S. aureus DNA Gyrase Product Background
Highly purified S. aureus DNA gyrase is a type II topoisomerase encoded by two genes GyrA and GyrB from staphylococcus aureus. DNA gyrase is an essential topoisomerase that supercoils DNA through a process of strand breakage/resealing and DNA wrapping (3). As a type II enzyme, gyrase is unique in its ability to negatively supercoil a relaxed plasmid DNA substrate. S. aureus DNA gyrase is also the target for quinolone-based antibacterial agents which act by subverting the enzyme into a DNA damaging agent. TopoGEN offers purified DNA gyrase from the gram positive bacterium, Staphylococcus aureus for use in all aspects of drug development and screening assays. The enzyme is very active in decatenating and supercoiling KDNA (TG2013) but will also negatively supercoil relaxed plasmid DNA. The S. aureus enzyme is a heterotetramer of GyrA2GyrB2 and is purified as His tagged subunits that are reassembled to make active enzyme (4).
S. aureus DNA Gyrase Quality Control Tests
DNA gyrase subunits were cloned overexpressed and purified using the proprietary company methods. A single band on SDS-PAGE was detected by CB staining for each subunit. Cross contamination by topo I was assessed by assaying for relaxation of supercoiled DNA under conditions optimized for type I activity. Under these conditions, after 2 hours of incubation with supercoiled plasmid DNA, no relaxation products were detectable.
A test for nuclease contamination was carried out by assaying for the formation of linear KDNA and linear plasmid DNA. Incubations of 1 µg of catenated KDNA or supercoiled DNA (4 hrs. at 37°C in the presence of 10 mM MgCl2) were performed. Linear DNA or breakdown products were not generated under these conditions.
The subunits are better than 95% pure based upon SDS-PAGE. Neither subunit is active in the absence of the other and neither subunit displays nucleolytic activity. These data show that host (E. coli) is not contributing to the activity of individual subunits of S. aureus gyrA or B. This was confirmed by Western blotting probings with anti-GyrA IgG specific to E. coli (data not shown).
TopoGEN provides samples of *three buffers that are used to assay S. aureus DNA Gyrase. The buffers are:
5X Assay Stock (0.5 ml provided) contains the following solutes:
- 375 mM Tris-HCl (pH 7.5)
- 37.5mM MgCl2
- 37.5mM DTT
- 0.375 mg/ml BSA
- 150mM KCl
- 10X ATP Stock: 20 mM ATP (0.25 mL). Enzyme requires 2.0 mM ATP FINAL CONCENTRATION
- 5X Potassium Glutamate Stock: 2.5M (0.25 mL). Enzyme requries 500 mM K-Glu FINAL CONCENTRATION.
*Note: These buffers are provided to allow the customer to establish a working control for the Gyrase assay. Additional buffer may be required. Store all buffers at -20°C.
Assays should be performed in a final volume of 20 ul by adding appropriate amounts of buffers, DNA enzyme (add enzyme last to initiate the assay). Typical assay would be:
(Enzyme diluted to working concentration using dilution buffer or titrate as 2 fold serial dilutions.)
Incubate 30 min 37°C, stop by addition of SDS to 1% (not included); add Bromophenol blue/glycerol loading dye, run 1% agarose gel with relaxed/supercoiled markers. Optional: digest with proteinase K and repurify DNA by phenol extraction. Gels should be stained with Ethidium Bromide (0.5 ug/ml 20-30 min), destained in water for 15-30 min at room temperature and photodocumented.
If necessary, dilute the enzyme using the following buffer (1x) supplied with the enzyme.
50 mM Tris-HCl [pH 7.5], 100 mM NaCl, 2mM b- DTT, 1 mM EDTA, 10% glycerol.
Modifications of the Gyrase Assay: Detecting DNA cleavages.
Gyrase is a target for several antibiotics that induce the enzyme to cleave the plasmid DNA substrate (3,5). Quinolone drugs are known to induce cleavages concurrent with covalent complex formation; thus, to detect such complexes in drug screening experiments, it is essential that proteinase K digestions be carried out and that reaction products repurified by phenol/chloroform extraction. Additionally, the conditions that are optimal for cleavage detection, differ slightly from those optimized for catalytic assays. For DNA Gyrase, we find that reduced potassium glutamate (200 mM final concentration) is ideal for cleavage complex formation.
To resolve nicked and linear DNA cleavages, we recommend running 1% agarose gels containing 0.5 u/ml EB in the gel and running buffer. Destain for 15 min prior to photodocumenting the results. It is essential to include linear DNA markers in these gels.
- Distilled water – 8ul
- 5X Assay stock – 4ul
- 10X ATP Stock – 2ul
- 5X K-Glu Stock – 4ul
- 0.1 ug supercoiled Plasmid DNA – 1ul
- Purified enzyme (variable) – 1ul Added last
- The 1x cleavage buffer contains:
- 75mM Tris-HCl (pH 7.5)
- 7.5mM MgCl2
- 7.5mM DTT
- 2 mM ATP
- 75 ug/ml BSA
- 30mM KCl
- 200mM K-Glu
Reviews and Citations
Phillips JW, Goetz MA, Smith SK, Zink DL, Polishook J, Onishi R, Salowe S, Wiltsie J, Allocco J, Sigmund J, Dorso K, Lee S, Skwish S, de la Cruz M, Martin J, Vicente F, Genilloud O, Lu J, Painter RE, Young K, Overbye K, Donald RGK, Singh SB: Discovery of Kibdelomycin, A Potent New Class of Bacterial Type II Topoisomerase Inhibitor by Chemical-Genetic Profiling in Staphylococcus aureus. Cell Chemistry and Biology 2011, 18: 955-965. doi: 10.1016/j.chembiol.2011.06.011.
Peng H, Marians K: DNA Topoisomerase Protocols Volume I 1999. p. 163 TGVi
Wang J: DNA Topoisomerases. Ann. Rev. Biochem 1996, 65:635-692
This enzyme is shipped on dry ice and should be stored at -70°C. It is stable undiluted for at least 6 months in this concentrated state. The enzyme can be aliquoted on first thawing to minimize damage from multiple freeze thaw cycles.