a process interrupted by quinolone inhibitors. In contrast, the B subunits bind ATP and are the locus of action of coumarin antibiotics such as novobiocin (11,20,30). The A and B subunits can be individually purified but must be combined to generate the topoisomerase activities of gyrase (14, 19

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“…In pBR322, analysis of oxo/gyrase-induced DNA cleavage sites in vivo has suggested a weak consensus sequence; 5Ј RNNNRNR(TG)GRYC(TG)YNYN(GT)NY 3Ј, with cleavage occurring 5Ј of the sequence GRYC (underlined) on both strands (Lockshon and Morris, 1985). A mutational analysis of the major site in pBR322 revealed that cleavage is sensitive to point mutations at the cleavage site (residues G and C in the sequence GRYC) (Fisher et al, 1986). However, it should be recalled that this consensus might not be exclusive as DNA gyrase binds efficiently and breaks DNA in the pSC101 par locus and in the central region of bacteriophage Mu at sites that differ from it (Wahle and Kornberg, 1988;Pato et al, 1990).…”

Section: Introductionmentioning

confidence: 99%

In vivo cleavage of Escherichia coli BIME‐2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site

Espéli

Boccard

1997

Molecular Microbiology

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SummaryThe Escherichia coli chromosome contains about 300 bacterial interspersed mosaic elements (BIMEs). These elements, located at the 3Ј end of genes, are composed of three types of alternating repetitive extragenic palindromes (REPs). Based on the type of REP they contain and on their ability to interact with the integration host factor (IHF), BIMEs are subdivided into two families: BIME-1 elements contain an IHF binding site flanked by converging Y and Z1 REPs, whereas BIME-2 elements contain a variable number of alternating Y and Z2 REPs without an IHF site. Although some BIMEs have been implicated in the protection of mRNA against 3Ј exonucleolytic degradation, the main role of elements belonging to both families remains to be elucidated. In this paper, we used oxolinic acid, a drug that reveals potential sites of DNA gyrase action, to demonstrate that DNA gyrase interacts in vivo with BIME-2 elements. The frequency of cleavage varied from one element to another, and the cleavage pattern observed in elements containing several REPs indicated that DNA gyrase cut DNA every two REPs. A single cleavage site has been identified in the Y REP in six out of seven instances, and the nucleotide sequence of a 44 bp fragment containing the scission point displayed conserved residues at six positions. The lack of one of the conserved residues accounted for the absence of cleavage in most of the Z2 REPs. Our results also showed that cleaved REPs were always associated with another REP, suggesting that a pair of diverging REPs constitutes the target of DNA gyrase. DNA gyrase cleavage at repetitive BIME-2 elements may have consequences for DNA topology and genomic rearrangements.

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

Cited by 57 publications

References 35 publications

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins

Nucleotide sequence of the Staphylococcus aureus gyrB-gyrA locus encoding the DNA gyrase A and B proteins

Cloning and characterization of a DNA gyrase A gene from Escherichia coli that confers clinical resistance to 4-quinolones

In vivo cleavage of Escherichia coli BIME‐2 repeats by DNA gyrase: genetic characterization of the target and identification of the cut site

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