Sequence specificity of Bacillus subtilis DNA gyrase in vivo

Abstract

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 scissions 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: [sequence: see text] 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 DNA-gate of Bacillus subtilis gyrase is predominantly in the closed conformation during the DNA supercoiling reaction

“…In the presence of oxolinic acid (OXO) or ciprofloxacin (CFX), the 60-bp DNA substrate was cleaved, indicating that the lack of cleavage products is due to efficient religation. The main product comprises 24 bases, confirming that cleavage occurs preferentially at the sequence identified by Bashkirov and Zvingila (23). Altogether, our results validate the 60-bp dsDNA as an adequate gDNA for B. subtilis gyrase.…”

“…In pt-DNA, a 3Ј-bridging pt-DNA was introduced in strand A at the cleavage site (italicized). All substrates contain a preferred cleavage site for B. subtilis gyrase (23) in the center (bold) (Fig. 1D).…”

“…Gyrases exhibit limited sequence specificity for DNA cleavage. To facilitate studies on the DNA gate conformation by FRET, we designed a 60-bp substrate for gyrase that contains a preferred cleavage site in the center (23), flanked by a donor (A488) and acceptor fluorophore (A546 or A555) (Fig. 1B).…”

DNA gyrase with a single catalytic tyrosine can catalyze DNA supercoiling by a nicking-closing mechanism

“…The conformation of the G-segment bound to gyrase was investigated in smFRET experiments using a 60 bp DNA (33) as a model G-segment (Figure 4A). This DNA contains a preferred gyrase binding site in the center of the sequence (49), flanked by donor and acceptor fluorophores (33). We have previously established that the 60 bp DNA exists in two different conformations when bound to gyrase (33).…”

Dissection of the Nucleotide Cycle of B. subtilis DNA Gyrase and its Modulation by DNA

“…As the DNA substrate, a 60 bp double strand comprising a preferred binding site for B. subtilis DNA gyrase was used. 27 Again, Michaelis-Menten behavior was observed when the DNA concentration was varied at saturating ATP concentrations (Figure 7(a)), with a k cat value of 0.32(± 0.003) s − 1 and a K M of 0.55(± 0.02) μM (corresponding to 33 μM base-pairs). While the k cat value is similar to the one determined in the presence of negatively supercoiled DNA (0.55 s − 1 ), the K M value for the short DNA duplex is higher than the corresponding one for supercoiled DNA (6.2 μM base-pairs), most likely reflecting a lack in affinity due to its limited size.…”